李杰章, 罗华伦, 谭光辉, 吴磊, 覃媛钰, 张依裕
贵州大学 动物科学学院 高原山地动物遗传育种与繁殖教育部重点实验室，贵州 贵阳 550025
收稿日期：2019-07-11；接收日期：2019-10-11
基金项目：国家自然科学基金(No. 31760663)，贵州省科技人才补助项目(No. [2017]5788)，贵州省千层次创新人才项目(No. 701030174401)资助

摘要：硬脂酰辅酶A去饱和酶-1 (Stearoyl-CoA desaturase-1，SCD-1)是催化单不饱和脂肪酸合成的关键性蛋白酶，Ca²⁺是生物体内重要阳离子，在生物体内发挥着重要作用。为探讨SCD-1基因与脂质指标和钙离子含量之间的关联性，通过构建pcDNA3.1(+)+SCD-1+Flag真核过表达载体和培养鸭子宫上皮细胞并共转染，通过载体上Flag标签检测SCD-1基因的过表达量，用Fluo-3/AM钙离子荧光标记法检测Ca²⁺浓度，用脂质指标试剂盒检测细胞内的脂质含量。结果表明，鸭SCD-1基因过表达量与甘油三酯(TG)和高密度脂蛋白胆固醇(HDL-C)含量呈负相关，与Ca²⁺浓度、总胆固醇(TC)含量、极低密度脂蛋白胆固醇(VLDL-C)含量和低密度脂蛋白胆固醇(LDL-C)含量呈正相关；Ca²⁺与TG、LDL-C和HDL-C的含量呈正相关，与TC和VLDL-C含量呈负相关，研究结果揭示了SCD-1基因过表达能够调控鸭子宫上皮细胞中Ca²⁺浓度和脂质合成与转运。
关键词：SCD-1基因鸭子宫上皮细胞Ca²⁺脂质指标
Effects of SCD-1 gene overexpression on the content of calcium ion and lipids in duck uterine epithelial cells
Jiezhang Li, Hualun Luo, Guanghui Tan, Lei Wu, Yuanyu Qin, Yiyu Zhang
Key Laboratory of Genetics, Breeding and Reproduction of Plateau Mountain Animals, Ministry of Education, College of Animal Science, Guizhou University, Guiyang 550025, Guizhou China
Received: July 11, 2019; Accepted: October 11, 2019
Supported by: National Natural Science Foundation of China (No. 31760663), Guizhou Province Science and Technology Talent Subsidy Project (No. [2017]5788), Guizhou Provincial Thousand-level Innovative Talent Project (No. 701030174401)
Corresponding author: Yiyu Zhang. Tel: +86-851-88298005; E-mail: zyy8yyc@163.com.

