,1,3Related genes and characteristic analysis of trophoblast cells during early embryo developmental cessation
Yue Zhang1, Ying Feng2, Fang Ma,1,3通讯作者: 马芳，教授，研究方向：糖生物学与生殖医学。E-mail:mafangmed@126.com
编委: 杜茁
收稿日期:2020-08-8修回日期:2020-10-2网络出版日期:2020-10-20
| 基金资助: |
Editorial board:
Received:2020-08-8Revised:2020-10-2Online:2020-10-20
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作者简介 About authors
张悦,在读硕士研究生,专业方向：母婴医学。E-mail:
摘要
滋养层细胞对维持正常胚胎植入、生长发育有重要作用。研究停育胚胎的滋养层细胞的基因表达差异有助于了解胚胎发育停止或不良妊娠结局的发生发展机制。本研究通过对26例正常妊娠、胚胎停育妇女的绒毛组织进行全转录组测序和初步生物信息学分析,发现胚胎停育组存在436个差异基因,其中406个mRNA为显著上调基因,32个mRNA为显著下调基因。基因富集分析显示这些基因显著富集于免疫相关功能、细胞间黏附等方面,如淋巴细胞激活、髓系细胞激活、细胞外基质及胶原连接等,其潜在调控通路富集到补体及凝血级联反应和细胞外基质降解等条目。此外,本研究利用WGCNA共表达分析得到和差异基因存在共表达关系的lncRNA。根据模块功能不同,绘制了两个网络图,可得4个关键基因,分别为VSIG4、C1QC、CD36和SPP1。本研究得到的这些差异基因可作为对胚胎停育具有潜在影响的关键分子,所富集到的条目可为深入了解胚胎发育停止或不良妊娠结局的病因及机制提供理论依据及方向。
关键词：
Abstract
Trophoblast cells play essential roles in the maintenance of normal embryo implantation, growth and development. The study of abnormal gene changes in trophoblastic cells from arrested embryos is helpful to understand the developmental mechanism of embryo developmental cessation or adverse pregnancy outcomes. In this study, we sequenced and analyzed the transcriptomes of the villi from ten women who have undergone abortion with either normal pregnancy or embryo development cessation. We found that there were 436 differentially expressed genes, of which 406 mRNA were significantly up-regulated and 32 mRNA were significantly down-regulated. Gene enrichment analysis showed that these genes were significantly enriched in immune-related functions and intercellular adhesion, such as lymphocyte activation, myeloid cell activation, extracellular matrix and collagen junction. And their potential regulatory pathways were enriched in terms of complement and coagulation cascade, extracellular matrix degradation. In addition, in this study the co-expression analysis of WGCNA was used to obtain the lncRNA with co-expression relationship with the differential genes. According to the different functions of the modules, two network diagrams were drawn, and four key genes were obtained, namely VSIG4, C1QC, CD36 and SPP1. These differential genes obtained in this study can be used as key molecules with potential effects on embryo development cessation. The enriched entries can provide a theoretical basis and new direction for further understanding of the etiology and mechanism of embryo development cessation or adverse pregnancy outcomes.
Keywords：
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本文引用格式
张悦, 冯颖, 马芳. 早期停育胚胎的滋养层细胞相关基因与特征分析. 遗传[J], 2020, 42(10): 1004-1016 doi:10.16288/j.yczz.20-144
Yue Zhang.
早期绒毛组织的细胞构成主要指滋养层细胞(trophoblast)[1]。根据细胞功能及形态的特点,滋养层细胞可分为细胞滋养层、合体滋养层和绒毛外滋养层细胞。其中,细胞滋养层分化为合体滋养层参与营养、分泌、代谢等功能[2],而绒毛外滋养层细胞主要参与迁移、侵袭作用入侵子宫内膜及血管,建立母胎循环及启动血管重塑[3]。绒毛滋养层细胞作为直接和母体子宫内膜接触的细胞,目前认为对维持胚胎正常发育有3大功能：侵袭迁移功能、内分泌功能(包括自分泌及旁分泌)及免疫调节功能。绒毛外滋养层细胞通过分泌黏附因子(整合素、钙黏素、选择素及免疫球蛋白超家族[4])对子宫内膜上皮细胞进行识别及黏附,并通过分泌水解酶(基质金属蛋白酶、纤维蛋白酶等)降解植入部位的细胞外基质进行迁移、浸润,以完成正常的胚胎植入。其中,整合素家族和子宫内膜容受性密切相关,整合素αvβ3表达不足会降低子宫内膜容受性,导致胚胎着床能力低下,引起流产或胚胎发育迟滞[5]。而过高的水解酶会导致滋养层细胞过度浸润,引起妊娠期高血压、侵入性胎盘疾病等病理妊娠[6]。滋养层细胞还通过分泌细胞因子(如表皮生长因子EGF[7]、转化生长因子TGF[8]及集落刺激因子CSF-1[9]等)调节自身增殖分化、能量代谢及侵袭功能等。其中,EGF表达异常可能造成滋养层细胞侵袭不足、胎盘血管重铸障碍,最终引起流产[7]。TGF的表达不足影响滋养层细胞分化,造成胎盘发育不良[8]。此外,滋养层细胞可分泌激素(人绒毛膜促性腺激素hCG、孕酮等)维持早期胚胎发育及妊娠。hCG能维持及促进母体卵巢分泌雌、孕激素,并作为早孕时判断胚胎发育停止的指标之一。因此,绒毛滋养层细胞对胚胎植入、发育及胎盘生长、妊娠结局等均有重要影响,而绒毛滋养层细胞的异常形态及功能障碍往往提示胚胎停育或不良妊娠结局。据报道[9],普通光学显微镜下观察可发现停育胚胎的绒毛组织形态异常,出现绒毛间质水肿,合体滋养层核固缩等病理改变。经凋亡小体检测发现,停育胚胎的绒毛滋养层细胞中细胞凋亡数目明显增多[10]。考虑到目前胚胎停育的病因复杂,且部分患者未能找到病因,深入研究异常滋养层细胞变化能在一定程度上对胚胎停育及不良妊娠结局做出解释。本研究借助全转录组测序技术,对停育胚胎的绒毛组织进行大规模的基因筛选及挖掘,寻找异常变化的基因,预测其变化对滋养层细胞生物学功能的潜在影响,为临床上不明原因性胚胎停育的病因及分子生物学机制做出一定解释。
1 材料与方法
1.1 样本收集及制备
26例患者的绒毛组织均来自华西第二医院门诊手术室,其中10例用于转录组测序,其中5例为无生育要求进行人工流产的患者(对照组),5例患者为经B超检测确诊胚胎停育进行清宫术的患者(胚胎停育组)。剩余16例用于验证测序结果,其中9例为无生育要求进行人工流产的患者(对照组),7例患者为经B超检测确诊胚胎停育进行清宫术的患者(胚胎停育组)。两组别中对照组患者平均年龄29.2岁,孕周46.4 d,胚胎停育组患者平均年龄30.6岁,孕周60.2 d,患者临床信息(参与转录组测序的临床样本)见表1。患者术后的绒毛组织立即置于4℃冰盒中防止RNA降解。加入10%中性福尔马林固定12 h,依次通过50%酒精、70%酒精梯度脱水,进行石蜡包埋。所有的总RNA提取、逆转录、扩增、文库构建由南京极光基因科技有限公司完成。本研究通过了四川大学华西第二医院伦理委员会的审查,参与研究的所有患者均签署了知情同意书。1.1 样本收集及测序
以Trizol法提取总RNA,每个样本取3 μg的总RNA作为起始量构建文库,使用试剂盒Ribo-ZeroTM GoldKits (北京New England Biolabs公司)去除样品中的rRNA,并在两端添加测序引物结合位点,在Illumina HiSeq2000平台上进行测序。测序结束后,通过去除低质量序列,去接头污染,去除rRNA后得到高质量序列(clean reads),后续分析基于高质量序列。采取HiSAT2将过滤后的RNA-seq数据同人基因组(hg19)进行比对。1.2 差异mRNA的筛选
采用FPKM (fragments per kilobase per millon mapped fragments)定量估计基因的总表达量,采用R包ggplot2对总基因数绘制箱式图进行比较。采用DEseq进行差异表达分析,定义P值≤0.05或者|log2 ratio|≥1的基因为差异mRNA。采用R包ggplot2对差异mRNA绘制火山图,采用R包pheatmap对差异mRNA绘制热图。1.3 差异mRNA的注释及富集分析
采用omicshare在线平台(https://www.omicshare. com/tools/home/soft/getsoft.html)对筛选出的基因进行了GO基因功能(gene ontology, GO)和KEGG通路(Kyoto encyclopedia of genes and genomes)的富集分析,以FDR<0.05作为显著性的阈值。1.4 加权共表达网络分析WGCNA筛选核心lncRNA
使用R包WGCNA构建加权基因共表达网络并进行模块划分[11]。首先准备基因矩阵,将差异mRNA按差异倍数(fold change, FC)排序,选择前20个差异mRNA及所有lncRNA纳入分析。同时,剔除了平均表达量低于0.5的低质量RNA,并利用函数hclust进行聚类,剔除了离群样本。采用pick soft threshold计算权重值,采用power=9,使用blockwiseModules构建无尺度网络[12],对基因进行模块分析。每个模块的最低基因容量为50,利用函数plot dendro and colors绘制树状图并对每个模块进行颜色可视化。最后,确定了差异mRNA所在的两个模块作为目标模块,进行后续分析。Table 1
表1
表1患者临床信息(参与转录组测序的临床样本)
Table 1
| 序号 | 临床诊断 | 年龄(岁) | 孕产次 | 孕周(d) | 是否可探胎心 | β-HCG(mIU/mL) |
|---|---|---|---|---|---|---|
| 1 | 人工流产 | 24 | G2P0+1 | 42 | 胎芽不清 | - |
| 2 | 人工流产 | 34 | G4P0+3 | 43 | 胎芽不清 | - |
| 3 | 人工流产 | 29 | G3P1+1 | 49 | 可见胎心搏动 | - |
| 4 | 人工流产 | 31 | G2P1 | 47 | 可见胎心搏动 | - |
| 5 | 人工流产 | 28 | - | 51 | 可见胎心搏动 | - |
| 6 | 胚胎停育 | 30 | 55 | 未见胎心 | 11,951.4 | |
| 7 | 胚胎停育 | 36 | G2P1 | 50 | 未见胎心 | 12,612.1 |
| 8 | 胚胎停育 | 27 | G1P0 | 57 | 未见胎心 | 69,220 |
| 9 | 胚胎停育 | 27 | G1P0 | 80 | 未见胎心 | - |
| 10 | 胚胎停育 | 33 | G1P0 | 59 | 未见胎心 | 32,594.4 |
新窗口打开|下载CSV
1.5 差异mRNA及lncRNA共表达网络的构建
选择WGCNA中富集到的两个模块,根据关联强度(weight)和关联数(edge)筛选最有可能和差异mRNA互作的lncRNA。lncRNA筛选标准为weight值>0.6且和差异mRNA至少存在一条直接连接。使用cytoscape软件绘制网络图。1.6 差异表达mRNAs和lncRNAs实时荧光定量PCR验证
为了验证测序结果的可靠性,选取了10个mRNAs (包括后续分析所得模块中的核心mRNA,VSIG4、C1QC、CD36和SPP1)和4个lncRNA进行实时荧光定量PCR（quantitative real-time PCR,qRT-PCR）验证。纳排标准与前期测序的样本一致,能有效保证实验结果的可靠性。根据Ensemble ((美国Thermo Fisher公司),引物序列见表2。以GAPDH作为内参基因,使用2-ΔΔCt法计算两组中差异表达mRNAs和lncRNAs的相对表达量。
Table 2
