李桢, 杨世雄, 牛胜, 张宁, 李欣, 张阳阳, 贾云飞, 田志雄, 宁官保, 张鼎, 田文霞^,山西农业大学动物科技学院,山西太谷 030801

Effect of Recombinant GSTA3 Protein on Expression of the Anti-Apoptotic Gene BAG-3 in Thiram-Induced Tibial Chondrodysplasia

LI Zhen, YANG ShiXiong, NIU Sheng, ZHANG Ning, LI Xin, ZHANG YangYang, JIA YunFei, TIAN ZhiXiong, NING GuanBao, ZHANG Ding, TIAN WenXia^,College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi

通讯作者: 田文霞,E-mail：wenxiatian@126.com

责任编辑: 林鉴非
收稿日期:2019-05-20接受日期:2019-12-26网络出版日期:2020-05-16

基金资助:

国家重点研发计划.2016YFD0500806 2017年地方高校国家级大学生创新创业训练计划项目.201710113001 2017年山西省高等学校大学生创新创业训练计划项目.2017081 山西省回国留学人员科研资助项目.2017-073 晋中市重点科技创新平台.P171002-3 国家自然科学基金.31072179 山西省科技攻关项目.20130311027-3

Received:2019-05-20Accepted:2019-12-26Online:2020-05-16
作者简介 About authors
李桢,E-mail：1091856050@qq.com。













摘要
【目的】肉鸡胫骨软骨发育不良（TD）是肉鸡常见的一种骨骼性疾病,研究重组GSTA3蛋白对福美双诱导的TD肉鸡软骨细胞中抗凋亡基因BAG-3表达的影响,为治疗TD提供新的思路和方法。【方法】将120羽1周龄肉雏鸡随机分为6组（编号为A、B、C、D、E、F组）。A、B、C组为基础日粮对照组,D、E、F组为添加福美双日粮诱导TD组。试验饲喂福美双2 d诱发TD,在添加福美双第1、3、5、7天,腿部肌肉注射重组鸡GSTA3蛋白和磷酸盐缓冲液,A组与D组注射（100 μg·kg ^-1）磷酸盐缓冲液;B组与E组注射低剂量（100 μg·kg ^-1）GSTA3;C组与F组注射高剂量（200 μg·kg ^-1）GSTA3。试验历时23 d。添加福美双后1、2、4、6、10和15 d采集胫骨生长板。通过Real-time qPCR检测BAG-3基因的mRNA水平,利用免疫组化来检测BAG-3蛋白表达水平。【结果】Real-time qPCR结果显示,TD损伤修复期内,相比较于基础日粮对照组,福美双对照组肉鸡胫骨生长板中BAG-3 mRNA的表达水平基本都显著上调（P<0.05）;相比较于福美双对照组,E和F组在第2、4、10、15天都有显著差异,且在第10和15天显著低于福美双对照组（P<0.05）,表明与D组相比恢复较快。免疫组化结果表明BAG-3蛋白在肉鸡胫骨软骨细胞的增殖区和前肥大区无表达,只在肥大区细胞质中表达;福美双组与空白对照组相比,BAG-3蛋白表达增加;福美双高低剂量组与未注射蛋白的福美双组相比,重组GSTA3增加了肥大区的蛋白表达水平（第10和15天）。【结论】在福美双诱导肉鸡发生TD的过程中,GSTA3重组蛋白能够通过调控BAG-3表达参与凋亡途径,抑制细胞凋亡。在TD损伤修复期,注射GSTA3后使抗凋亡基因BAG-3蛋白表达增强,从而可参与细胞凋亡来缓解TD损伤,使得肉鸡TD生长板功能较快地恢复正常。
关键词： 谷胱甘肽S-转移酶A3（GSTA3）;胫骨软骨发育不良;BAG-3基因;抗凋亡;肉鸡

Abstract
【Objective】Tibial chondrodysplasia (TD) is a common skeletal disease in broilers. The study was conducted to investigate the effect of recombinant GSTA3 protein on the expression of anti-apoptotic gene BAG-3 in the chondrocytes of broilers induced by thiram, so as to provide a new idea and method for the treatment of TD. 【Method】120 one-week-old broiler chicks were randomly divided into six groups (A, B, C, D, E and F), in which Group A, B and C were basic diet groups, and group D, E and F were thiram-containing diet groups. TD was induced by thiram for 2 days. After thiram was added for days 1, 3, 5 and 7, recombinant chicken GSTA3 protein and phosphate buffer were injected into the leg muscles. Group A and group D were injected with PBS (100 μg·kg ^-1); group B and group E were injected with low dose (100 μg·kg ^-1) GSTA3; group C and group F were injected with high dose (200 μg·kg ^-1) GSTA3. The experiment lasted 23 days. Tibia growth plates were collected at the 1st, 2nd, 4th, 6th, 10th and 15th days after thiram treatment. The mRNA level of BAG-3 gene was detected by quantitative PCR, and the protein expression of BAG-3 gene was observed by immunohistochemistry. 【Result】Real-time PCR results showed that the expression level of BAG-3 mRNA in tibia growth plate of broilers in the thiram-containing diet group was significantly increased, compared with that in the basal diet control group during the repair period of TD injury (P<0.05). Compared with the thiram-containing diet group, E and F groups had significant differences on days 2, 4, 10 and 15, and were significantly lower than that under the thiram-containing diet group on days 10 and 15 (P<0.05), indicating a faster recovery compared with group D. Immunohistochemical results showed that BAG-3 protein was not expressed in the proliferation area and the pre-hypertrophic area of the chicken tibial chondrocytes, but only in the cytoplasm of the hypertrophic area. Compared with the control group A, the expression of BAG-3 protein in the thiram-containing diet group was increased. The recombinant GSTA3 induced the protein expression level in the hypertrophic area in the high and low dose of GSTA3 group compared with the thiram-containing diet group D (days 10 and 15). 【Conclusion】In conclusion, GSTA3 recombinant protein could regulate the expression of BAG-3 to participate in the apoptosis pathway and inhibit the apoptosis during the induction of TD in broilers. During the TD-injury repair period, after GSTA3 injection, the expression of anti-apoptotic gene BAG-3 protein was enhanced, which could participate in cell apoptosis to alleviate the TD-injury and make the TD growth plate function of broilers return to normal quickly.
Keywords：glutathione S-transferase A3 (GSTA3);tibial dyschondroplasia;BAG-3;antiapoptotic;broiler


