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  

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

作　　者：李桢[1] 杨世雄 牛胜 张宁[1] 李欣[1] 张阳阳 贾云飞 田志雄 宁官保[1] 张鼎[1] 田文霞[1] 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)

机构地区：[1]山西农业大学动物科技学院,山西太谷030801

出　　处：《中国农业科学》2020年第9期1921-1930,共10页Scientia Agricultura Sinica

基　　金：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)。

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(100μg·kg^-1)磷酸盐缓冲液;B组与E组注射低剂量(100μg·kg^-1)GSTA3;C组与F组注射高剂量(200μg·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。【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(100μg·kg^-1);group B and group E were injected with low dose(100μg·kg^-1)GSTA3;group C and group F were injected with high dose(200μg·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

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 

分 类 号：S858.31[农业科学—临床兽医学]