表2
表2实时荧光定量PCR引物序列表
Table 2
| 序列 | 类型 | 基因名称 | 上游序列(5?→3?) | 下游序列(5?→3?) |
|---|---|---|---|---|
| 1 | mRNA | C1QC | ATCCTGGGAAAAATGGCCCC | GAGGACCGCGTTGAATCTGA |
| 2 | VSIG4 | TCCAGCAGGCAAAGTACCAG | GTTTCTGGACACGGAGCTCA | |
| 3 | SPP1 | GATGACCATGTGGACAGCCA | AACCACACTATCACCTCGGC | |
| 4 | CD36 | GACCGAGGAAGCCACTTTGA | TAAGCAGGTCTCCAACTGGC | |
| 5 | NCKAP1L | TGTCTTCCACTCCCGAATGC | TGCAGTGGACAAAGTGAGCA | |
| 6 | FGD2 | TGCTACGCATTCCTCACTGG | ATAGAGCACGAGGGGGTCAT | |
| 7 | LILRB5 | ACCCTGCTGTGTCAGTCATG | GTAGGACCTGATTGCGCTGT | |
| 8 | FOLR2 | GCACCACAAGACAAAGCCAG | CAGGTTGGGTGAGCACTCAT | |
| 9 | DPPA3 | CCAGGGTCTCCACAAATGCT | ATTTCCCTGAGGACTGCTGC | |
| 10 | MX1 | TCGGAGGCTACAGGAAGACT | TTTGCGATGTCCACTTCGGA | |
| 11 | lncRNA | CLRN1-AS1 | GAAAGTCTGAAGCCAGGCCT | CTTTGGGCTTGCACAGTCAC |
| 12 | AC104809.4 | CGTGGGCTCGTCTAAGTGTT | GCACTGAGCTGTTTGCAGTC | |
| 13 | LINC01136 | ACCTCAGAGGCTACCCACAT | AGAAGAAATCCAGGGGCTGC | |
| 14 | USP27X-AS1 | TGCAACCAGAGGAACTGCAA | AGGTGGACCTATGGGCTTCT |
新窗口打开|下载CSV
1.7 统计学方法
采用R语言(3.6.1)进行算法分析及可视化图形。患者的临床信息采用描述性统计,两组别间所有mRNA采用独立样本t检验,所有数据分析采用双侧检验,以P≤0.05表示差异有统计学意义。2 结果与分析
2.1 正常与胚胎停育组绒毛组织存在436个差异mRNA、41个差异lncRNA
经高通量测序、数据过滤后,10例绒毛样本的基因总体表达值(图1A),log2(FPKM+1)值集中于2~3,说明各样本间基因数均一,测序质量良好,具有良好的可比性。经过差异表达分析后,发现有436个mRNA在两组间具有统计学差异,胚胎停育组相较于正常对照有406个mRNA显著上调,32个mRNA显著下调(图1B)。此外,发现41个lncRNA在两组间具有统计学差异,胚胎停育组相较于正常对照有28个lncRNA显著上调,13个lncRNA显著下调(图1C)。对差异mRNA及lncRNA进行表达聚类分析,从蓝到黄表示表达水平从低到高,发现各组均可形成独立的簇,说明两组别间生物学重复较好。图1
新窗口打开|下载原图ZIP|生成PPT图1差异基因的表达情况
A：10例样本的基因总体表达量(箱式图);B：两组别的差异mRNA分析(热图);C：两组别的差异lncRNA分析(热图)。
Fig. 1Expression of differential genes
2.2 代表性差异mRNA及lncRNA的qRT-PCR验证结果与转录组结果一致
为了确定测序结果的正确性,分别选择了10个mRNA (包括后续分析所得模块中的核心mRNA, VSIG4、C1QC、CD36和SPP1)和4个lncRNA进行实时荧光定量PCR (图2)。图2A、B结果是通过转录组测序所得的差异mRNA和lncRNA相对表达量。在胚胎停育组中,C1QC、VSIG4、SPP1、CD36、NCKAP1L、FGD2、LILRB5、FOLR2基因的mRNA水平显著高于对照组,DPPA3、MX1基因的mRNA水平显著低于对照组。对于lncRNA而言,CLRN1- AS1、AC104809.4、LINC01136、USP27X-AS1的水平显著高于对照组。图2 C、D结果是通过实时荧光定量PCR所得的差异mRNA和lncRNA相对表达量。在胚胎停育组中,C1QC、VSIG4、SPP1、CD36、NCKAP1L、FGD2、LILRB5、FOLR2基因的mRNA水平显著高于对照组,DPPA3、MX1基因的mRNA水平显著低于对照组。对于lncRNA而言,CLRN1- AS1、AC104809.4、LINC01136、USP27X-AS1的水平显著高于对照组。此结果与转录组测序结果相符合。为了更为直观地比较两种方法所得结果的一致性,计算了两组间的相对表达比值(胚胎停育组相对于对照组的差异倍数) (图2,E和F),在转录组结果中显著上调/下调的mRNA和lncRNA在实时荧光定量PCR中也呈现显著上调/下调的趋势。由此推断,这些差异mRNA及lncRNA在qRT-PCR中的表达模式与转录组结果一致,说明本研究的测序结果具有一定的可信度。图2
新窗口打开|下载原图ZIP|生成PPT图2差异mRNA及lncRNA的qRT-PCR、转录组验证结果
A：两组别间差异mRNA相对表达量(转录组测序);B：两组别间差异lncRNA相对表达量(转录组测序);C：两组别间差异mRNA相对表达量(实时荧光定量PCR);D：两组别的差异lncRNA相对表达量(实时荧光定量PCR);E：差异mRNA相对表达比值=胚胎停育组/对照组(取log2对数后);F：差异lncRNA相对表达比值=胚胎停育组/对照组(取log2对数后)。
Fig. 2qRT-PCR and transcriptome validation among differential mRNAs and lncRNAs
2.3 差异mRNA功能富集于细胞外基质黏附、免疫功能类别
为了深入分析差异mRNA的具体功能,选择436个mRNA进行GO分析,共富集到54个条目(FDR<0.05) (图3A)。其中,分别富集到生物学过程(biological process, BP)、细胞组分(cellular component, CC)和分子功能(molecular function, MF)的基因数分别为26条、17条及11条。在BP条目下的富集程度最高前3个的条目为细胞内过程(cellular process)、生物调节(biological regulation)和生物学过程调节(regulation of biological process)。在CC条目下的富集程度最高前3个的条目为细胞(cell)、细胞组分(cell part)和细胞器(organelle)。在MF条目下的富集程度最高前3个的条目为连接(binding)、催化过程(catalytic activity)和分子传导过程(molecular transducer activity)。436个mRNA进行KEGG富集分析,共富集到36个条目(FDR<0.05) (图3B)。其中,代谢相关通路富集程度最高的是脂质代谢(lipid metabolism)和碳水化合物代谢(carbohydrate metabolism),其他二级信号通路富集程度最高前3个分别是免疫系统(immune system)、感染性疾病(infectious diseases)和信号传导(signal transduction)。为进一步明确二级信号通路下属的具体条目,按置信度从低到高将所有信号通路进行排列,列出了前10条富集程度最高的信号通路(表3)。从表3可知,胚胎停育组中差异基因富集于补体及凝血级联反应、细胞外基质、吞噬体、血小板活及微生物感染相关的通路。已有的研究已经表明,过度的补体系统激活[13]、凝血系统激活[14]、细胞外基质降解[15]、血小板激活[16]均引起免疫系统过度攻击胚胎并导致流产的发生,而富集于微生物感染相关通路可能是由于免疫失衡后,机体对胚胎的攻击和感染性疾病类似,且胚胎停育后植入部位易引起感染。图3
新窗口打开|下载原图ZIP|生成PPT图3差异mRNA的GO功能和KEGG通路富集分析
A：两组别差异mRNA的GO功能分析;B：两组别差异mRNA的KEGG功能分析。
Fig. 3GO function enrichment and KEGG pathway analysis of differential mRNAs
2.4 筛选细胞外基质黏附、免疫功能模块下具有共表达关系的6个核心lncRNA
为筛选和差异mRNA有共表达关系的lncRNA,采用加权共表达网络WGCNA (weighted gene co- expression network analysis)分析差异mRNA及lncRNA的相关性。WGCNA是基于待测基因间的共表达关系对基因进行模块分类．从而呈现基因的全局表达规律,并通过寻找与样本性状相关的模块和候选基因,为下游机制方面的研究提供方向。在本研究中,根据差异mRNA及lncRNA的共表达关系,可聚类为4个聚类树,树的一个分支代表一簇表达量高度相关的基因模块(图4A)。通过动态剪切树法对各模块进行区分,并根据模块相似度(0.8)合并相似模块,最终得到4个模块,分别为Blue模块(1236个)、Brown模块(1227个)、Green模块(642个)和Turquoise模块(1292个) (图4B)。在Brown和Turquoise这两个模块中,按照筛选标准：连接程度大于0.6且和差异mRNA至少存在一条直接连接,确定了两个模块中的lncRNA,并对其进行网络图的绘制(图5)。图5A是Brown模块的网络图,包括58个节点(node)和201条边(edge)。蓝色圆形符号包括差异基因TFEC、IGSF6、GPR34、NCF2、TYROBP、VSIG4、CYTH4、C1QC、LILRB5和FOLR2,红色矩形符号代表与其相连的lncRNA,其中连接度最高的lncRNA是RP11-212I21.4、LINC00954、RP11- 327J17.9。图5B是Turquoise模块的网络图,包括55个节点(node)和285条边(edge)。蓝色圆形符号包括差异基因WDFY4、CD36、HAVCR2、CTSS、TIMD4、SPP1、MS4A4A、MPEG1、DOCK2和NCKAP1L,红色矩形符号代表与其相连的lncRNA,其中连接度最高的lncRNA是CTD-2201G3.1、RP11-147L13.11、RP11-541N10.3。
Table 3
表3
表3差异mRNA的KEGG富集分析(置信度最高的前10条信号通路)
Table 3
| 通路二级分类(B类) | 通路 | P值 | q值 | 通路号 | 富集到的基因 |
|---|---|---|---|---|---|
| 免疫系统 (immune system) | 补体及凝血级联反应(complement and coagulation cascades) | 3.70E-07 | 9.07E-05 | ko04610 | C7、CLU、F13A1、VSIG4、C1QC、ITGB2、ITGAM、C3AR1、C1QB、C1QA、C5AR1、CFD、CR1 |
| 信号分子与相互作用 (signaling molecules and interaction) | 细胞外基质 (cell adhesion molecules) | 3.78E-07 | 1.16E-03 | ko04514 | ITGAL、SPP1、CD4、PTPRC、SIGLEC1、NRCAM、MAG、CD36、LRRC4、VTCN1、ITGB2、NFASC、ITGAM、HLA-DOA、HLA-DRA、HLA-C、HLA-G、HLA-A、HLA-B |
| 运输和分解代谢 (transport and catabolism) | 吞噬体 (phagosome) | 2.54E-06 | 2.07E-04 | ko04145 | MSR1、CORO1A、COMP、NCF2、CD36、TLR2、FCGR2A、FCGR1A、PLA2R1、ITGB2、CTSS、CYBB、ITGAM、CD14、HLA-DOA、HLA-DRA、HLA-C、HLA-G、HLA-A、HLA-B、MRC1、RAB7B |
| 感染性疾病 (infectious diseases) | 金黄色葡萄球菌感染 (Staphylococcus aureus infection) | 3.84E-06 | 2.35E-04 | ko05150 | ITGAL、FCGR2A、FCGR1A、C1QC、ITGB2、ITGAM、FPR2、FPR1、C3AR1、C1QB、C1QA、C5AR1、CFD、HLA-DOA、HLA-DRA |
| 感染性疾病 (infectious diseases) | 阿米巴病 (amoebiasis) | 6.45E-06 | 3.16E-04 | ko05146 | GNA15、LAMB4、LAMA1、TLR2、PLCB2、ITGB2、CXCL1、PRKCB、ITGAM、CD14、PIK3CD、RAB7B |
| 免疫系统 (immune system) | 血小板激活 (platelet activation) | 1.59E-05 | 6.48E-04 | ko04611 | LCP2、PTGS1、P2RX1、PLCB2、PIK3R5、FCGR2A、FCER1G、PTGIR、GUCY1A3、SYK、PIK3CD、RASGRP1、PLCG2 |
| 感染性疾病 (infectious diseases) | 肺结核 (tuberculosis) | 3.59E-05 | 1.16E-03 | ko05152 | CD74、CORO1A、LSP1、TLR2、FCGR2A、FCGR1A、PLA2R1、FCER1G、ITGB2、CTSS、SYK、ITGAM、CD14、TLR1、CR1、HLA-DOA、HLA-DRA、MRC1 |