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本文引用格式
李桢, 杨世雄, 牛胜, 张宁, 李欣, 张阳阳, 贾云飞, 田志雄, 宁官保, 张鼎, 田文霞. 重组GSTA3蛋白对福美双诱导的肉鸡TD中抗凋亡基因BAG-3表达的影响[J]. 中国农业科学, 2020, 53(9): 1921-1930 doi:10.3864/j.issn.0578-1752.2020.09.018
LI Zhen, YANG ShiXiong, NIU Sheng, ZHANG Ning, LI Xin, ZHANG YangYang, JIA YunFei, TIAN ZhiXiong, NING GuanBao, ZHANG Ding, TIAN WenXia. Effect of Recombinant GSTA3 Protein on Expression of the Anti-Apoptotic Gene BAG-3 in Thiram-Induced Tibial Chondrodysplasia[J]. Scientia Acricultura Sinica, 2020, 53(9): 1921-1930 doi:10.3864/j.issn.0578-1752.2020.09.018

0 引言

【研究意义】肉鸡胫骨软骨发育不良（tibial dyschondroplasia, TD）是一种肉鸡常见的骨骼疾病,胫骨生长板形成无血管的软骨栓,使肉鸡的胫骨软骨内成骨受阻^[1]。TD是目前危害肉鸡业的主要代谢病之一,其致病机理尚不清楚,目前仍无有效的防治方法,TD的发病机制可能与生长板软骨细胞相关凋亡基因的表达调控有关^[2]。【前人研究进展】RATH等研究了3种二硫代氨基甲酸二甲酯诱导TD发生,其中福美双效果最为显著,通过饲喂福美双的方法,可在短时间内建立与自发TD临床表现相似的动物模型^[3]。谷胱甘肽S-转移酶（glutathione S-transferases,GSTs）是机体内一类十分重要的酶,由BOOTH等在1961年首次发现^[4]。GSTs有3个亚族,分别为微粒体GSTs、细胞质GSTs、细菌磷霉毒抗性GSTs^[5]。当机体在恶劣条件下,它通过催化某些内源性或外来有害物质的电子基团与还原型谷胱甘肽的疏基结合,保护细胞使其不受损伤^[6]。GSTA3蛋白与已知的GSTs其他酶对催化类固醇激素生物合成的过程相比更有效^[7]。田文霞等基因芯片检测结果发现GSTA3基因表达下调,福美双可能影响了GSTs的解毒功能,从而导致肉鸡TD的发生^[8]。Bcl-2家族蛋白作为细胞凋亡的主要调控因子,主要定位于核膜、内质网和线粒体外膜上^[9],Bcl-2相关抗凋亡蛋白3（Bcl-2associated athanogene 3,BAG-3）也被称为Bis或CAIR- 1,是抗凋亡基因BAG家族的成员之一,Bcl-2相关抗凋亡家族的C末端均有一段高度保守的功能区域^[10],因此其进化高度保守。BAG-3通过免疫共沉淀、酵母双杂交及免疫染色等研究发现BAG-3能够与Bcl-2相互作用,共同发挥抗调亡作用^[11]。BAG-3是一种生存蛋白,在细胞对高温、重金属、蛋白酶体抑制和人类免疫缺陷病毒1（HIV-1）感染等应激条件的反应过程中被激活而发挥作用^[12]。在急性应激和细胞老化过程中,BAG-3可激活蛋白质量控制（PQC）系统使细胞蛋白质稳定,也可参与清除与年龄相关的神经退行性疾病相关的蛋白^[13]。BAG-3是骨骼肌机械转导的必要条件,在免疫系统中发挥重要调节作用^[14]。RATH等与山西农业大学动物科技学院兽医生物制品实验室的前期研究发现福美双可引起血管内皮细胞及软骨细胞凋亡,但是BAG-3在TD肉鸡中软骨细胞的表达情况、GSTA3与细胞凋亡及BAG-3存在什么关系,尚未见相关研究。【本研究切入点】本研究团队前期研究结果显示,重组GSTA3蛋白可能在福美双诱导的TD中发挥重要作用,但其对抗软骨细胞凋亡的作用如何,有待进一步研究。【拟解决的关键问题】本试验通过免疫组织化学技术和Real-time PCR技术,研究福美双诱导的肉鸡TD中抗凋亡基因BAG-3的表达以及鸡重组GSTA3蛋白对BAG-3表达的影响,探讨GSTA3与BAG-3的关系,以及对TD恢复的作用,为后期对TD的治疗提供新的思路和方法。

