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

doi:10.3864/j.issn.0578-1752.2020.09.018

摘要/Abstract

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.

Key words: glutathione S-transferase A3 (GSTA3), tibial dyschondroplasia, BAG-3, antiapoptotic, broiler

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.

Zhen LI,ShiXiong YANG,Sheng NIU,Ning ZHANG,Xin LI,YangYang ZHANG,YunFei JIA,ZhiXiong TIAN,GuanBao NING,Ding ZHANG,WenXia TIAN. Effect of Recombinant GSTA3 Protein on Expression of the Anti-Apoptotic Gene BAG-3 in Thiram-Induced Tibial Chondrodysplasia[J]. Scientia Agricultura Sinica, 2020, 53(9): 1921-1930.

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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^-1recombinant 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

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