| 免疫系统 (immune system) | FcγR 介导的吞噬作用 (Fc gamma R-mediated phagocytosis) | 2.01E-04 | 2.07E-04 | ko04666 | WAS、PTPRC、HCK、DOCK2、BIN1、FCGR2A、FCGR1A、PLPP3、SYK、PRKCB、PIK3CD、PLCG2 |
| 生长发育 (development) | 破骨细胞分化 (osteoclast differentiation) | 0.000123966 | 3.37E-03 | ko04380 | TYROBP、LCP2、SPI1、TREM2、LILRB1、LILRB5、NCF2、FCGR2A、FCGR1A、SYK、PIK3CD、CSF1、PLCG2、LILRA6 |
| 感染性疾病 (infectious diseases) | 疟疾 (malaria) | 0.000277305 | 6.79E-03 | ko05144 | ITGAL、COMP、CCL2、CD36、TLR2、ITGB2、CR1 |
新窗口打开|下载CSV
Brown模块以VSIG4和C1QC为核心基因,根据表3可知,VSIG4和C1QC是KEGG富集分析中补体及凝血级联反应(complement and coagulation cascades, P=3.70E-07)通路下的基因,故本研究推测VSIG4和C1QC可能协同该模块其他基因及共表达lncRNA介导补体及凝血级联反应。已有的研究表明[17,18],生理状态下的补体成分激活对妊娠及胎儿发育是有利的,但是过度激活可导致炎症损伤,免疫紊乱。因此,滋养层细胞中过度升高的VSIG4和C1QC可能协同该模块其他基因过度激活了补体系统,可能导致植入部位免疫应激状态,最后引起母胎免疫耐受失衡。同时,凝血系统的激活容易引发凝血-抗凝机制或纤溶活性失衡,引起微血栓形成,使得绒毛或胎盘发育受限。
Turquoise模块以CD36和SPP1为核心基因,根据表3可知,CD36和SPP1是KEGG富集分析中细胞外基质(cell adhesion molecules, P=3.78E-07)通路下的基因,因此推测CD36和SPP1可能协同该模块其他基因及共表达lncRNA对胚胎停育时细胞外基质的分泌和降解有关。细胞外基质对于维持植入部位的正常细胞间连接及细胞-基质黏附非常重要,同时也是调节免疫应答的场所,过度的细胞外基质降解不利于绒毛的早期发育和免疫耐受的建立,可能引起妊娠丢失。
图4
新窗口打开|下载原图ZIP|生成PPT图4基因聚类系统树状图及模块划分
A：基于拓扑重叠网络的聚类树;B：动态剪切法得到的基因模块。每一个颜色代表一类表达程度相似的基因,共4个模块,分别为Blue模块(1236个基因)、Brown模块(1227个基因)、Green模块(642个基因)和Turquoise模块(1292个基因)。
Fig. 4Gene cluster dendrograms and module division
图5
新窗口打开|下载原图ZIP|生成PPT图5候选mRNA及相关lncRNA共表达网络
A：Brown模块所富集到的差异mRNA及共表达lncRNA;B：Turquoise模块所富集到的差异mRNA及共表达lncRNA。节点椭圆形为mRNA,长方形为lncRNA。节点颜色越深表示关联程度越高。
Fig. 5Co-expression networks of candidate mRNAs and related lncRNAs
3 讨论
目前研究发现,滋养层细胞对胚胎发育早期有重要作用,承担内分泌、迁移侵入及免疫调节等多种功能[19,20],且和宫内生长受限[21]、妊娠期高血压、子痫[22]和早产[23]等妊娠疾病密切相关。在胚胎发育早期,滋养层细胞的结构或功能异常可能引起胚胎植入失败、胚胎发育停止,可能导致流产的发生[24,25]。但目前胚胎停育原因复杂,具体机制未明。通过研究滋养层细胞的异常变化可以帮助理解胚胎停育的发生机制,找到可能对胚胎停育存在潜在影响的关键分子。因此,本研究通过收集临床胚胎停育患者的绒毛组织,寻找可能引起滋养层细胞功能改变的重要的基因,共筛选到436个差异基因,并通过GO、KEGG富集分析对差异基因进行功能注释及信号通路预测,发现停育胚胎的绒毛组织中存在过度的补体反应和细胞外基质降解不足的情况,可能引起母体对胎儿细胞的过度攻击或胚胎发育障碍,从而导致胚胎停育。同时,为了寻找可能对mRNA存在调控的lncRNA,本研究利用加权共表达网络进行分析,发现Green和Turquoise模块富集到了较多的差异mRNA,并找到了和差异mRNA呈高共表达关系的lncRNA。通过差异表达分析,本研究在对照组和胚胎停育组间共发现了436个差异基因,胚胎停育组相较于正常对照显著上调的基因较多,406个mRNA显著上调,32个mRNA显著下调。基于此进行文献回顾后发现,Yang等[26]通过对绒毛组织全转录组测序发现早期胚胎丢失(early embryonic arrest, EEA)相较于对照组也存在大量mRNA上调的情况。Pan等[27]的研究显示通过对妊娠丢失患者绒毛的蛋白组学发现早期胚胎丢失相较于对照组存在大量蛋白上调的情况。由此可知,本研究结果和以往类似研究结果趋势一致,说明本研究结果具有一定可信度。其中FBN1、VSIG4、CD180、NCF2均被报道和胚胎停育、母胎界面免疫异常或滋养层细胞功能障碍有关[28]。本研究发现这些基因和以往研究相类似,说明本研究结果较为可信。通过对差异mRNA进行GO富集分析发现,在细胞组分条目下差异mRNA显著富集于细胞外基质、胶原连接等,富集到该条目的基因有ADAMTSL2、CDON、COL12A1、ELN、LAMA1、MMP9等。在分子功能条目下,差异mRNA显著富集于淋巴细胞激活、髓系细胞激活等,说明当发生胚胎停育时,绒毛组织中存在过度免疫反应,可能和母胎免疫耐受失衡、启动流产发生有关,富集到该条目的基因有TYROBP、PTPRC、NLRP3、FTL、CYBB等。
通过KEGG的信号通路分析,本研究发现差异mRNA显著富集在免疫系统中的信号通路数目较多,包括补体及凝血级联反应、血小板激活,FcγR介导的吞噬作用等(表3),提示停育后的绒毛组织中存在母胎免疫耐受异常和过度免疫炎性反应。本研究筛选高置信度的前20个基因,发现其中2个基因可能和绒毛组织中的免疫反应密切相关,分别是VSIG4、C1QC。其中VSIG4 (log2FC=3.791, P=0.016)和C1QC (log2FC=4.142, P=0.011)在胚胎停育组中表达水平较对照组显著升高,且富集到补体及凝血级联反应这一信号通路中。VSIG4是免疫球蛋白结构域遏制蛋白4,和B7家族共抑制分子具有一定的同源性,主要表达于肺、胎盘组织中。目前主要认为VSIG4可作为T细胞受体的负向调节靶点[29],同时在清除补体调理的病原或其他颗粒如自体成分[30]中发挥作用。VSIG4被报道和免疫抑制密切相关,在胚胎停育组织中异常升高提示可能介导母胎界面的免疫抑制紊乱。C1QC是C1Q的其中一个亚基,而C1Q是经典补体系统的重要组成部分,能够通过激活补体下游成分,诱导免疫复合物介导的杀伤作用并提高吞噬作用[31]。补体广泛分布于体内,在滋养层细胞表面有高水平的表达[32]。具有凝血酶的C5组分可直接激活补体系统,介导炎症损伤,影响胚胎及胎盘发育障碍,最终导致妊娠失败[33]。在动物实验中,补体过度活化可引起小鼠胎盘病理改变、炎症因子增加和血栓形成[34]。由此推测,绒毛细胞中差异基因VSIG4和C1QC可能介导了过度的补体激活,引起免疫耐受失衡,从而导致胚胎损伤,引起发育停止[35]。
同时,CD36 (log2FC=3.026, P=0.006)和SPP1 (log2FC=3.357, P=0.002)作为本研究中置信度相对较高的基因,在KEGG分析中被富集到细胞外基质这一信号通路,提示可能滋养层细胞对子宫内膜细胞外基质降解出现异常,可能存在组织蛋白酶、金属蛋白酶分泌异常的情况[36]。CD36是广泛表达于单核细胞、巨噬细胞等多种血细胞表面的一类跨膜糖蛋白受体[37],可作为脂类受体、血小板受体及胶原受体调控多种生物学功能,如免疫调节[38]、炎症[39]、癌症[40]的发生与转移等。SPP1又称骨桥蛋白,主要参与细胞黏附、细胞间基质附着或免疫调节等多种生物学过程,是母胎界面粘附和信号转导所需的重要分子[41]。正常的胚胎着床和黏附需要多种基质酶促使植入部位的子宫内膜疏松而富有粘性[42]。当胎儿的滋养层细胞分泌能力下降时,植入部位的细胞外基质可能因过于致密导致植入失败或胚胎发育障碍,从而导致胚胎停育或流产的发生[43]。在研究复发性流产时,SPP1可作为评价子宫内膜容受性的指标,其水平的异常提示植入失败或胚胎发育障碍。
此外,本研究还发现差异mRNA集中于炎性反应和感染性疾病中,富集到了金黄色葡萄球菌感染、阿米巴病和肺结核等的信号通路中,推测原因可能是胚胎发育异常、停育后母胎免疫逐渐失衡,母体免疫系统开始将绒毛组织作为外来物进行强烈的免疫排斥,并启动免疫反应开始攻击胎儿,此时的绒毛组织中也存在大量炎性反应及免疫因子,这种胚胎停育后的攻击状态和感染性疾病类似。
同时,本研究通过WGCNA算法构建了共表达模块,寻找了和差异mRNA具有相关性的lncRNA。在Brown模块的网络图中,连接度最高的是RP11- 212I21.4、LINC00954和RP11-327J17.9;在Turquoise模块的网络图,连接度最高的是CTD-2201G3.1、RP11-147L13.11和RP11-541N10.3。这6个lncRNA和核心基因具有相类似的表达模式,其机制可能是由于差异mRNA及lncRNA共同参与了某条通路或者受到同样的调控,有可能协同发挥同一生物学功能。目前,由于大部分lncRNA的功能未明确,通过WGCNA共表达分析探究和差异mRNA具有相关性的lncRNA能从一定程度上预测其功能,同时从lncRNA的角度对两组间出现的差异mRNA做出解释,例如lncRNA通过选择性剪切模式改变mRNA的亚型种类及表达水平或合成内源性siRNA进行转录干扰,从而影响mRNA的转录水平[44,45]。
本研究也存在些许不足。首先,本次研究样本量有限,仅纳入14例人工流产患者作为对照和12例胚胎停育患者。因此本研究的结果还需要进一步的大样本测序结果来验证。本研究对于筛选到的lncRNA的功能未进一步研究,希望在今后能开展相关实验,以深入分析lncRNA对差异基因的确切的调控机制。其次,本研究使用的转录组测序流程依次为：首先去除rRNA,进行文库构建(mRNA片段化、逆转录、双链合成、末端补齐、末尾加A、加测序接头、PCR富集)和Illumina测序,最后采用FPKM对转录本进行相对定量分析[46,47]。但由于文库PCR扩增偏好性,可能存在所有序列不会被同比例放大的情况,可能对结果带来一定程度的偏倚。
综上所述,本研究通过全转录测序及初步生物信息学分析,发现了数个可能对滋养层细胞的生物学功能存在潜在影响的重要差异基因及相关lncRNA网络。深入研究这些基因能从母胎免疫失衡和滋养层细胞植入不足等原因对胚胎停育出一定解释,并为不明原因流产提供新的思路和方向。
参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
DOI:10.1016/j.biocel.2018.09.016URLPMID:30266525 [本文引用: 1]
The placenta is the first organ to be created during mammalian development. As the main link between the mother and the fetus it has more diverse functions than any other organ, serving as a digestive, excretory, respiratory, endocrine, and immune system. The outer layer of the placenta, the trophoblast, plays a key role in fetal development by orchestrating all these functions. Recent research has associated perturbations of maternal conditions (such as malnutrition, stress or inflammation) with alterations of the trophoblasts' endocrine, transport and metabolic processes. As reviewed here, adaptations to these conditions enable the fetus to survive, but at the cost of permanently changing its physiology and structure. Moreover, these adaptations trigger fetal programming that increases predisposition to various pathological conditions in adult life, typically metabolic, cardiovascular or CNS diseases.