1 材料与方法

1.1 材料

1.1.1 实验动物 120只1日龄艾维茵（AA）肉雏鸡购于山西大象农牧集团有限责任公司。

1.1.2 试验试剂 福美双（美国Amresco公司）;重组鸡GSTA3蛋白^[15]（实验室自行制备）;BAG-3一抗（Bioworld生物公司）;RNAiso plus（Trizol）（AA909-1）（大连宝生物工程公司）、prime script TM RT Reagent Kit（AK3020）（大连宝生物工程公司）、SYBR? Premix Ex Taq Ⅱ（Tli RNaseH Plus）（AK6003）(大连宝生物工程公司);DEPC（Amresco公司）;6×DNA loading buffer（中科瑞泰生物科技有限公司）、DNA ladder（中科瑞泰生物科技有限公司）;50×TAE Buffer（北京博奥拓达科技有限公司）、Agarose-G10（Biowest公司）;甲醛、异丙醇、异戊醇、乙醚、氯仿、氯化钠、无水乙醇、盐酸、二甲苯、EDTA均为国产分析纯试剂。

1.1.3 试验地点 试验于2017年7月至9月,在山西农业大学动物科技学院兽医生物制品实验室完成。

1.2 方法

1.2.1 肉鸡TD模型建立及生长板的采集 120羽一日龄AA肉鸡饲养7 d,随机分六组（编号为A、B、C、D、E、F组）。A、B、C组为基础日粮组,D、E、F组为福美双日粮组（第8、9日龄在饲料中添加福美双）。第8、10、12、14天,腿部肌肉注射重组鸡GSTA3蛋白,A组与D组注射（100 μg·kg^-1）磷酸盐缓冲液;B组与E组注射低剂量（100 μg·kg^-1）GSTA3;C组与F组注射高剂量（200 μg·kg^-1）GSTA3（表1）。试验历时23 d。攻毒后,分别在第1、2、4、6、10、15天,每组选3羽,取胫骨生长板,一部分固定用于免疫组化;一部分冻存,用于定量PCR检测。

Table 1
表1
表1试验分组情况
Table 1Test grouping

分组Group	日粮Daily diet	注射物Injectant
A	基础日粮Basic diet	PBS
B	基础日粮Basic diet	100 μg·kg-1重组GSTA3蛋白100 μg·kg-1 recombinant GSTA3 protein
C	基础日粮Basic diet	200 μg·kg-1重组GSTA3蛋白200 μg·kg-1 recombinant GSTA3 protein
D	福美双日粮Thiram diet	PBS
E	福美双日粮Thiram diet	100 μg·kg-1重组GSTA3蛋白100 μg·kg-1 recombinant GSTA3 protein
F	福美双日粮Thiram diet	200 μg·kg-1重组GSTA3蛋白200 μg·kg-1 recombinant GSTA3 protein

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1.2.2 免疫组织化学技术检测BAG-3蛋白的表达 将各处理组的胫骨生长板用4%的甲醛溶液固定24 h,修块后固定24 h,置于10%的EDTA中脱钙24 h,用梯度酒精（50%、70%、75%、80%、90%、100%）脱水2 h,在无水乙醇与二甲苯的1﹕1混合液中20 min,用二甲苯Ⅰ和二甲苯Ⅱ依次透明35 min后,包埋,制片,染色,利用显微镜观察BAG-3蛋白定位及阳性表达结果。

1.2.3 总RNA提取及反转录 采用Trizol改进法进行RNA的提取,具体操作方法参考喻进^[16]的方法进行。根据prime script ^TM RT Reagent Kit的说明书进行反转录,去除基因组DNA,所有操作在冰上完成,最后置于-80℃保存。

1.2.4 Real-time qPCR检测BAG-3基因mRNA的表达 采用Real-time qPCR对BAG-3基因mRNA的表达进行检测（表2）。Real-time qPCR反应体系如下：5.0 μL SYBR Premix Ex Taq II（2×）,0.25 μL PCR Forward Primer（10 μmol·L^-1）,0.25 μL PCR Reverse Primer（10 μmol·L^-1）,0.1 μL ROX Reference DyeⅡ（50×）,1.0 μL RT反应液（cDNA溶液）,3.4 μL ddH₂O。反应条件：预变性反应 1 cycle,95℃ 3 min;PCR反应 42 cycles,95℃ 30 s,55℃ 30 s;溶解曲线分析1 cycle,55℃ 30 s,95℃ 30 s。然后利用2.0% 的琼脂糖凝胶电泳对PCR反应产物进行鉴定。以18S rRNA作为内参基因,并用2^-△△Ct法计算目的基因在胫骨生长板的相对表达量。

Table 2
表2
表2Real-time PCR所用引物
Table 2Primer design for Real-time PCR

基因Gene	引物序列（5′→3′）Primer Sequence（5′→3′）	片段Size (bp)	退火温度Annealing temperature (℃)
BAG-3	F: TTCTGATTCTCCAACGGTTT R: TTCATGGATCACAGGAATTG	100	55
18S rRNA	F：TTCCGATAACGAACGACAC R：GACATCTAAGGGCATCACAG	139	55

F代表上游引物;R代表下游引物
F means forward primers, R means reverse primers

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1.2.5 数据分析 用SPSS Statistics 17.0软件对计算出的各组数据进行差异显著性分析,P<0.05即为差异显著,差异性的结果按照统计学标注方法来区分显著性。