Abstract: Stearoyl-CoAdesaturase-1 (SCD-1) is a key regulator of monounsaturated fatty acid synthesis. It plays a vital role in lipid synthesis and metabolism. Ca²⁺ is an important cation in the body and plays an important role in the organism. The aims of this study were to investigate the correlation of SCD-1 gene overexpression with lipid indexes and calcium ion level. The pcDNA3.1 (+) + SCD-1 +Flag eukaryotic expression vector and cultured duck uterine epithelial cells were co-transfected. The overexpression of SCD-1 gene was measured using the Flag Label Detection Kit. Ca ions and lipid contents were detected through Fluo-3/AM Calcium Ion Fluorescence Labeling method and Lipid Measuring Kit, respectively. SCD-1 gene overexpression was negatively correlated with triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C), and positively correlated with Ca ion, total cholesterol (TC), very low-density lipoprotein cholesterol (VLDL-C) and low density lipoprotein cholesterol (LDL-C) levels. Meanwhile, Ca ion was positively correlated with TG, LDL-C and HDL-C contents, and negatively correlated with TC and VLDL-C levels. Overexpression of SCD-1 gene could regulate Ca ion secretion, as well as lipid synthesis and transport in duck uterine epithelial cells.
Keywords: SCD-1 geneduckuterine epithelial cellsCa²⁺lipid indexes
钙的代谢是畜禽机体中一个极其复杂的一项生命活动，能调节生物体内的脂质代谢，对脂肪球细胞的分化具有重要调节作用，还可以作为生物体内第二信使来调节其他生命活动^[1-2]，适当给动物补充膳食钙能有效降低血脂含量和改善血脂紊乱^[3-5]。Ca²⁺可以调节子宫上皮细胞的增殖、分化或凋亡，对参与调节哺乳动物繁殖系统生理和病理生理过程具有重要影响^[6]。至今，关于Ca²⁺与脂质代谢之间的调控关系已有相当多的报道，但关于调控脂质代谢的相关基因在调控脂质合成及转运过程中如何影响Ca²⁺水平的报道相当缺乏。