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Until recently, trophoblast invasion during human placentation was characterized by and restricted to invasion into uterine connective tissues and the uterine spiral arteries. The latter was explained to connect the arteries to the intervillous space of the placenta and to guarantee the blood supply of the mother to the placenta. Today, this picture has dramatically changed. Invasion of endoglandular trophoblast into uterine glands, already starting at the time of implantation, enables histiotrophic nutrition of the embryo prior to perfusion of the placenta with maternal blood. This is followed by invasion of endovenous trophoblasts into uterine veins to guarantee the drainage of fluids from the placenta back into the maternal circulation throughout pregnancy. In addition, invasion of endolymphatic trophoblasts into the lymph vessels of the uterus has been described. Only then, invasion of endoarterial trophoblasts into spiral arteries takes place, enabling hemotrophic nutrition of the fetus starting with the second trimester of pregnancy. This new knowledge paves the way to identify changes that may occur in pathological pregnancies, from tubal pregnancies to recurrent spontaneous abortions.
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The placenta is a transient organ that plays a critical role in sustaining pregnancy and supporting fetal growth and nutrition. The placental epithelium is comprised of trophoblast cells. Trophoblast cells are the first cell type to differentiate during embryogenesis and ultimately diversify into a heterogeneous population of cells specializing in distinct functions essential for placentation. The emergence of the trophoblast lineage and subsequent specialization into distinct trophoblast sublineages is tightly regulated by transcription factors. This chapter will provide an overview of transcription factors that regulate trophoblast development and function. The chapter is divided into three sections. In the first section, a generalized outline of trophoblast ontogeny and a functional description of different trophoblast sublineages will be provided. In the second section, transcription factors involved in emergence of the trophoblast lineage and maintenance of trophoblast stem cells will be discussed. In the third section, transcription factors implicated in the formation and function of villous and extravillous cytotrophoblast lineages will be described.
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Abstract
At the tips of anchoring villi, cytotrophoblast (CTB) proliferation leads to a process of multilayering in which cells lose their attachment to the villous basement membrane and develop into columns, within which they adhere to one another using desmosomes, with associated intermediate filament bundles. Non-desmosomal cadherins, tight junction proteins and other adhesion molecules are also present, suggesting that actin-associated adhesions contribute to placental anchorage. In the distal columns, cell–cell interactions diminish, cells upregulate β1 integrins and bind to a provisional fibrinoid extracellular matrix, eventually detaching to migrate into the decidual stroma and myometrium, where interstitial and endovascular extravillous trophoblast (EVT) populations show distinct repertoires of adhesion molecules.DOI:10.1016/j.bbrc.2015.12.103URLPMID:26723254 [本文引用: 1]
The embryo implantation including adhesion between trophoblast and endometrium is a crucial process for the successful pregnancy. LIF and adhesion molecules including integrins are known as significant factors for embryo implantation. However, the function of LIF on the regulation of adhesion molecule expression and promotion of trophoblast adhesion to endometrial cells has not been fully elucidated. Here we show that LIF significantly induced mRNA expression of ITGAV, ITGB3, and ITGB5 in endometrial cells, as evidenced by RT-PCR and qRT-PCR analysis. Based on the results from treatment of antagonist for LIF receptor (hLA), LIF positively regulates expression of integrin alphaV, beta3, and beta5, and adhesion of the human trophectoderm-derived JAr cells to endometrial Ishikawa cells. Furthermore, the adhesion between trophoblastic cells and LIF-stimulated endometrial cells was significantly reduced by neutralization of LIF-mediated integrin beta3 and beta5 expression on endometrial cell surface with integrin subunit beta3 and beta5 antibodies. Taken together, we firstly demonstrate that LIF enhances the adhesion of trophoblastic cells to endometrial cells by up-regulating expression of integrin heterodimer alphaVbeta3 and alphaVbeta5, indicating the promotion of endometrial receptivity for embryo implantation.
DOI:10.3390/ijms21010289URL [本文引用: 1]
DOI:10.1371/journal.pone.0178269URLPMID:28542650 [本文引用: 2]
Invasion of trophoblast cells is spatio-temporally regulated by various cytokines and growth factors. In pregnancy, complications like preeclampsia, shallow invasion of trophoblast cells and low amounts of epidermal growth factor (EGF) have been reported. In the present study, regulatory mechanisms associated with EGF-mediated invasion in HTR-8/SVneo trophoblastic cells have been delineated. Treatment of HTR-8/SVneo cells with EGF (10 ng/ml) led to eight fold increase (p < 0.05) in invasion. Increased invasion of HTR-8/SVneo cells by EGF was associated with an increase in phosphorylation of ERK(1/2). In addition, significant phosphorylation of STAT1 (ser 727) and STAT3 (both tyr 705 and ser 727 residues) was also observed, accompanied by a decrease in total STAT1. Inhibition of ERK(1/2) phosphorylation by U0126 (10 muM) led to a significant decrease in EGF-mediated invasion with simultaneous decrease in the phosphorylated forms of STAT3 and STAT1. Decrease in total STAT1 was also reversed on inhibition of ERK(1/2). Interestingly, inhibition of STAT3 by siRNA led to a significant decrease in EGF-mediated invasion of HTR-8/SVneo cells and phosphorylation of STAT1, but it did not have any effect on the activation of ERK(1/2). On the other hand, inhibition of STAT1 by siRNA, also led to a significant decrease in the EGF-mediated invasion of HTR-8/SVneo cells, showed concomitant decrease in ERK(1/2) phosphorylation and STAT3 phosphorylation at ser 727 residue. These results suggest cross-communication between ERK(1/2) and JAK-STAT pathways during EGF-mediated increase in invasion of trophoblast cells; phosphorylation at ser 727 residue of both STAT3 and STAT1 appears to be critical.