2 结果

2.1 肉鸡生长板BAG-3蛋白表达的情况

免疫组化结果发现,BAG-3蛋白主要在肉鸡胫骨软骨细胞的肥大区表达,增殖区和前肥大区均未表达,定位于细胞质中。试验第1天,与空白对照组（A）相比,低剂量和高剂量的基础日粮组（B和C）及低剂量和高剂量福美双组（E和F）无明显变化,而福美双对照组（D）表达明显增强（图1）;试验第2天,A组与B、C组表达无明显变化,D、E与F组表达一致（图2）;试验第4天,E组相比D、F组相对减少,趋于正常,和A、B、C组表达情况一致（图3）;试验第6天,D表达稍弱,其他无明显差异（图4）;试验第10天,A、B、C表达无明显差异,D表达增强,E、F相比D表达较弱（图5）;试验第15天,A、B、C表达无明显差异,D强于E、F（图6）。

图1

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图1第1天肉鸡生长板BAG-3表达变化

P.增殖区;PH.前肥大区;H.肥大区。下图同
Fig. 1Expression changes of BAG-3 in tibial growth plate on day 1

P: Proliferative zone; PH: Prehypertrophic zone; H: Hypertrophic zone. The same as below

图2

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图2第2天肉鸡生长板BAG-3表达变化

Fig. 2Expression changes of BAG-3 in tibial growth plate on day 2

图3

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图3第4天肉鸡生长板BAG-3表达变化

Fig. 3Expression changes of BAG-3 in tibial growth plate on day 4

图4

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图4第6天肉鸡生长板BAG-3表达变化

Fig. 4Expression changes of BAG-3 in tibial growth plate on day 6

图5

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图5第10天肉鸡生长板BAG-3表达变化

Fig. 5Expression changes of BAG-3 in tibial growth plate on day 10

图6

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图6第15天肉鸡生长板BAG-3表达变化

Fig. 6Expression changes of BAG-3 in tibial growth plate on day 15

2.2 重组GSTA3蛋白对TD肉鸡中BAG-3 mRNA水平的调节

A.基础日粮+注射PBS;B.基础日粮+100 μg·kg^-1重组GSTA3蛋白;C.基础日粮+200 μg·kg^-1重组GSTA3蛋白;D.福美双日粮+注射PBS;E.福美双日粮+100 μg·kg^-1重组GSTA3蛋白;F.福美双日粮+200 μg·kg^-1重组GSTA3蛋白;不同的小写字母表示差异有统计学意义（P<0.05）。

图7表明,TD损伤修复期内,相比较于基础日粮对照组,福美双对照组肉鸡胫骨生长板中BAG-3 mRNA的表达水平基本都显著上调（P<0.05）;相比较于福美双对照组,E和F组在第2、4、10、15天都有显著差异,且在第10和15天显著低于福美双对照组（P<0.05）,表明与D组相比恢复较快,并与蛋白表达相一致。

图7

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图7重组鸡GSTA3蛋白对TD肉鸡胫骨生长板BAG-3 mRNA表达的影晌

A. 基础日粮组+ PBS; B. 基础日粮组+100 μg·kg^-1 重组GSTA3蛋白; C. 基础日粮组+200 μg·kg^-1重组GSTA3蛋白; D. 福美双日粮组+ PBS; E. 福美双日粮组+100 μg·kg^-1重组GSTA3蛋白;F. 福美双日粮组+200 μg·kg^-1重组GSTA3蛋白;不同小写字母表示差异有统计学意义（P<0.05）
Fig. 7Expression of BAG-3 mRNA in growth plate of the TD broiler injected with different dosage of chGSTA3 protein

A. Basic diet + PBS; B. Basic diet +100 μg·kg^-1 recombinant GSTA3 protein; C. Basic diet +200 μg·kg^-1 recombinant GSTA3 protein; D. Diet containing thiram + PBS injection; E. Diet containing thiram +100 μg·kg^-1 recombinant GSTA3 protein; F. Diet containing thiram +200 μg·kg^-1 recombinant GSTA3 protein; Different small letters indicate significant statistics difference(P<0.05)

3 讨论

田文霞等和张宁等用福美双诱导TD后,胫骨近端软骨增殖区增厚,前肥大区和肥大区加长,软骨细胞急剧增生、大量调亡^[2,17]。RATH等采取TUNEL法研究发现,在22日龄TD明显时,有大量的毛细血管内皮细胞和软骨细胞凋亡^[18]。细胞凋亡由不同群体执行者和调控分子及其特异性功能所调控^[19]。抗凋亡基因BAG-3表达越多,表明越能抵抗细胞凋亡。