硬脂酰辅酶A去饱和酶-1 (Stearoyl-CoA desaturase-1，SCD-1)有助于催化形成单不饱和脂肪酸，而单不饱和脂肪酸又是甘油三酯蜡和膜磷脂的重要组成成分^[7]。最近在山羊乳腺上皮细胞(GMEC)中证实了SCD-1基因过表达对三酰甘油合成和脂肪酸组成具有重要的促进作用^[8]。SCD-1基因的活性在畜禽体内受温度波动、饮食、激素、再生过程、酒精、金属离子、过氧化物酶体增殖物和酚类化合物的影响^[9-10]。动物机体糖脂代谢紊乱导致的疾病如Ⅱ-型糖尿病、肥胖等均与SCD-1基因过表达有关^[11]。在人卵丘细胞上的研究表明，卵丘细胞中的SCD-1活性有助于卵母细胞的成熟，对卵丘细胞脂质的储存和类固醇的生成起重要作用^[12]。鞘脂类的合成对小鼠子宫上皮细胞具有增殖的作用^[13]。在猪子宫中胆固醇类激素参与子宫内的表达并参与把孕体固定到子宫中层侧^[14]。在人的膳食中补充钙能有效减轻体脂和体重，在人类预防肥胖病中发挥着重要作用^[15]。在家兔的饮食中增加胆固醇类和钙的含量可以降低发生动脉粥样硬化的风险^[16-17]。Ca²⁺浓度可以促进脂肪合成，抑制脂肪分解，在脂肪细胞代谢中起着重要作用，Ca²⁺浓度增加，会促进SCD-1的表达和脂肪酸转化酶(Fatty acid synthase，FAS)的活性，进一步促进脂肪酸的合成代谢^[18]。本研究通过体外培养鸭子宫上皮细胞，构建SCD-1基因真核表达载体，探讨SCD-1基因过表达对鸭子宫上皮细胞内Ca²⁺和脂质含量的效应，为进一步深入阐释鸭子宫上皮细胞中脂质和钙分泌路径提供了理论基础。
1 材料与方法1.1 试验材料随机选取饲养于贵州大学科研鸡场的产蛋高峰期(45周龄)的三穗鸭3只，用于鸭子宫上皮细胞的分离培养。大肠杆菌DH5α感受态细胞和无内毒素质粒小提试剂盒购自天根公司；限制性内切酶、Ex Taq均购自TaKaRa公司；T4 DNA Digase购自Promega公司；琼脂糖凝胶DNA纯化回收试剂盒、Thermo Fisher cDNA逆转录试剂盒均购自Invitrogen公司，支原体抑制剂购自GenDEPOT公司；青链霉素混合液、DMEM/F12培养基、Opti-MEM培养基、胎牛血清、0.05% Trypsin- EDTA、HEPES均购自于Gibco公司；PBS缓冲液Pluronic F-127、Hepes溶液均购自Solarbio公司；RIPA裂解液(强)购自于康为世纪公司；钙离子载体A23187半钙盐、Fluo-3/AM均购自Sigma公司；Lipofectamine^?3000、二甲基亚风(DMSO)均购自Invitrogen公司；甘油三酯(TG)试剂盒、高密度脂蛋白胆固醇(HDL-C)试剂盒、低密度脂蛋白胆固醇(LDL-C)试剂盒、总胆固醇(TC)试剂盒均购自于南京建成生物工程研究所；鸭Flag ELISA检测试剂盒、鸭极低密度脂蛋白胆固醇(VLDL-C) ELISA检测试剂盒购自生工生物工程(上海)股份有限公司。
1.2 方法1.2.1 子宫上皮细胞的培养将鸭猝死，在无菌条件下取出子宫，同时剔除子宫表面的薄膜，分成两份，一份用于细胞分离培养，一份用于总RNA提取。将预处理后的子宫用含3×双抗的HBSS (Hank’s Balanced Salt Solution——Hank’s平衡盐溶液)冲洗2–3次，将子宫剪碎，加入0.2% Ⅳ型胶原酶，置于37 ℃水浴锅中消化20 min后，加入终止液将消化液用400目和200目不锈钢细胞过滤筛过滤，用5%的普通培养液重悬细胞，37 ℃、5% CO₂条件下培养2–3 h，去掉上清液，用15% DMEM/F12完全培养基(100 mg/mL肝素钠、200 mmol/L L-谷氨酰胺、1 g/mL表皮生长因子、双抗、2.5 mg/mL胰岛素、5 μg/mL支原体预防剂)重悬沉淀，进行细胞铺板置于37 ℃、5% CO₂细胞培养箱中进行培养，取0.5 mL细胞悬液用台盼蓝染色检测细胞活性，每天显微镜观察细胞形态活性。
1.2.2 总RNA提取、cDNA合成和PCR扩增用Trizol法提取鸭子宫总RNA，根据Thermo Fisher cDNA逆转录试剂盒操作说明逆转录合成cDNA，–80 ℃保存。根据鸭SCD-1基因mRNA序列(GenBank登录号NW_004677643.1)，设计特异性引物(表 1)，单下划线区域为KOZAK序列，双下划线区域为加入的Flag标签，加粗碱基为保护碱基，上游引物中斜体碱基为KpnⅠ酶切位点，下游引物中斜体碱基为BamHⅠ酶切位点，虚线区域碱基上游为起始密码子下游为终止密码子，引物由生工生物工程(上海)股份有限公司合成。
表 1 试验所用引物Table 1 Primers used this study