DOI:10.18632/oncotarget.16826URLPMID:28432277 [本文引用: 2]
Transforming growth factor (TGF)-beta1 is involved invasion of human trophoblasts. However, the underlying mechanisms remain unclear. In this study, we performed Transwell assay and found that TGF-beta1 promoted the invasion of trophoblast cell line JEG-3. Treatment with TGF-beta1 up-regulated the expression of receptor-regulated Smad transcription factors Smad2 and Smad3, and two invasive-associated genes, namely, matrix metallopeptidase (MMP)-9 and MMP-2, in JEG-3 cells. Over-expressing activin receptor-like kinase (ALK) 5, the TGF-beta type I receptor (TbetaRI) enhanced the up-regulation of Smad2, Smad3, MMP-9, and MMP-2 induced by TGF-beta1, whereas application of TbetaRI inhibitor SB431542 diminished the stimulatory effects of TGF-beta1 on these genes. Furthermore, transfection of Smad3 and ALK-5 seperately or in combination into JEG-3 cells before TGF-beta1 treatment significantly increased the expression of MMP-9 and MMP-2. By contrast, silencing Smad3 and Smad2 by siRNAs significantly decreased the expression of MMP-9 and MMP-2, with Smad3 silence having a more potent inhibitory effect. Inhibiting TbetaRI with SB431542 or knockdown of Smad3, but not Smad2, abolished the stimulatory effect of TGF-beta1 on the invasion of JEG-3 cells. Taken together, the results indicate that TGF-beta1 activates the Smads signaling pathway in JEG-3 trophoblast cells and Smad3 play a key role in TGF-beta1-induced invasion of JEG-3 and up-regulation of MMP-9 and MMP-2 expression.
DOI:10.1016/bs.ctdb.2016.10.004URLPMID:28236968 [本文引用: 2]
Macrophages are found in all tissues and regulate tissue morphogenesis during development through trophic and scavenger functions. The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) is the major regulator of tissue macrophage development and maintenance. In combination with receptor activator of nuclear factor kappaB (RANK), the CSF-1R also regulates the differentiation of the bone-resorbing osteoclast and controls bone remodeling during embryonic and early postnatal development. CSF-1R-regulated macrophages play trophic and remodeling roles in development. Outside the mononuclear phagocytic system, the CSF-1R directly regulates neuronal survival and differentiation, the development of intestinal Paneth cells and of preimplantation embryos, as well as trophoblast innate immune function. Consistent with the pleiotropic roles of the receptor during development, CSF-1R deficiency in most mouse strains causes embryonic or perinatal death and the surviving mice exhibit multiple developmental and functional deficits. The CSF-1R is activated by two dimeric glycoprotein ligands, CSF-1, and interleukin-34 (IL-34). Homozygous Csf1-null mutations phenocopy most of the deficits of Csf1r-null mice. In contrast, Il34-null mice have no gross phenotype, except for decreased numbers of Langerhans cells and microglia, indicating that CSF-1 plays the major developmental role. Homozygous inactivating mutations of the Csf1r or its ligands have not been reported in man. However, heterozygous inactivating mutations in the Csf1r lead to a dominantly inherited adult-onset progressive dementia, highlighting the importance of CSF-1R signaling in the brain.
DOI:10.3892/ijmm.2019.4146URLPMID:30942389 [本文引用: 1]
Macrophages can induce Fas ligand (FasL)mediated apoptosis, and the deregulation of apoptosis is known to be associated with recurrent miscarriage (RM). The aim of the present study was to investigate the possible involvement of FasL in macrophagemediated trophoblast apoptosis and its potential role in RM. Human decidual and placental villous tissues were collected from 81 women (21 for the RM group, 26 for the spontaneous abortion group and 34 for the control group) at 79 weeks of gestation. The distribution changes of macrophages and the expression of FasL on macrophages were evaluated by immunohistochemical, immunofluorescence and western blot analyses. A macrophage and trophoblast coculture model was used to determine the effects of FasL on the apoptosis of trophoblasts. The results indicated that CD86+ macrophage populations in decidual tissues were significantly increased, accompanied by reduced CD163+ macrophages in the abortion and RM groups. Furthermore, the distribution of CD68+ macrophages was also significantly altered in specimens from the abortion and RM groups, and they were observed to have infiltrated into the trophoblast cells. In addition, elevated expression of FasL on CD68+ and CD86+ macrophages in the decidua was observed in the spontaneous abortion and RM groups of patients, and FasL was demonstrated to mediate the induction of trophoblast apoptosis by macrophages in coculture. These results indicate that the aberration of macrophageinduced FasLmediated apoptosis may represent one of the causes of RM.
DOI:10.1371/journal.pone.0077261URLPMID:24130868 [本文引用: 1]
Plants are simultaneously exposed to multiple stresses resulting in enormous changes in the molecular landscape within the cell. Identification and characterization of the synergistic and antagonistic components of stress response mechanisms contributing to the cross talk between stresses is of high priority to explore and enhance multiple stress responses. To this end, we performed meta-analysis of drought (abiotic), bacterial (biotic) stress response in rice and Arabidopsis by analyzing a total of 386 microarray samples belonging to 20 microarray studies and identified approximately 3100 and 900 DEGs in rice and Arabidopsis, respectively. About 38.5% (1214) and 28.7% (272) DEGs were common to drought and bacterial stresses in rice and Arabidopsis, respectively. A majority of these common DEGs showed conserved expression status in both stresses. Gene ontology enrichment analysis clearly demarcated the response and regulation of various plant hormones and related biological processes. Fatty acid metabolism and biosynthesis of alkaloids were upregulated and, nitrogen metabolism and photosynthesis was downregulated in both stress conditions. WRKY transcription family genes were highly enriched in all upregulated gene sets while 'CO-like' TF family showed inverse relationship of expression between drought and bacterial stresses. Weighted gene co-expression network analysis divided DEG sets into multiple modules that show high co-expression and identified stress specific hub genes with high connectivity. Detection of consensus modules based on DEGs common to drought and bacterial stress revealed 9 and 4 modules in rice and Arabidopsis, respectively, with conserved and reversed co-expression patterns.
DOI:10.1093/bioinformatics/btp616URLPMID:19910308 [本文引用: 1]
SUMMARY: It is expected that emerging digital gene expression (DGE) technologies will overtake microarray technologies in the near future for many functional genomics applications. One of the fundamental data analysis tasks, especially for gene expression studies, involves determining whether there is evidence that counts for a transcript or exon are significantly different across experimental conditions. edgeR is a Bioconductor software package for examining differential expression of replicated count data. An overdispersed Poisson model is used to account for both biological and technical variability. Empirical Bayes methods are used to moderate the degree of overdispersion across transcripts, improving the reliability of inference. The methodology can be used even with the most minimal levels of replication, provided at least one phenotype or experimental condition is replicated. The software may have other applications beyond sequencing data, such as proteome peptide count data. AVAILABILITY: The package is freely available under the LGPL licence from the Bioconductor web site (http://bioconductor.org).
DOI:10.1016/j.imbio.2014.01.001URL [本文引用: 1]
The complement system is one component of innate immunity that could participate in fetal loss. We have already reported that adipsin, a complement activator in the alternative pathway, is stably expressed in the placenta and that an increase in this expression is related to spontaneous abortion. However, complement inhibitor Crry was concurrently expressed in the placenta, and the role of complement factors during pregnancy was not clear. In the present study, we examined the endogenous regulation of complement factors in placenta and serum by using another model mouse for spontaneous abortion and studied the effect of exogenous complement disruption on pregnancy. Compared to control mice, the CBA/J x DBA/2 model mice had higher expression levels of adipsin in the placenta and serum. Adipsin and complement C3 were localized in the metrial gland and labyrinth regions, and both positive reactive ranges were limited in the maternal blood current in normal implantation sites. These results suggest that extrauterine adipsin hematogenously reaches the placenta, activates complement C3, and promotes destruction of the feto-maternal barrier in aborted implantation sites. Crry was consistently expressed in the placenta and serum and reduced in the resorption sites of CBA/J x DBA/2 mice as compared to normal sites. Injection of recombinant adipsin increased the resorption rate and changed the expression of Th-type cytokines toward a Th1 bias. The present study indicates that adipsin could induce the fetal loss that accompanies the Thl bias and may be a crucial cause of spontaneous abortion. In addition, the local expression of Crry prevents complement activation in placenta in response to a systemic increase of adipsin. (C) 2014 Elsevier GmbH.
DOI:10.1530/REP-16-0574URLPMID:28283674 [本文引用: 1]
The endometrium becomes receptive to the embryo only in the mid-luteal phase, but not in the other stages of the menstrual cycle. Endometrial factors play an important role in implantation. Women with recurrent miscarriage and recurrent implantation failure have both been reported to have altered expression of receptivity markers during the window of implantation. We aimed to compare the gene expression profiles of the endometrium in the window of implantation among women with unexplained recurrent implantation failures (RIF) and unexplained recurrent miscarriages (RM) by RNA sequencing (RNA-Seq). In total 20 patients (9 RIF and 11 RM) were recruited. In addition 4 fertile subjects were included as reference. Endometrium samples were precisely timed on the 7th day after luteal hormone surge (LH + 7). All the 24 endometrium samples were extracted for total RNA. The transcriptome was determined by RNA-Seq in the first 14 RNA samples (5 RIF, 6 RM and 3 fertile). Differentially expressed genes between RM and RIF were validated by quantitative real-time PCR (qPCR) in all 24 RNA samples (9 RIF, 11 RM and 4 fertile). Transcriptomic profiles of RM and RIF, but not control samples, were separated from each other by principle component analysis (PCA) and support vector machine (SVM). Complementary and coagulation cascades pathway was significantly up-regulated in RIF while down-regulated in RM. Differentially expressed genes C3, C4, C4BP, DAF, DF and SERPING1 in complement and coagulation cascade pathway between RM and RIF were further validated by qPCR. This study compared endometrial transcriptome among patients with RIF and RM in the window of implantation; it identified differential molecular pathways in endometrium between RIF and RM, which potentially affect the implantation process.