BAG-3主要分布于细胞质,在机体中起抵抗细胞癌变,提高细胞存活率的作用^[20] ,参与细胞凋亡调控是BAG-3重要生物学功能之一,其作用为保护细胞抵御重金属^[21]、高温^[22]及某些药物的刺激,其机制之一是通过介导蛋白的传递过程从而干扰细胞色素c的释放、凋亡小体的组装等过程来调节细胞凋亡^[23]。研究表明,在癌症和肌病的发生发展过程中多功能BAG-3蛋白起着决定性的作用^[24],在胰腺癌细胞中BAG-3基因表达水平较高,可能通过Bcl-2和BAG-3的协调作用使细胞发生抗凋亡^[25];在结肠癌细胞中BAG-3基因也呈现高表达,促进细胞存活^[26];在膀胱癌细胞中BAG-3是协同诱导其凋亡的联合治疗的合适靶点^[27];在心肌细胞中BAG-3促进细胞增殖并抑制细胞凋亡^[28]。魏莉^[29]等报道,BAG-3在子宫颈癌细胞和组织中呈现高表达,从而参调控细胞凋亡。有试验研究指出,BAG-3在红细胞中的表达与TD的发生发展密切相关^[30]。本研究结果表明,在诱导TD发生后的第1、2和4天,BAG-3表达量相比健康组低,而在第10、15天BAG-3表达量则较高,说明机体发生TD后会产生一种防御机制来抵抗细胞的非正常性凋亡。试验第1、2、4、10天,D、E和F组BAG-3蛋白的阳性表达是相同的;第6天E组阳性表达量高于D组,而F组与D组表达一致;第15天,E和F组表达情况均高于D组。说明低剂量和高剂量重组GSTA3后期对机体都有轻微的解毒作用。Real-time PCR试验表明,TD发生后,BAG-3基因mRNA表达水平也显著上调;注射GSTA3蛋白后,第2、4、10、15天有显著差异,说明肉鸡TD生长板软骨细胞的凋亡受BAG-3基因调控,而重组GSTA3蛋白可能通过调控BAG-3基因的表达,调节软骨细胞凋亡。

4 结论

重组GSTA3蛋白可能通过调控抗凋亡蛋白3抗凋亡因子的表达,抑制细胞凋亡。在胫骨软骨发育不良损伤修复期,注射GSTA3后使抗凋亡基因抗凋亡蛋白3蛋白表达增强,从而可能参与细胞凋亡来缓解胫骨软骨发育不良损伤,使胫骨软骨发育不良生长板的功能恢复。因此,GSTA3对预防肉鸡胫骨软骨发育不良的发生具有重要的指导意义和很好的临床应用前景。

参考文献 View Option 原文顺序
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120羽1日龄健康AA肉鸡预饲1周后随机分为2组,对照组饲以基础日粮,试验组饲以基础日粮添加100mg/kg福美双,进行了肉鸡胫骨长度、生长板厚度、肉鸡胫骨软骨发育不良(TD)指数及TD发病率等指标的检测,并进行了形态学和组织病理学观察.结果显示,患病鸡胫骨长度、生长板厚度和TD指数均有显著变化(P＜0.01),TD发病率显著上升(P＜0.05);病鸡胫骨近端的纵切面有玉白色楔状软骨团块深入干骺端甚至骨髓腔,呈现典型的胫骨软骨发育不良病理学变化.结果提示,100 mg/kg福美双可显著提高AA肉鸡TD发病率,并引起相应的组织病理学变化,为TD分子机理的研究提供了一个理想的实验动物模型.
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Tibial dyschondroplasia (TD) is a metabolic cartilage disease of young poultry in which endochondral bone formation is disrupted leading to the retention of a non-calcified, avascular plug of cartilage in the tibial growth plate. Chicks aged 7 days were fed either a control diet or one containing thiram 100 ppm for 48 h to induce TD. Cell multiplication in the growth plate was determined thereafter with bromodeoxyuridine (BrdU) labelling, and metabolic changes by measuring alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), and glutathione (GSH) activities. The effect on chondrocyte maturation was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and DNA fragmentation were used to determine the effects of thiram on cell survival. The results showed that thiram-induced TD was not due to the multiplication of cells in the post-proliferative zones. Thiram did not affect ALP activity, which would have indicated a loss of calcification potential, but it reduced both TRAP and the glutathione concentrations, suggesting that the growth plate metabolism and remodelling functions were adversely affected. Thiram appeared to have no effect on the expression of type X collagen, transglutaminase, RUNX2, or matrix metalloproteinase-2 (MMP) genes suggesting that it did not alter the maturation potential of chondrocytes. On the contrary, the expressions of MMP-13 and vascular endothelial growth factor (VEGF) genes were &amp;quot;up-regulated,&amp;quot; suggesting that thiram has pro-angiogenic activity. However, TUNEL assay showed that thiram induced endothelial cell apoptosis in the capillary vessels of the growth plates, as early as 10 days of age, when TD was not visually evident. The vascular death increased on subsequent days accompanied by massive death of chondrocytes in the transition zone of the growth plate. The induction of apoptosis in the growth plate was also demonstrated by DNA fragmentation. It was concluded that thiram induced TD not through an increase in the multiplication of chondrocytes in the transition zone and not by altering the expression of genes causing the arrest of chondrocytes in a prehypertrophic state, but by creating a metabolic dysfunction which led to the destruction of blood capillaries in the transition zone chondrocytes.

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Cell Death & Disease, 2011,2(4):e141.
DOI:10.1038/cddis.2011.24URLPMID:21472004 [本文引用: 1]
Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110-124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression.