Primer	Primer sequence (5′–3′)	Size (bp)
F	CGCGGTACCGCCACCATGGACTACAAAGACGATGAC GACAAGGATGAGACCTACCGTGAGAAGG	815
R	GGGGATCCTCATCAATAGTCATAGGGGAAGGTGTGG
Note: the single underlined region is the KOZAK sequence, the double underlined region is the added Flag tag, and the bold is the protection base. The italic site in the upstream primer is the Kpn I restriction site, the downstream primer italic is the BamH I restriction site, and the dotted region is the start codon and the stop codon.

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根据设计的特异性引物，以CDS为模板进行扩增，该区域共编码253个氨基酸。利用TaKaRa Ex Taq进行PCR扩增，PCR反应体系：Ex Taq 5 μL，上下游引物各0.5 μL (10 μmol/L)，模板cDNA 0.5 μL，DNase/RNase-Free Water (无酶无菌水)补充至10 μL。反应程序：预变性94 ℃ 7 min；变性95 ℃ 60 s，退火62 ℃ 40 s，延伸72 ℃ 1 min，35个循环；最后终延伸72 ℃ 8 min。将目的片段进行胶回收纯化，将其连接至pUCm-T载体中，转化到大肠杆菌DH5α，挑取白色阳性菌落进行扩繁，获得重组质粒，同时用BamHⅠ和KpnⅠ双酶切验证，回收酶切产物。
1.2.3 真核表达载体构建在回收的酶切产物中选取具有相同粘性末端的目的片段连接到pcDNA3.1(+)载体上，构建得到pcDNA3.1(+)+SCD-1+Flag真核表达载体，再次转化大肠杆菌DH5α进行培养，挑选白色阳性菌落进行菌液扩增，用无内毒素质粒提取试剂盒提取质粒DNA，进行双酶切验证。
1.2.4 转染子宫上皮细胞对子宫上皮细胞始浓度调整为1×10⁶个/mL进行培养，用Lipofectamine^?3000转染试剂分别加入5 μL pcDNA3.1(+)+SCD-1+Flag和pEGFP-C1共转染到子宫上皮细胞，待转染48 h后观察pEGFP-C1的荧光转染效率。未转染组设为空白组，在空白组中加入与转染相同的等体积水，保障空白组细胞受到相同的刺激。
1.2.5 子宫上皮细胞Ca²⁺荧光负载待真核表达载体转染到鸭子宫上皮细胞48 h后，将细胞稀释成1×10⁶个/mL，用胰蛋白酶消化细胞悬液，按照钙离子荧光探针Fluo-3/AM检测试剂盒说明对钙离子进行荧光负载，37 ℃、5% CO₂细胞培养箱中进行避光孵育45 min，设计7个重复组，对每组检测3次。[Ca²⁺]i浓度计算公式：[Ca²⁺]i=K_d*(F–F_min)/(F_max–F)^[19]，其中，F为实时荧光强度，Fluo-3/AM探针K_d为荧光剂与Ca²⁺形成复合物的解离常数，K_d=400 nmol/L。
1.2.6 脂质含量和基因表达量检测对转染48 h的鸭子宫上皮细胞用0.25%胰蛋白酶进行消化，将细胞稀释成1×10⁶个/mL，进行离心沉淀，用RIPA细胞裂解液进行裂解。设计7个重复组对每组检测3次，分别用TG、TC、HDL-C、LDL-C、VLDL-C脂质指标试剂盒和鸭Flag ELISA试剂盒检测脂质指标和Flag的表达量。同时，以空白组作为对照组。
1.3 数据统计采用Excel 2016电子表格整理数据，形成数据库，将数据导入SPSS 17.0统计软件，计算所有检测指标数据的均值和标准误及各指标间的相关系数。
2 结果与分析2.1 鸭子宫上皮细胞的培养分离鸭子宫上皮细胞在倒置显微镜下观察可见到单个的圆形细胞，散乱分布(图 1A)，台盼蓝染色活性鉴定(图 1B)，细胞生长良好存活率较高，活细胞呈透明状态，可以进行下一步试验。对细胞继续培养18 h后，在倒置显微镜下观察到细胞开始分裂，成团生长(图 2A)。培养48 h后观察到细胞贴壁成块向四周生长，呈不规则形态(图 2B)。培养72 h后，细胞开始出现扁平形状，多数细胞处于核分裂状态且细胞生长速度快(图 2C)。继续培养至96 h，细胞核明显，细胞与细胞之间紧密相连向四周生长，大部分形状规则，间隙清晰，细胞生长面积不断扩大(图 2D、2E)。培养5 d后，细胞基本铺满培养皿，细胞间隙明显，细胞呈现高度分裂状态，部分区域出现细胞重叠现象，此时可进行细胞转染及后续实验(图 2F)。

图 1 鸭子宫上皮细胞处分离和活性鉴定 Fig. 1 Isolation and activity identification of uterus epithelial cells in ducks. (A) Primary isolated uterine epithelial cells. (B) Tymphenol blue staining (Yellow arrow indicates dead cells, purple arrows indicate living cells).

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图 2 鸭子宫上皮细胞培养 Fig. 2 Growth state of duck primary uterus epithelial cells.

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2.2 SCD-1基因真核表达载体的构建根据设计的特异性引物，以cDNA为模板进行PCR扩增，用琼脂糖凝胶电泳检测后，在815 bp左右处出现单一条带，将片段进行回收纯化之后连接到pUCm-T载体，用KpnⅠ和BamHⅠ双酶切T克隆重组质粒和pcDNA3.1(+)空载体，将目的基因与pcDNA3.1(+)载体连接，转化到大肠杆菌DH5α，挑选白色阳性菌落进行菌液PCR检验(图 3A)和提取质粒进行双酶切电泳(图 3B)验证，测序结果显示，目的片段序列与载体序列大小一致，表明SCD-1基因真核表达载体构建成功，可用于后续试验。

图 3 琼脂糖凝胶电泳检测结果 Fig. 3 Agarose gel photograph. (A) Gel electrophoresis of PCR product. M : DM2000DNA marker; 1–6 : pcDNA3.1 (+) +SCD-1+Flag. (B) Double digestion validation map. M: SuperDNA marker; 1–2: pcDNA3.1(+)+SCD-1+Flag.