DOI:10.1080/00016340802177412URLPMID:18607815 [本文引用: 1]
BACKGROUND: Early placental development is associated with complex regulatory mechanisms, and molecular communication problems that arise during the developmental process are dangerous for continuation of the pregnancy. As studies on the process of invasion and migration of trophoblast cells have shown the importance of cell-cell and cell-matrix interactions, we examined the effects of adhesion molecules on the mechanism(s) of spontaneous abortions and compared them to elective abortion materials using histopathological and immunohistochemical methods. To the best of our knowledge, this is the first study to investigate adhesion molecules in spontaneous abortions. METHODS: Curettage materials from abortions were examined retrospectively in the Department of Pathology, Zonguldak Karaelmas University School of Medicine, Zonguldak, Turkey. CD31/PECAM-1 (endothelial cell marker), CD44v (variant 3), E-cadherin, CD54/ICAM-1, and CD106/VCAM-1 expression profiles were evaluated by immunohistochemistry, and cellular localization was determined under light microscopy. The results of spontaneous abortions were compared to those of elective abortions. RESULTS: The staining percentages of CD31, CD44, CD106, and E-cadherin decreased in cases of spontaneous abortion, but CD54 (ICAM-1) expression increased. Statistically significant differences were detected between spontaneous and elective abortion materials with regard to cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs) with the anti-CD31 antibody (p=0.0001). In addition, CD54 (p=0.007 and p=0.002) and E-cadherin (p=0.002 and p=0.02) expression in CTs and STs, respectively, were significantly different. Furthermore, CD44 expression (p=0.003) in decidual (D) cells and CD106 (p=0.0001) expression in vessels of endometrial (E) and villous tissues were also significantly different. CONCLUSIONS: Decreased CD31 expression in CTs that invade the spiral arterioles and mimic E cells in spontaneous abortion cases suggests that CD31/PECAM-1 is an important molecule in uteroplacental adequacy. Moreover, diminished expression of CD44 in D cells caused impaired stroma-villous connections. Enhancement of ICAM-1 in placental and invading STs may be useful as a diagnostic marker for patients who may have a tendency to have spontaneous abortions. A down-regulation of E-cadherin was observed, which may be responsible for impaired CT differentiation and loss of the pregnancy. Furthermore, decreased VCAM-1 expression in spontaneous abortions may be consistent with the importance of VCAM-1 in trophoblast-endothelial cell interactions. Many adhesion molecules are known to be effective in the normal development of a pregnancy, and the analysis of adhesion molecules in spontaneous abortions will provide useful information for clarifying the physiopathology of spontaneous abortions.
DOI:10.1080/01443615.2016.1174823URLPMID:27183899 [本文引用: 1]
The aim of this study was to compare platelet parameters between abortus groups with gestational trophoblastic disease (GTD) (molar pregnancy, invasive mole, choriocarcinoma, etc) and without disease according to pathological result. The study population consisted of patients with GTD (n = 53) and aborted patients without disease as a control group (n = 53) who were seen in our clinic between January 2010 and December 2013. In this retrospective study, age, gravidity, levels of haemoglobin, white blood cell count, platelets, platelet parameters (mean platelet volume (MPV), platelet distrubition width (PDW), platelet crit (PCT), which shows platelet functions were recorded. The pathological diagnosis of GTD was recorded. The mean platelet count, MPV, PDW and PCT levels were similar between the groups. There is no statistically significiant difference between types of GTN in these parameters according to pathological diagnosis. According to our study results, platelet count and levels of MPV, PDW ve PCT in GTD patients were similar to aborted patients without disease.
DOI:10.1080/0891693031000067322URLPMID:12765467 [本文引用: 1]
In the United States, between 1 and 3% of women suffer recurrent miscarriages; 50-70% of all conceptions fail. Although in the majority of affected women the cause of recurrent miscarriages is unknown, an immune mechanism involving the inappropriate and subsequently injurious recognition of the conceptus by the mother's immune system has been proposed. Murine models have recently been developed that are relevant to this issue. We and others have identified a novel role for complement as an early effector in the pathway leading to pregnancy loss associated with placental inflammation. Indeed, it appears that inhibition of complement activation is an absolute requirement for normal pregnancy, and that in the antiphosphospholid syndrome overwhelming activation of complement triggered by antibodies (Ab) deposited in placenta leads to fetal injury. Identification of complement activation as a mediator of pregnancy loss and definition of the complement components necessary to trigger such injury is likely to lead to a better understanding of its pathogenesis and to new and improved treatments.
DOI:10.3389/fimmu.2015.00317URLPMID:26175731 [本文引用: 1]
Complement protein C1q, the recognition molecule of the classical pathway, performs a diverse range of complement and non-complement functions. It can bind various ligands derived from self, non-self, and altered self and modulate the functions of immune and non-immune cells including dendritic cells and microglia. C1q involvement in the clearance of apoptotic cells and subsequent B cell tolerance is more established now. Recent evidence appears to suggest that C1q plays an important role in pregnancy where its deficiency and dysregulation can have adverse effects, leading to preeclampsia, missed abortion, miscarriage or spontaneous loss, and various infections. C1q is also produced locally in the central nervous system, and has a protective role against pathogens and possible inflammatory functions while interacting with aggregated proteins leading to neurodegenerative diseases. C1q role in synaptic pruning, and thus CNS development, its anti-cancer effects as an immune surveillance molecule, and possibly in aging are currently areas of extensive research.
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DOI:10.1016/j.ajog.2017.11.577URLPMID:29422210 [本文引用: 1]
Placental-related fetal growth restriction arises primarily due to deficient remodeling of the uterine spiral arteries supplying the placenta during early pregnancy. The resultant malperfusion induces cell stress within the placental tissues, leading to selective suppression of protein synthesis and reduced cell proliferation. These effects are compounded in more severe cases by increased infarction and fibrin deposition. Consequently, there is a reduction in villous volume and surface area for maternal-fetal exchange. Extensive dysregulation of imprinted and nonimprinted gene expression occurs, affecting placental transport, endocrine, metabolic, and immune functions. Secondary changes involving dedifferentiation of smooth muscle cells surrounding the fetal arteries within placental stem villi correlate with absent or reversed end-diastolic umbilical artery blood flow, and with a reduction in birthweight. Many of the morphological changes, principally the intraplacental vascular lesions, can be imaged using ultrasound or magnetic resonance imaging scanning, enabling their development and progression to be followed in vivo. The changes are more severe in cases of growth restriction associated with preeclampsia compared to those with growth restriction alone, consistent with the greater degree of maternal vasculopathy reported in the former and more extensive macroscopic placental damage including infarcts, extensive fibrin deposition and microscopic villous developmental defects, atherosis of the spiral arteries, and noninfectious villitis. The higher level of stress may activate proinflammatory and apoptotic pathways within the syncytiotrophoblast, releasing factors that cause the maternal endothelial cell activation that distinguishes between the 2 conditions. Congenital anomalies of the umbilical cord and placental shape are the only placental-related conditions that are not associated with maldevelopment of the uteroplacental circulation, and their impact on fetal growth is limited.
URLPMID:32580159 [本文引用: 1]
DOI:10.3409/fb58_1-2.79-83URL [本文引用: 1]
URLPMID:29944223 [本文引用: 1]
The first in a series on improving embryo implantation is presented with emphasis on embryo attachment and trophoblast invasion. PURPOSE: To present knowledge of events needed for embryo attachment to the endometrium and subsequent trophoblast invasion and uterine remodeling leading to successful pregnancy. MATERIALS AND METHODS: Based on normal events, some practical suggestions are proposed as to possible means of improving pregnancy rates by enhancing possible embryo attachment and trophoblast invasion. RESULTS: Potential benefits of achieving adequate serum estradiol levels at peak follicular maturation, and the benefits of progesterone in the luteal phase are discussed. Also the potential benefits of purposeful endometrial injury is considered. CONCLUSIONS: Knowledge of the events leading to embryo attachment and trophoblast invasion could lead to novel research ideas helping to improve pregnancy rates in addition to proper hormone supplementation and endometrial biopsy.
DOI:10.1016/j.placenta.2017.06.007URLPMID:28651899 [本文引用: 1]
Many complications of pregnancy have their pathophysiological roots in the early stages of placentation. Impaired trophoblast invasion and deficient remodelling of the maternal spiral arteries are a common feature. While malperfusion of the placenta may underpin cases of fetal growth restriction and early-onset pre-eclampsia, the mechanistic links to spontaneous miscarriage, pre-term labour and premature rupture of the membranes are less obvious. Here, we speculate that formation of a well-developed cytotrophoblastic shell at the maternal-fetal interface is crucial for pregnancy success. Initially, extravillous trophoblast cells differentiate from the outer layer of the shell in contact with the endometrium. Impaired development may thus contribute to reduced invasion and deficient remodelling. In addition, the extent of the shell influences the timing and spatial configuration of onset of the maternal arterial circulation. A thin and fragmentary shell results in premature and disorganised onset, leading to spontaneous miscarriage. In less severe cases it may predispose to haemorrhage at the interface and formation of intrauterine haematomas. If pregnancy continues, these haematomas may act as a source of oxidative stress, promoting senescence and weakening of the membranes, and stimulating inflammation in the uterine wall and premature contractions. Formation of the shell is dependent on proliferation of cytotrophoblast progenitor cells during the first weeks after implantation, when the developing placenta is supported by histotrophic nutrition from endometrial glands. Hence, we propose the fitness of the endometrium prior to conception, and the peri-conceptional dialogue between the endometrium and the trophoblast is critical for avoidance of later complications of pregnancy.
