福美双诱导的肉鸡胫骨软骨发育不良软骨细胞凋亡

摘要

为研究肉鸡胫骨软骨发育不良(TD)软骨细胞的凋亡分布及凋亡相关基因的表达水平,将80羽1日龄健康AA肉鸡基础日粮饲养1周后,随机分为对照组和处理组,处理组饲喂添加100 mg·kg1福美双的日粮,采用HE染色和TUNEL法分别对肉鸡胫骨生长板软骨细胞的形态学和凋亡情况进行观察,同时采用Real-time PCR方法和免疫组化技术分别对软骨细胞中凋亡相关基因的mRNA转录水平及蛋白质表达水平进行检测.结果显示,福美双诱发TD后第1、2、4和6天,肉鸡生长板软骨栓形成逐渐增大,异常软骨细胞增加,TD病变区软骨细胞,尤其是肥大区软骨细胞凋亡程度明显增加.Real-time PCR检测结果显示,与对照组比较,TD生长板促凋亡基因Bax在试验第1、2、4和6天表达量极显著上调(P＜0.01);Caspase-3在第1、2和4天表达量极显著增加(P＜0.01);抗凋亡基因BAG-1表达下调,在试验第1和2天差异极显著(P＜0.01).免疫组化结果显示,第1和4天处理组Bax的蛋白表达量明显高于对照组.本研究提示肉鸡TD生长板软骨细胞凋亡与促凋亡基因的表达增强和抗凋亡基因的表达下降有关.%In order to investigate the region of apoptosis and the expression of genes related to apoptosis in cartilage cells of Tibial Dyschondroplasia (TD) in broiler chickens,TD model was established,in which eighty AA broiler chickens were divided into two groups;control group and experiment group (given thiram 100 mg · kg-1 in feed).The morphology of chondrocytes and chondrocytes apoptosis in tibial growth plate of broilers were determined by HE (hematoxylin and eosin) staining and TUNEL technology.Real-time PCR and immunohistochemistry respectively were used to detect the mRNA transcription level of apoptosis-related genes in chondrocytes and protein expression levels.The HE staining result revealed that obvious cartilage plug formed and gradually bigger in the tibial growth plate after day 1,2,4 and 6 in TD broilers induced by thiram;and the abnormity of chondrocyte also increased.The TUNEL technology result showed that apoptosis levels of the chondrocytes significantly increased in cartilage lesion parts,especially in the hypertrophic zone.The Real-time PCR results showed that the mRNA transcription level of Bax was up-regulated from day 1 to 6 significantly (P＜ 0.01).The Caspase-3 gene transcription significantly increased on day 1,2 and 4 in TD broiler tibial growth plate (P＜0.01).The transcription of anti-apoptosis gene BAG-1 was significantly decreased on day 1 and 2 in TD broiler tibial growth plate (P＜0.01).The results of immunohistochemistry showed that the expression of Bax protein was significantly higher in the treated group than in the control group on day 1 and 4.It is concluded that the apoptosis of chondrocytes in TD broilers growth plate was related to the enhancement of pro-apoptotic gene expression and decreased of anti-apoptotic gene expression.