大鼠椎间盘软骨终板细胞凋亡体外模型的建立  

Establishment of the model of rat disc chondrocytes apoptosis in vitro

作　　者：李德芳[1] 吴靖平[1] 朱斌[1] 丁磊[1] 卢伟[1] 

机构地区：[1]复旦大学附属金山医院骨科,上海200540

出　　处：《中国矫形外科杂志》2013年第2期173-177,共5页Orthopedic Journal of China

基　　金：上海市金山区科委基金项目(编号:2009-3-14区科委)

摘　　要：[目的]建立大鼠椎间盘软骨终板细胞凋亡体外模型。[方法]为了模拟椎间盘内部低营养低代谢环境,采用低胎牛血清培养法培养椎间盘软骨终板细胞分别含0%,1%,3%,5%,8%,10%胎牛血清,设置不同浓度梯度筛选最佳浓度,检测凋亡率、凋亡蛋白表达及caspase酶活性。[结果]低胎牛血清培养组细胞发生形态改变,DAPI染色阳性细胞增多;流式细胞仪检测凋亡率随着FBS浓度降低而升高,1%为最有效诱导凋亡浓度;Western blot示FAS、caspase-3、PARP、细胞色素C表达在1%FBS组明显高于10%时,同时caspase-3/8/9酶活性增高。[结论]低胎牛血清培养法能诱导体外培养的软骨终板细胞发生凋亡,最终会引起细胞功能丧失和椎间盘退变,caspase家族可能参与了这一过程。[ Objective] To develop an apoptosis model of cartilage endplate using a cellular culture. [ Method ] To mimic the normal internal disc milieu（ decreased nutrients and low metabolism）, chondrocytes derived from SD rat endplate cartilage were cultured under serum limiting conditions, to select the optimum concentration ceils were treated by culture medium with 0%, 1% ,3% ,5% ,8% , 10% fetal bovine serum（FBS） separately. Apoptosis was assessed by flow cytometry and by DAPI stai- ning. The expression of apoptosis-related pro^ein was assessed by Western blot. The enzyme activity changes of caspase in the process of low serum-induced apoptosis were ~tetected by caspase colorimetric assay kit. [ Result] Being treated with low FBS re- suiting in changes in cellular morphology, DAPI-positive cells increased with the decrese of FBS, apoptosis quantified by flow cy- tometry also increased significantly （P 〈 O. 05 ） and shown that 1% FBS was the optimum concentration, there was strong increas- ing in the expression of apoptosis-related proteins comprise FAS, caspase-3, PARP, cytochrome C （ cyt C） in 1% FBS group. Activity of caspase-8 ,9 ,3 was elevated compared with those in the 10% fetal bovine serum. [ Conclusion] Low serum condition would induce the apoptosis of cartilage endplate chondrocytes,later may ultimately compromise cell function and induce interver- tebral degeneration. The activation of caspase family would involve in the process.

关 键 词：软骨终板细胞 凋亡 营养夺获 CASPASES 

分 类 号：R681.53[医药卫生—骨科学]