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2.3 Fluo-3/AM荧光标记法对钙离子进行荧光负载检测Fluo-3/AM荧光标记法对钙离子进行荧光负载，37 ℃、5% CO₂细胞培养箱中进行避光孵育45 min后，在倒置荧光显微镜下观察到点状绿色荧光(图 4)，说明Fluo-3/AM荧光探针成功负载到鸭子宫上皮细胞Ca²⁺上。

图 4 Fluo-3/AM荧光探针负载子宫上皮细胞Ca2+ Fig. 4 Ca2+ labeled in uterus epithelial cells using Fluo-3/AM fluorescent probe

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2.4 pcDNA3.1(+)+SCD-1+Flag和pEGFP-C1共转染鸭子宫上皮细胞将pcDNA3.1(+)+SCD-1+ Flag和pEGFP-C1共转染鸭子宫上皮细胞如图 5所示，pEGFP-C1能正常发出荧光，说明转染成功，同时也说明了pcDNA3.1(+)+SCD-1+Flag能够在同一条件下成功转染到鸭子宫上皮细胞。

图 5 pcDNA3.1(+)+SCD-1+Flag和pEGFP-C1共转染子宫上皮细胞 Fig. 5 pcDNA3.1(+)+SCD-1+Flag and pEGFP-C1 co-transfection uterus epithelial cells. (A) Dark field. (B) Bright field.

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2.5 鸭SCD-1基因过表达与子宫上皮细胞内Ca²⁺及脂质指标的相关性Fluo-3/AM荧光探针对鸭子宫上皮细胞内钙离子负载成功后，在激发光488 nm下检测荧光值。通过公式[Ca²⁺]i计算出子宫上皮细胞内Ca²⁺浓度，根据鸭Flag标签检测试剂盒和脂质指标检测试剂盒检测SCD-1基因在鸭子宫上皮细胞中的过表达量和相应的脂质指标含量，结果见表 2。由表 2可知，子宫上皮细胞内Ca²⁺浓度最高为57.45 nmol/L，最低为12.58 nmol/L。所检测的5个脂质指标的含量与SCD-1基因过表达增高不呈规律性变化。空白对照组通过Flag标签检测SCD-1基因过表达为0，脂质指标TG和HDL-C含量都低于7个实验组平均值，TC、VLDL-C、LDL-C和Ca²⁺浓度高于平均值。
表 2 鸭SCD-1基因在子宫上皮细胞中的过表达量与脂质指标含量和Ca²⁺浓度Table 2 Overexpression of duck SCD-1 gene, lipid content, and Ca²⁺ concentration in uterine epithelial cells

Group	SCD-1 (ng/mL)	TG (mmol/gprot)	TC (mmol/gprot)	HDL-C (mmol/gprot)	VLDL-C (mg/mL)	LDL-C (mmol/gprot)	[Ca2+]i (nmol/L)
1	0	0.141 0±0.001 2	0.269 5±0.000 4	1.817 2±0.034 5	10.817 2±0.007 8	0.193 4±0.000 4	31.683±6.427 5
2	4.571 9±0.002 3	0.204 7±0.000 3	0.192 3±0.000 1	2.538 5±0.025 9	10.125 4±0.000 9	0.101 9±0.000 7	18.81±2.625 8
3	4.945 7±0.002 0	0.304 8±0.001 6	0.134 1±0.000 2	4.134 0±0.001 5	11.175 5±0.002 1	0.121 4±0.000 3	40.75±4.860 5
4	4.954 6±0.005 1	0.191 6±0.000 3	0.110 7±0.000 3	3.070 4±0.014 5	8.181 8±0.006 9	0.150 7±0.000 1	57.45±9.117 4
5	5.008 0±0.007 0	0.235 1±0.000 7	0.215 7±0.000 1	2.774 9±0.033 7	9.498 4±0.004 4	0.113 9±0.000 3	12.58±2.010 6
6	5.372 9±0.001 7	0.047 9±0.000 3	0.518 7±0.001 3	3.755 8±0.010 1	9.232 0±0.017 0	0.186 8±0.000 6	19.73±0.852 6
7	5.586 6±0.007 9	0.230 8±0.000 1	0.169 0±0.000 2	3.354 0±0.024 3	10.235 1±0.002 8	0.182 8±0.001 1	36.28±3.717 3
8	5.969 3±0.002 4	0.204 7±0.000 1	0.145 7±0.000 1	2.361 3±0.011 0	11.426 3±0.001 8	0.109 9±0.000 3	29.80±2.497 7
Mean	5.201 3±0.004 0	0.202 8±0.000 5	0.212 3±0.000 3	3.141 2±0.017 3	9.982 1±0.005 1	0.138 1±0.000 5	30.77±3.67
TG: triglyceride; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; VLDL-C: low density lipoprotein cholesterol; HDL-C: high density lipoprotein cholesterol.