[21] PAGLIUCA M G, LEROSE R, CIGLIANO S, LEONE A . Regulation by heavy metals and temperature of the human BAG‐3 gene, a modulator of Hsp70 activity
Febs Letters, 2003,541(1):11-15.
[本文引用: 1]

[22] LIAO Q, OZAWA F, FRIESS H, ZIMMERMANN A, TAKAYAMA S, REED J C, KLEEFF J, BUCHLER M W . The anti-apoptotic protein BAG-3 is overexpressed in pancreatic cancer and induced by heat stress in pancreatic cancer cell lines
Febs Letters, 2001,503(2):151-157.
DOI:10.1016/s0014-5793(01)02728-4URLPMID:11513873 [本文引用: 1]
Pancreatic cancer cells are usually resistant to apoptosis mediated by intrinsic or extrinsic factors. BAG-3 (Bis, CAIR), which was identified as a BAG-1-related protein, is a novel modulator of cellular anti-apoptotic activity that functions through its interaction with Bcl-2. In this study we analyzed BAG-3 expression in human pancreatic cancer tissues and cell lines. BAG-3 mRNA was expressed at moderate to high levels in all pancreatic cancer samples, but at low levels in normal pancreas tissues. In situ hybridization and immunohistochemistry analysis revealed that BAG-3 was present in the cancer cells within the pancreatic tumor mass. When BAG-3 mRNA was analyzed in other gastrointestinal cancers (hepatocellular carcinoma; esophageal, stomach and colon cancer), no difference was found from their corresponding normal controls. In pancreatic cancer cells, BAG-3 mRNA expression levels were strongly induced after heat stress, but not in response to members of the tumor necrosis factor (TNF)-alpha family (TNF-alpha, TRAIL, FasL). These findings indicate that in pancreatic cancer, in contrast to other gastrointestinal malignancies, increased levels of BAG-3 might function to block apoptosis. This characteristic of pancreatic cancer might contribute to its more aggressive growth behavior and poor responsiveness to treatment in vivo.

[23] ROSATI A, AMMIRANTE M, GENTILELLA A, BASILE A, FESTA M, PASCALE M, MARZULLO L, BELISARIO M A, TOSCO A, FRANCESCHELLI S, MOLTEDO O, PAGLIUCA G, LEROSE R, TURCO M C . Apoptosis inhibition in cancer cells: a novel molecular pathway that involves BAG3 protein
International Journal of Biochemistry & Cell Biology, 2007,39(7):1337-1342.
DOI:10.1016/j.biocel.2007.03.007URLPMID:17493862 [本文引用: 1]
Stress-induced apoptosis regulates neoplasia pathogenesis and response to therapy. Indeed, cell transformation induces a stress response, that is overcome, in neoplastic cells, by alterations in apoptosis modulators; on the other hand, antineoplastic therapies largely trigger the apoptosis stress pathway, whose impairment results in resistance. Therefore, the study of the roles of apoptosis-modulating molecules in neoplasia development and response to therapy is of key relevance for our understanding of these processes. Among molecules that regulate apoptosis, a role is emerging for BAG3, a member of the BAG co-chaperone protein family. Proteins that share the BAG domain are characterized by their interaction with a variety of partners (heat shock proteins, steroid hormone receptors, Raf-1 and others), involved in regulating a number of cellular processes, including proliferation and apoptosis. BAG3, also known as CAIR-1 or Bis, forms a complex with the heat shock protein (Hsp) 70. This assists polypeptide folding, can mediate protein delivery to proteasome and is able to modulate apoptosis by interfering with cytochrome c release, apoptosome assembly and other events in the death process. It has been recently shown that, in human primary lymphoid and myeloblastic leukemias and other neoplastic cell types, BAG3 expression sustains cell survival and underlies resistance to therapy, through downmodulation of apoptosis. This review summarizes findings that assign an apoptotic role to BAG3 in some neoplastic cell types and identify the protein as a candidate target of therapy.

[24] 何丽囡, 李文君, 黄韬, 林成源, 尹俊林, 樊保敏, 曾广智, 卞兆祥 . Bag3 P215L基因突变小鼠的繁殖与基因型鉴定
云南民族大学学报(自然科学版), 2019(5):433-438.
[本文引用: 1]

HE L N, LI W J, HUANG T, LIN C Y, YIN J L, FAN B M, ZENG G Z, BIAN Z X . Reproduction and genotype identification of Bag3 P215L
Journal of Yunnan Nationalities University (Natural Sciences Edition), 2019(5):433-438. (in Chinese)
[本文引用: 1]

[25] 廖泉, 胡亚, 赵玉沛 . 胰腺癌细胞中抗凋亡蛋白BAG-3的过度表达
中华肝胆外科杂志, 2003(8):33-37.
[本文引用: 1]

LIAO Q, HU Y, ZHAO Y P . Overexpression of anti-apoptotic protein BAG-3 in pancreatic cancer cells
Chinese Journal of Hepatobiliary Surgery, 2003(8):33-37. (in Chinese)
[本文引用: 1]

[26] 曹艳莎, 李婳, 陈明红, 刘宝琴, 王华芹, 李宁 . 组织芯片技术检测BAG3蛋白在结肠癌组织中的表达及其临床意义
吉林大学学报:医学版, 2017,43(6):1177-1181.
[本文引用: 1]

CAO Y S, LI H, CHEN M H, LIU B Q, WANG H Q, LI N . Tissue chip technology to detect BAG3 protein expression in the colon cancer tissue and its clinical significance
Journal of Jilin University: Medical Science Edition, 2017,43(6):1177-1181. (in Chinese)
[本文引用: 1]

[27] MANI J, ANTONIETTI P, RAKEL S, BLAHETA R, BARTSCH G, HAFERKAMP A, KOGEL D . Knockdown of BAG3 sensitizes bladder cancer cells to treatment with the BH3 mimetic ABT-737
World Journal of Urology, 2016,34(2):197-205.
DOI:10.1007/s00345-015-1616-2URLPMID:26100943 [本文引用: 1]
BAG3 is overexpressed in several malignancies and mediates a non-canonical, selective form of (macro)autophagy. By stabilizing pro-survival Bcl-2 proteins in complex with HSP70, BAG3 can also exert an apoptosis-antagonizing function. ABT-737 is a high affinity Bcl-2 inhibitor that fails to target Mcl-1. This failure may confer resistance in various cancers.