DOI:10.1016/j.gene.2018.12.084URLPMID:30776465 [本文引用: 1]
Early Embryonic Arrest (EEA) is one of the major causes of female infertility. Genetic factors including specific genes and miRNAs may play pivotal roles on EEA. However, it is not well defined what genes and micro RNAs participate the pathophysiological alterations of EEA. In this work, we compared the Transcriptome -Seq and microRNA profiles from three pairs of villi (three EEA patients and three normal pregnancy, NP). We first confirmed the array data by qPCR with ten randomly selected differentially expressed genes and ten differentially expressed miRNAs in villi from 20 EEA and 20 NP controls. We next applied Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis and found that these differentially expressed genes enriched in the PI3K-Akt signaling pathway, Jak-STAT signaling pathway, MAPK signaling pathway, Complement and coagulation cascades, Hypertrophic cardiomyopathy (HCM), Dilated cardiomyopathy (DCM). Interestingly, hsa-miR-6515-5p and its target genes NLRP3, UGP2 may regulate the Immune system and carbohydrate metabolism. Hsa-miRNA 518 and its target gene EGR1 may regulate cell proliferation, angiogenesis, and cell apoptosis to impact early embryonic development. Moreover, novel-m0045-5p and its target gene RMDN3 may regulate microtubule formation on the development of EEA. Our research provides novel biomarkers for EEA and establishes a foundation for further study of the mechanism of EEA.
DOI:10.1016/j.placenta.2017.11.001URLPMID:29277264 [本文引用: 1]
INTRODUCTION: Recurrent miscarriage (RM) affects 5% of women, it has an adverse emotional impact on women. Because of the complexities of early development, the mechanism of recurrent miscarriage is still unclear. We hypothesized that abnormal placenta leads to early recurrent miscarriage (ERM). The aim of this study was to identify ERM associated factors in human placenta villous tissue using proteomics. Investigation of these differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying recurrent miscarriage (RM). METHODS: To gain more insight into mechanisms of recurrent miscarriage (RM), a comparative proteome profile of the human placenta villous tissue in normal and RM pregnancies was analyzed using iTRAQ technology and bioinformatics analysis used by Ingenuity Pathway Analysis (IPA) software. RESULTS: In this study, we employed an iTRAQ based proteomics analysis of four placental villous tissues from patients with early recurrent miscarriage (ERM) and four from normal pregnant women. Finally, we identified 2805 proteins and 79,998 peptides between patients with RM and normal matched group. Further analysis identified 314 differentially expressed proteins in placental villous tissue (>/=1.3-fold, Student's t-test, p < 0.05); 209 proteins showed the increased expression while 105 proteins showed decreased expression. These 314 proteins were analyzed by Ingenuity Pathway Analysis (IPA) and were found to play important roles in the growth of embryo. Furthermore, network analysis show that Angiotensinogen (AGT), MAPK14 and Prothrombin (F2) are core factors in early embryonic development. We used another 8 independent samples (4 cases and 4 controls) to cross validation of the proteomic data. DISCUSSION: This study has identified several proteins that are associated with early development, these results may supply new insight into mechanisms behind recurrent miscarriage.
DOI:10.1016/j.autrev.2010.09.003URL [本文引用: 1]
Problem: The aim of this study was to investigate anti-fibrillin-1 autoantibody in patients with a history of recurrent pregnancy loss (RPL) and during normal pregnancy.
Method of Study: Anti-fibrillin-1 IgG and IgM antibodies were measured by a home made ELISA in serum samples of 48 medically and obstetrically normal pregnant women, classified to three trimester groups, 15 female patients with RPL and 26 healthy non-pregnant women classified to two control subgroups: (a) women who had already had at least one previous successful pregnancy and (b) women who had never been pregnant. Differences in anti-fibrillin-1 autoantibodies between the groups were analyzed for statistical significance (P<0.05) with one-way analysis of variance (ANOVA) and multiple comparison test Post Hoc test, Least Significant Difference method.
Results: Anti-fibrillin-1 IgM autoantibodies were significantly decreased in the second and third trimester pregnant women compared to the nulligravida controls. RPL patients had significantly increased anti-fibrillin-1 IgM antibody compared to control group (a).
Conclusion: Fibrillin-1 degradation seems to be decreased during the second and third trimester of normal pregnancy. Increased anti-fibrillin-1 IgM antibodies in RPL patients may be a secondary phenomenon of increased fibrillin-1 degradation and contribute to the pathogenesis of pregnancy losses. (C) 2010 Elsevier B.V.
DOI:10.1172/JCI25673URLPMID:17016562 [本文引用: 1]
T cell activation by APCs is positively and negatively regulated by members of the B7 family. We have identified a previously unknown function for B7 family-related protein V-set and Ig domain-containing 4 (VSIG4). In vitro experiments using VSIG4-Ig fusion molecules showed that VSIG4 is a strong negative regulator of murine and human T cell proliferation and IL-2 production. Administration to mice of soluble VSIG4-Ig fusion molecules reduced the induction of T cell responses in vivo and inhibited the production of Th cell-dependent IgG responses. Unlike that of B7 family members, surface expression of VSIG4 was restricted to resting tissue macrophages and absent upon activation by LPS or in autoimmune inflammatory foci. The specific expression of VSIG4 on resting macrophages in tissue suggests that this inhibitory ligand may be important for the maintenance of T cell unresponsiveness in healthy tissues.
DOI:10.1016/j.cell.2005.12.039URLPMID:16530040 [本文引用: 1]
The complement system serves an important role in clearance of pathogens, immune complexes, and apoptotic cells present in the circulation. Complement fragments deposited on the particle surface serve as targets for complement receptors present on phagocytic cells. Although Kupffer cells, the liver resident macrophages, play a dominant role in clearing particles in circulation, complement receptors involved in this process have yet to be identified. Here we report the identification and characterization of a Complement Receptor of the Immunoglobulin superfamily, CRIg, that binds complement fragments C3b and iC3b. CRIg expression on Kupffer cells is required for efficient binding and phagocytosis of complement C3-opsonized particles. In turn, Kupffer cells from CRIg-deficient mice are unable to efficiently clear C3-opsonized pathogens in the circulation, resulting in increased infection and mortality of the host. CRIg therefore represents a dominant component of the phagocytic system responsible for rapid clearance of C3-opsonized particles from the circulation.
DOI:10.1182/blood-2008-01-134304URLPMID:18524992 [本文引用: 1]
Complement activation on human platelets is known to cause platelet degranulation and activation. To evaluate how normal platelets escape complement attack in vivo, we studied the fate of murine platelets deficient in 2 membrane complement regulatory proteins using an adoptive transfer model. We show here that deficiency of either decay-accelerating factor (DAF) or complement receptor 1-related gene/protein y (Crry) on murine platelets was inconsequential, whereas DAF and Crry double deficiency led to rapid clearance of platelets from circulation in a complement- and macrophage-dependent manner. This finding contrasted with the observation on erythrocytes, where Crry deficiency alone resulted in complement susceptibility. Quantitative flow cytometry showed DAF and Crry were expressed at similar levels on platelets, whereas Crry expression was 3 times higher than DAF on erythrocytes. Antibody blocking or gene ablation of the newly identified complement receptor CRIg, but not complement receptor 3 (CR3), rescued DAF/Crry-deficient platelets from complement-dependent elimination. Surprisingly, deficiency of CRIg, CR3, and other known complement receptors failed to prevent Crry-deficient erythrocytes from complement-mediated clearance. These results show a critical but redundant role of DAF and Crry in platelet survival and suggest that complement-opsonized platelets and erythrocytes engage different complement receptors on tissue macrophages in vivo.
DOI:10.1038/mi.2016.87URLPMID:27731323 [本文引用: 1]
The complement subunit C1q was recently identified as a marker for monocyte-derived regulatory dendritic cells supporting the differentiation of interleukin (IL)-10-secreting CD4(+) T cells with a suppressive activity. Furthermore, C1q expression is upregulated in peripheral blood mononuclear cells of allergic patients in the course of successful allergen immunotherapy. Herein, we investigated a potential direct role of C1q in downregulating allergic inflammation. In mice with ovalbumin (OVA) or birch pollen (BP)-induced allergic asthma, C1q is as efficacious as dexamethasone to reduce both airway hyperresponsiveness (AHR), eosinophil, and ILC2 infiltrates in bronchoalveolar lavages, as well as allergen-specific T helper 2 cells in the lungs. Administration of C1q does not expand IL-10(+)/Foxp3(+) regulatory T cells in the lungs, spleen, or in the blood. Depletion of plasmacytoid dendritic cells (pDCs) abrogates the capacity of C1q to reduce AHR and eosinophilic infiltrates in OVA-sensitized mice. Also C1q treatment inhibits the activation of human and mouse pDCs by CpGs, thereby demonstrating a critical role for pDCs in the anti-inflammatory activity of C1q. We conclude that regulatory dendritic cells can mediate a potent direct anti-inflammatory activity via the expression and/or secretion of molecules such as C1q, independently of their capacity to expand the pool of regulatory T cells.
DOI:10.1080/08820130802191615URLPMID:18716942 [本文引用: 1]
The paternal antigens presented by the fetus could be considered foreign by the mother's immune system and elicit an immune response. Here we show that the complement system functions as an effector in fetal rejection in two different mouse models of pregnancy loss. In a mouse model of fetal loss and growth restriction (IUGR) induced by antiphospholipid antibodies (aPL), we found that complement activation is a crucial and early mediator of pregnancy loss. We demonstrated that C5a-C5aR interaction and neutrophils are key mediators of fetal injury. We identified tissue factor (TF) as a critical intermediate that, acting downstream of C5 activation, enhances neutrophil activity and trophoblast injury. In an antibody-independent mouse model of spontaneous miscarriage and IUGR (CBAxDBA) we also identified C5a as an essential mediator. Complement activation caused dysregulation of the angiogenic factors (deficiency of free vascular endothelial growth factor (VEGF) and elevated levels of soluble VEGF receptor 1) required for normal placental development. Inhibition of complement activation prevented angiogenesis failure and rescued pregnancies. Our studies in antibody-dependent and antibody-independent models of pregnancy complications identified complement activation as the crucial mediator of damage and will allow development of new interventions to prevent pregnancy loss and IUGR.
DOI:10.1016/j.molimm.2011.04.011URL [本文引用: 1]
Pregnancy is a most intriguing feature of biology, not at least because of the need to regulate the maternal immune response against fetal antigens. The mammalian embryo expresses paternal antigens foreign to the mother's immune system and thus elicits an immune response that can lead to fetal rejection and bad pregnancy outcomes such as recurrent miscarriages and preeclampsia. More effective strategies to prevent these pregnancy complications should be forthcoming once the underlying pathophysiological mechanisms that are involved in fetal rejection are completely understood. Our goal in writing this review is to discuss the crucial role of the complement system as an effector mechanism in placental and fetal damage that leads to bad pregnancy outcomes. Important information about the role of excessive complement activation and bad pregnancy outcomes was obtained from animal models. That uncontrolled complement activation puts at risk the survival of the fetus was reported in mouse models of recurrent miscarriages and preeclampsia. Interestingly, several observations described in the mouse models were confirmed in humans. Increased circulating levels of complement proteins, and their activation fragments were found in patients with preeclampsia, recurrent miscarriages and intrauterine growth restriction.