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通过对测定的值进一步分析SCD-1基因过表达、钙离子和脂质指标之间的关系，结果见表 3。结果表明，鸭子宫上皮细胞中SCD-1基因过表达量与TG和HDL-C的含量呈负相关，与TC、VLDL-C和LDL-C的含量及[Ca²⁺]i呈正相关，说明SCD-1基因过表达对细胞中TG和HDL-C的含量有抑制分泌的趋势，对TC、VLDL-C和LDL-C含量及[Ca²⁺]i的浓度有促进分泌的趋势；Ca²⁺浓度与细胞内TC和VLDL-C含量呈负相关，与细胞内TG、HDL-C和LDL-C的含量呈正相关，表明细胞内Ca²⁺浓度升高，对细胞内TG、LDL-C和HDL-C的分泌有促进作用，对细胞TC和VLDL-C的分泌有抑制的作用；TG的含量与VLDL-C的含量呈正相关，与HDL-C和LDL-C的含量呈负相关，与TC的含量呈显著负相关关系，表明TG的含量与VLDL-C在细胞内协调分泌，与其他脂质指标相互抑制分泌；TC的含量与VLDL-C的含量呈负相关关系，与HDL-C和LDL- C的含量呈正相关；HDL-C的含量与VLDL-C的含量呈负相关，与LDL-C呈正相关；VLDL-C与LDL-C呈负相关，揭示SCD-1基因过表达能够影响子宫上皮细胞中Ca²⁺水平和脂质含量。
表 3 鸭SCD-1基因过表达量及其与脂质指标和[Ca²⁺]i的相关性Table 3 Overexpression of duck SCD-1 gene and its correlation with lipid index and [Ca²⁺]i

Index	SCD-1 overexpression	TG	TC	HDL-C	VLDL-C	LDL-C	[Ca2+]i
SCD-1 overexpression	1	–0.205	0.106	–0.108	0.383	0.326	0.032
TG		1	–0.852*	–0.005	0.479	–0.548	0.273
TC			1	0.316	–0.267	0.524	–0.529
HDL-C				1	–0.303	0.543	0.283
VLDL-C					1	–0.427	–0.198
LDL-C						1	0.237
[Ca2+]i							1
*P < 0.05 was significantly different. TG: triglyceride; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; VLDL-C: low density lipoprotein cholesterol; HDL-C: high density lipoprotein cholesterol.