[28] ZHANG J, HE Z, XIAO W, NA Q, WU T, SU K, CUI X . Overexpression of BAG3 attenuates hypoxia-induced cardiomyocyte apoptosis by inducing autophagy
Cellular Physiology and Biochemistry, 2016,39(2):491-500.
DOI:10.1159/000445641URLPMID:27383426 [本文引用: 1]
Hypoxia is a well-known factor in the promotion of apoptosis, which contributes to the development of numerous cardiac diseases, such as heart failure and myocardial infarction. Inhibiting apoptosis is an important therapeutic strategy for the treatment of related heart diseases caused by ischemia/hypoxic injury. Previous studies have demonstrated that BAG3 plays an important role in cardiomyocyte apoptosis and survival. However, the role of BAG3 in hypoxia-induced cardiomyocyte apoptosis remains to be clarified. Here, we demonstrate that BAG3 is induced by hypoxia stimuli in cultured cardiomyocytes.

[29] 魏莉, 秦晓鹏, 赵兴波, 王伟 . Bcl-2结合抗凋亡基因3参与子宫颈癌上皮间质转化的机制研究
中华妇产科杂志, 2017,52(8):551-557.
DOI:10.3760/cma.j.issn.0529-567X.2017.08.009URLPMID:28851173 [本文引用: 1]
Objective: To investigate the expression of Bcl-2 associated athanogene 3 (BAG3) in cervical cancer tissues and cells and its role in epithelial mesenchymal transition (EMT) of cervical cancer. Methods: (1) Cervical cancer samples were collected from September 2015 to March 2017 in the Qilu Hospital of Shandong University and Shangdong Provincial Hospital. While, 50 normal tissues were collected from August 2015 to March 2017 in the Dezhou Municiple Hospital, which were obtained from patients with uterine myoma underwent hysterectomy and patients with cervical biopsy. Reverse transcription (RT)-PCR and western blot were used to detect the expression of BAG3 mRNA and protein, and their clinical significances were analyzed. (2) The expression of BAG3 mRNA and protein was detected using RT-PCR and western blot method in HeLa and SiHa cell lines and normal cervical epithelial cells. The experiment was divided into two groups, BAG3 small interfering RNA transfected group (si-BAG3) and the control group transfected with small interfering RNA (siRNA). Cell counting kit 8 (CCK-8) analysis was used to detect cell proliferation of two groups. Wound-healing and transwell assay were used to detect the migration and invasion ability of HeLa and SiHa cells. The xenograft model of cervical cancer in nude mice was used to observe the effect of BAG3 on tumor xenografts and the tumor-related biomarkers were tested by western blot. Results: (1) The expression levels of BAG3 mRNA and protein in cervical carcinoma tissues were 1.20±0.15 and 1.10±0.16, which were significantly higher than that in normal cervical tissue, 0.23±0.04 and 0.29±0.03 (both P&lt;0.01). The results showed that the expression levels of BAG3 mRNA and protein were significantly correlated with cervical carcinoma staging and lymph node metastasis (P&lt;0.05).However, its expression was not correlated with the patient's age, pathological grade, and diameter of tumor (all P&gt;0.05). (2) Compared with normal cervical epithelial cells, the expression of BAG3 mRNA and protein levels in HeLa and SiHa cells were significantly increased (P&lt;0.01), the expression levels of BAG3 mRNA and protein in HeLa and SiHa cells transfected with si-BAG3 were significantly lower than that in control group (all P&lt;0.01). After post-transfected 72 hours, A value of HeLa and SiHa with transfection were significantly lower than those in control group [(0.88±0.08) vs (1.22±0.13), (0.92±0.09) vs (1.35±0.12); both P&lt;0.01]. After post-transfected 24 hours, the migration level of HeLa and SiHa cells with transfection were significantly lower than those in the control group [(20.1±2.1)% vs (58.6±5.6)%, and (21.1±2.1)% vs (61.7±5.4)%; both P&lt;0.01]. The transmembrane cell number in HeLa and SiHa cells with transfection were 76±11 and 71±8, which were significantly less than those in control group (131±12 and 129±14; both P&lt;0.01). After the inoculation into nude mice, tumor formation time of HeLa and SiHa cells with transfection were (9.5±0.5) and (10.5±1.3) days, respectively, which were significantly longer than those in control group [(4.5±0.5) and (5.2±1.1) days; both P&lt;0.05]. Compared with those in the control group, the expression level of Slug, N-cadherin and matrix metalloproteinase-2 (MMP-2) protein in HeLa and SiHa cells with transfected in tumor tissues were significantly decreased (all P&lt;0.01), while the expression level of E-cadherin protein was significantly increased (P&lt;0.01). Conclusion: BAG3 could be involved in the proliferation, migration and invasion of cervical cancer cells by affecting cervical cancer EMT, and BAG3 may be an effective target for the treatment of cervical cancer.
WEI L, QIN X P, ZHAO X B, WANG W . Mechanism of Bcl-2 combined with anti-apoptotic gene 3 in the epithelial mesenchymal transformation of cervical cancer
Chinese Journal of Obstetrics and Gynecology, 2017,52(8):551-557. (in Chinese)
DOI:10.3760/cma.j.issn.0529-567X.2017.08.009URLPMID:28851173 [本文引用: 1]
Objective: To investigate the expression of Bcl-2 associated athanogene 3 (BAG3) in cervical cancer tissues and cells and its role in epithelial mesenchymal transition (EMT) of cervical cancer. Methods: (1) Cervical cancer samples were collected from September 2015 to March 2017 in the Qilu Hospital of Shandong University and Shangdong Provincial Hospital. While, 50 normal tissues were collected from August 2015 to March 2017 in the Dezhou Municiple Hospital, which were obtained from patients with uterine myoma underwent hysterectomy and patients with cervical biopsy. Reverse transcription (RT)-PCR and western blot were used to detect the expression of BAG3 mRNA and protein, and their clinical significances were analyzed. (2) The expression of BAG3 mRNA and protein was detected using RT-PCR and western blot method in HeLa and SiHa cell lines and normal cervical epithelial cells. The experiment was divided into two groups, BAG3 small interfering RNA transfected group (si-BAG3) and the control group transfected with small interfering RNA (siRNA). Cell counting kit 8 (CCK-8) analysis was used to detect cell proliferation of two groups. Wound-healing and transwell assay were used to detect the migration and invasion ability of HeLa and SiHa cells. The xenograft model of cervical cancer in nude mice was used to observe the effect of BAG3 on tumor xenografts and the tumor-related biomarkers were tested by western blot. Results: (1) The expression levels of BAG3 mRNA and protein in cervical carcinoma tissues were 1.20±0.15 and 1.10±0.16, which were significantly higher than that in normal cervical tissue, 0.23±0.04 and 0.29±0.03 (both P&lt;0.01). The results showed that the expression levels of BAG3 mRNA and protein were significantly correlated with cervical carcinoma staging and lymph node metastasis (P&lt;0.05).However, its expression was not correlated with the patient's age, pathological grade, and diameter of tumor (all P&gt;0.05). (2) Compared with normal cervical epithelial cells, the expression of BAG3 mRNA and protein levels in HeLa and SiHa cells were significantly increased (P&lt;0.01), the expression levels of BAG3 mRNA and protein in HeLa and SiHa cells transfected with si-BAG3 were significantly lower than that in control group (all P&lt;0.01). After post-transfected 72 hours, A value of HeLa and SiHa with transfection were significantly lower than those in control group [(0.88±0.08) vs (1.22±0.13), (0.92±0.09) vs (1.35±0.12); both P&lt;0.01]. After post-transfected 24 hours, the migration level of HeLa and SiHa cells with transfection were significantly lower than those in the control group [(20.1±2.1)% vs (58.6±5.6)%, and (21.1±2.1)% vs (61.7±5.4)%; both P&lt;0.01]. The transmembrane cell number in HeLa and SiHa cells with transfection were 76±11 and 71±8, which were significantly less than those in control group (131±12 and 129±14; both P&lt;0.01). After the inoculation into nude mice, tumor formation time of HeLa and SiHa cells with transfection were (9.5±0.5) and (10.5±1.3) days, respectively, which were significantly longer than those in control group [(4.5±0.5) and (5.2±1.1) days; both P&lt;0.05]. Compared with those in the control group, the expression level of Slug, N-cadherin and matrix metalloproteinase-2 (MMP-2) protein in HeLa and SiHa cells with transfected in tumor tissues were significantly decreased (all P&lt;0.01), while the expression level of E-cadherin protein was significantly increased (P&lt;0.01). Conclusion: BAG3 could be involved in the proliferation, migration and invasion of cervical cancer cells by affecting cervical cancer EMT, and BAG3 may be an effective target for the treatment of cervical cancer.