Studies performed in animals and humans demonstrated the deleterious effect of complement activation on pregnancy outcomes. However, we also described in this article the strategic role of complement component C1q in normal placentation. C1q deserves special consideration for its role in promoting trophoblast invasion of deciduas, a crucial step in normal placental development.
In conclusion, in this review we discussed all the available results of basic and clinical studies on the role of the complement system in pregnancy to expand the understanding of the pathophysiology of pregnancy complications. (C) 2011 Elsevier Ltd.
DOI:10.1016/j.smim.2019.101337URLPMID:31757607 [本文引用: 1]
Preeclampsia is a serious vascular complication of the human pregnancy, whose etiology is still poorly understood. In preeclampsia, exacerbated apoptosis and fragmentation of the placental tissue occurs due to developmental qualities of the placental trophoblast cells and/or mechanical and oxidative distress to the syncytiotrophoblast, which lines the placental villi. Dysregulation of the complement system is recognized as one of the mechanisms of the disease pathology. Complement has the ability to promote inflammation and facilitate phagocytosis of placenta-derived particles and apoptotic cells by macrophages. In preeclampsia, an overload of placental cell damage or dysregulated complement system may lead to insufficient clearance of apoptotic particles and placenta-derived debris. Excess placental damage may lead to sequestration of microparticles, such as placental vesicles, to capillaries in the glomeruli of the kidney and other vulnerable tissues. This phenomenon could contribute to the manifestations of typical diagnostic symptoms of preeclampsia: proteinuria and new-onset hypertension. In this review we propose that the complement system may serve as a regulator of the complex tolerance and clearance processes that are fundamental in healthy pregnancy. It is therefore recommended that further research be conducted to elucidate the interactions between components of the complement system and immune responses in the context of complicated and healthy pregnancy.
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DOI:10.1007/s00011-010-0241-1URL [本文引用: 1]
Objectives and design Microbial products can act via stress-induced signaling cascades to link dysregulated endogenous microbiota to immune activation (e.g., macrophages) and pregnancy loss. Our previous studies demonstrated that mice deficient in the macrophage pattern recognition scavenger receptors, SR-A and CD36, are more susceptible to inflammatory complications including gut leakiness and experimental colitis. We hypothesized that bacterial penetration of the maternal mucosal surfaces and replication in embryonic fluids compromise the fetal status and can result in miscarriage.
Materials and methods Eighty pregnant ICR and SR-A/CD36-deficient mice were injected via tail vein or intraperitoneally with commensal bacteria (Streptococcus cricetus and/or Actinobacillus sp.) or sham controls. Dams were monitored daily for physical distress, pain and abortion.
Results Dams injected with single dose bacterial inoculum did not develop clinical symptoms. Day old pups injected with bacteria developed internal focal abscesses, lost weight but recovered after 1 week. Dams receiving a second bacterial inoculum delivered dead fetuses. However, SR-A/CD36-deficnet dams demonstrated 100% fetal death via aborted fetuses, and significant up-regulation of the proinflammatory markers (IL-6, serum Amyloid A) 24-74 h after single inoculum.
Conclusions These data indicate that macrophage scavenger receptors are required for the fetal protection against microbial attack and support that maternal transfer of innate immunity contributes to this protection.
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DOI:10.1016/j.bbalip.2016.03.015URLPMID:27004753 [本文引用: 1]
CD36 is a multifunctional immuno-metabolic receptor with many ligands. One of its physiological functions in the heart is the high-affinity uptake of long-chain fatty acids (FAs) from albumin and triglyceride rich lipoproteins. CD36 deletion markedly reduces myocardial FA uptake in rodents and humans. The protein is expressed on endothelial cells and cardiomyocytes and at both sites is likely to contribute to FA uptake by the myocardium. CD36 also transduces intracellular signaling events that influence how the FA is utilized and mediate metabolic effects of FA in the heart. CD36 transduced signaling regulates AMPK activation in a way that adjusts oxidation to FA uptake. It also impacts remodeling of myocardial phospholipids and eicosanoid production, effects exerted via influencing intracellular calcium (iCa(2+)) and the activation of phospholipases. Under excessive FA supply CD36 contributes to lipid accumulation, inflammation and dysfunction. However, it is also important for myocardial repair after injury via its contribution to immune cell clearance of apoptotic cells. This review describes recent progress regarding the multiple actions of CD36 in the heart and highlights those areas requiring future investigation. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.
DOI:10.7150/thno.36037URLPMID:31410189 [本文引用: 1]
CD36, a scavenger receptor expressed in multiple cell types, mediates lipid uptake, immunological recognition, inflammation, molecular adhesion, and apoptosis. CD36 is a transmembrane glycoprotein that contains several posttranslational modification sites and binds to diverse ligands, including apoptotic cells, thrombospondin-1 (TSP-1), and fatty acids (FAs). Beyond fueling tumor metastasis and therapy resistance by enhancing lipid uptake and FA oxidation, CD36 attenuates angiogenesis by binding to TSP-1 and thereby inducing apoptosis or blocking the vascular endothelial growth factor receptor 2 pathway in tumor microvascular endothelial cells. Moreover, CD36-driven lipid metabolic reprogramming and functions in tumor-associated immune cells lead to tumor immune tolerance and cancer development. Notable advances have been made in demonstrating the regulatory networks that govern distinct physiological properties of CD36, and this has identified targeting CD36 as a potential strategy for cancer treatment. Here, we provide an overview on the structure, regulation, ligands, functions, and clinical trials of CD36 in cancer.
DOI:10.1095/biolreprod.103.020651URLPMID:12890718 [本文引用: 1]
Osteopontin (OPN) is an acidic member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family of extracellular matrix proteins/cytokines that undergoes extensive posttranslational modification, including phosphorylation, glycosylation, and cleavage, yielding molecular mass variants ranging in size from 25 to 75 kDa. The result is a versatile protein(s) with multiple functions arising from its role as a mediator of cell-cell and cell-extracellular matrix (ECM) communication that encompass both normal and tumorigenic developmental processes, immunological responses during inflammation and wound healing, and biomineralization. Studies in primates, pigs, sheep, and rodents have revealed that OPN is a major constituent of the uterine-placental microenvironment with influence as 1) a component of histotroph required for adhesion and signal transduction at the uterine-placental interface throughout pregnancy, 2) a gene product expressed by uterine stroma contributing to a decidualization-like transformation that correlates with the degree of conceptus invasiveness, and 3) a product of resident uterine and placental immune cells that may regulate their behavior and cytokine production. This minireview summarizes information regarding uterine and placental expression of OPN that has accumulated over the past 15 yr, and we briefly describe structural/functional properties of this protein that are likely relevant to its role(s) during pregnancy. Comparative studies have offered insights into the potential hormonal/cytokine, cellular, and molecular mechanisms underlying OPN-mediated adhesion, remodeling, and cell-cell/cell-ECM communication within the uterus and placenta. OPN has the potential to profoundly impact pregnancy, and investigators are now challenged to focus on the mechanistic nature of the functions of this multifaceted and major component of the uterine-placental microenvironment.
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URLPMID:12698769 [本文引用: 1]
Initiation of implantation is due not to passive growth pressure but to an active biochemical process that requires a blastocyst to interact with a carefully prepared endometrium. This versatile and dynamic process requires a variety of different molecules secreted by human trophoblast as well as endometrial cells that play a unique role. Several molecules have been shown to regulate, by an autocrine and paracrine manner, the cross-talk between the implanting blastocyst and the endometrial epithelium. Particularly, the molecular dialogue involves either cell-to-cell or cell-to-extracellular matrix interactions, mediated by matrix metalloproteinases, cytokines and growth factors. The present overview of the literature reports on the most significant molecules involved in the implantation process and describes the mechanisms of interaction and control. Since impaired blastocyst implantation is a significant cause of natural and in vitro fertilization pregnancy failure, a better understanding of the aforementioned molecular dynamics would be useful in improving the chance of viable pregnancy.
DOI:10.1038/nsmb.3005URLPMID:25849144 [本文引用: 1]
Alternative pre-mRNA splicing is a highly cell type-specific process essential to generating protein diversity. However, the mechanisms responsible for the establishment and maintenance of heritable cell-specific alternative-splicing programs are poorly understood. Recent observations point to a role of histone modifications in the regulation of alternative splicing. Here we report a new mechanism of chromatin-mediated splicing control involving a long noncoding RNA (lncRNA). We have identified an evolutionarily conserved nuclear antisense lncRNA, generated from within the human FGFR2 locus, that promotes epithelial-specific alternative splicing of FGFR2. The lncRNA acts through recruitment of Polycomb-group proteins and the histone demethylase KDM2a to create a chromatin environment that impairs binding of a repressive chromatin-splicing adaptor complex important for mesenchymal-specific splicing. Our results uncover a new function for lncRNAs in the establishment and maintenance of cell-specific alternative splicing via modulation of chromatin signatures.
DOI:10.1038/s41594-018-0054-4URLPMID:29662218 [本文引用: 1]
A recent revolution in RNA biology has led to the identification of new RNA classes with unanticipated functions, new types of RNA modifications, an unexpected multiplicity of alternative transcripts and widespread transcription of extragenic regions. This development in basic RNA biology has spawned a corresponding revolution in RNA-based strategies to generate new types of therapeutics. Here, I review RNA-based drug design and discuss barriers to broader applications and possible ways to overcome them. Because they target nucleic acids rather than proteins, RNA-based drugs promise to greatly extend the domain of 'druggable' targets beyond what can be achieved with small molecules and biologics.
URLPMID:32944911 [本文引用: 1]
DOI:10.1038/nmeth.4220URLPMID:28263961 [本文引用: 1]
Single-cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, thereby revealing new cell types and providing insights into developmental processes and transcriptional stochasticity. A key question is how the variety of available protocols compare in terms of their ability to detect and accurately quantify gene expression. Here, we assessed the protocol sensitivity and accuracy of many published data sets, on the basis of spike-in standards and uniform data processing. For our workflow, we developed a flexible tool for counting the number of unique molecular identifiers (https://github.com/vals/umis/). We compared 15 protocols computationally and 4 protocols experimentally for batch-matched cell populations, in addition to investigating the effects of spike-in molecular degradation. Our analysis provides an integrated framework for comparing scRNA-seq protocols.