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3 讨论SCD-1基因是一种普遍存在的亚型，在组织中广泛表达，该基因的过表达水平与代谢紊乱(如肥胖和胰岛素抵抗)相关，近年研究表明SCD-1被确定是细胞生长、程序性细胞死亡和致癌的关键调节因子^[20-21]。还有研究表明SCD-1基因在人类癌症、致癌物诱导肿瘤和病毒转化细胞中过度表达，导致细胞膜流动性增强^[22-24]。SCD-1基因有助于形成单不饱和脂肪酸，硬脂酸盐和棕榈酸酯的积累可以改变细胞膜流动性，进而控制细胞增殖、分化和凋亡^[25-26]，膜层中增加的棕榈酸酯浓度可降低上皮细胞膜的流动性，从而导致细胞功能受损或细胞死亡^[27]。SCD-1基因的活性的增加被证明可以保护人类动脉内皮细胞免受饱和脂肪酸的毒性影响^[28]。SCD-1基因在肺内质网中过度表达时，其表达水平与胆固醇调节元件结合蛋白水平呈正相关^[29]。SCD-1在小鼠卵母细胞中参与饱和脂肪酸底物向甘油三酯代谢，SCD-1的活性与TG之间存在功能性间接联系^[30]，但是过多的不饱和脂肪酸和饱和脂肪酸都不利于正常胎盘形成和子宫内膜的生长^[31]。本研究发现鸭子宫上皮细胞中SCD-1基因过表达与TG和HDL呈负相关，与TC、VLDL和LDL呈正相关，表明SCD-1活性在鸭子宫上皮细胞与脂质指标之间存在一定的功能性联系。
钙是动物机体功能发挥的重要矿物质。Daugherty等^[32]通过研究发现，钙通道阻滞剂可以抑制巨噬细胞内胆固醇酯的沉积。在小鼠脂肪细胞中Ca²⁺内流增加能刺激脂肪生成，抑制脂肪分解，从而影响细胞的TG的积累^[33]。Ma等^[34]研究结果表明，通过降低饮食中的钙，可以导致血浆中总胆固醇(TC)和甘油三酯(TG)的含量降低，使高密度脂蛋白胆固醇(HDL-C)的含量增加，进一步说明Ca²⁺的浓度对脂质含量具有一定的调节作用。Baran等^[35]通过刺激膜联蛋白与Ca²⁺结合促进脂肪细胞内TG含量增多。在基质金属蛋白酶(MMPs)中能降低TG的含量，钙离子浓度增加进一步促进VLDL-C和LDL-C被脂蛋白脂肪酶(Lipoprotin lipase，LPL)水解^[36]。本研究鸭SCD-1基因过表达量及其与脂质指标和[Ca2+]i的相关性表明，Ca²⁺浓度与细胞内TG、HDL-C和LDL-C的含量呈正相关，与Baran等^[35]研究结果一致，表明随着细胞内Ca²⁺浓度升高，对细胞分泌TG、LDL-C和HDL-C有促进趋势，说明细胞内Ca²⁺浓度与细胞内脂质含量有着密切的联系。
在关联性分析结果中，SCD-1基因的过表达与Ca²⁺浓度呈正相关关系，表明过表达可以促进子宫上皮细胞内钙离子浓度升高。对SCD-1基因过表达和Ca²⁺浓度对脂质含量进行相关性分析，分析结果显示，SCD-1基因过表达和Ca²⁺浓度与LDL-C含量呈现正相关关系，对其他脂质指标的含量呈负相关，在鸭子宫上皮细胞中SCD-1基因过表达可能是在细胞内分泌Ca²⁺增加的基础上促进LDL-C的分泌，说明SCD-1基因过表达对细胞中Ca²⁺的浓度有促进分泌的趋势，SCD-1基因过表达对Ca²⁺具有正向调控的作用。本研究通过探究SCD-1基因过表达测量鸭子宫上皮细胞钙离子和脂质的参数，探究SCD-1在子宫上皮的作用，由于对原代细胞的利用有限，只测试了与该基因相关的指标，而要完全探究功能需要根据过表达、基因敲除和克服原代细胞培养的局限性，因此鸭子宫上皮细胞SCD-1基因的作用有待进一步研究。
4 结论本研究成功构建了pcDNA3.1(+)+SCD-1+Flag真核表达载体，实现其对鸭子宫上皮细胞的转染；相关性分析表明，鸭子宫上皮细胞中SCD-1基因过表达与甘油三酯(TG)和高密度脂蛋白胆固醇(HDL-C)含量呈负相关，与子宫上皮细胞内Ca²⁺浓度、总胆固醇(TC)含量、极低密度脂蛋白胆固醇(VLDL-C)含量和低密度脂蛋白胆固醇(LDL-C)含量呈正相关；Ca²⁺浓度与TG、LDL-C和HDL-C的含量呈正相关，与VLDL-C和TC含量呈负相关，进一步说明了SCD-1基因过表达能够调控子宫上皮细胞Ca²⁺分泌和脂质合成与转运。
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