[30] WANG C X, NIU S, JAHEJO A R, JIA F J, LI Z, ZHANG N, NING G B, ZHANG D, LI H Q, MA H L, HAO W F, GAO W W, GAO S M, LI J H, LI G L, YAN F, GAO R K, ZHAO Y J, CHEN H C, TIAN W X . Identification of apoptosis-related genes in erythrocytes of broiler chickens and their response to thiram-induced tibial dyschondroplasia and recombinant glutathione-S-transferase A3 protein
Research in Veterinary Science, 2018,120:11-16.
DOI:10.1016/j.rvsc.2018.08.001URLPMID:30165245 [本文引用: 1]
Thiram, a carbamate pesticide, is known to induce tibial dyschondroplasia (TD) in broiler chickens. This study used a thiram-induced TD model to explore whether apoptosis-related genes were expressed in erythrocytes of broiler chickens and the impacts of thiram-induced TD and the recombinant GSTA3 protein in regulating these genes expression. In this study, mRNA and protein expression of six types of apoptosis-related genes (Bcl-2, Bax, Murine double minute MDM2, Bcl-2-associated athanogene BAG-1, BAG-3, STAT3) were identified in erythrocytes of broiler chickens by real-time PCR and immunohistochemistry, and we also found that thiram-induced TD induced the decreased expression of these antiapoptotic genes in the initial stage of TD and promoted their expression in TD recovery, which suggested that the expression of these apoptosis-related genes in erythrocytes is highly related to the development of TD. Further, the recombinant GSTA3 protein promoted the expression of all apoptosis-related genes in the initial stage of TD and recovered the normal expression of these genes in the recovery stage of TD, which indicated that the recombinant GSTA3 protein may participate in the recovery of TD. Further studies are needed to elucidate the mechanism of the response of erythrocytes to thiram-induced TD and the recombinant protein GSTA3 in broiler chickens.