Caspase-3在间质干细胞分化过程中的基因芯片分析及体外作用  

Assessment of the expression profile of Casepase-3 during the chondrogenesis of mice by microarray and significance in induced anoikis of mesenchymal stem cells in vitro

作　　者：唐欣[1] 冯建军 陈雨辰[3] 李奇[3] 许伟华[1] 杨操[1] 李进[1] 杨述华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,武汉430022 [2]新疆自治区人民医院骨科 [3]生物芯片上海国家工程研究中心

出　　处：《中华实验外科杂志》2008年第12期1640-1642,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30471753）

摘　　要：目的观察Caspase-3在小鼠胚胎肢芽间质干细胞分化过程中的作用，并将其运用于体外间质干细胞模型中进行验证。方法用基因芯片技术检测Caspase-3在小鼠胚胎肢芽间质干细胞分化过程中的基因表达，分析其表达规律和可能作用；应用Caspase-3活性检测试剂盒，荧光比色法和Western blot法检测体外培养的骨髓间质干细胞诱导失巢凋亡中Caspase-3活性的改变；借助流式细胞仪分析骨髓间质干细胞凋亡的变化。结果Caspase-3在小鼠胚胎肢芽间质干细胞向软骨分化的关键期（E12）出现显著的表达下调；Caspase-3的活性以及蛋白表达水平随诱导凋亡时间的延长而明显升高，且与细胞凋亡率相关。结论Caspase-3蛋白在体内和体外诱导间质干细胞凋亡的过程中均发挥着重要作用；胚胎发育过程中，通过下调Caspase-3的表达以促进软骨形成，而抑制Caspase-3的活性可以有效降低细胞凋亡率。Objective To examine the gene expression profile of Casepase-3 during entoehondrostosis of mice and explore the expression rules and effects of it ;investigate the significance of Caspase-3 in anoikis induced by preventing mesenchymal stem cells （MSCs） from adherence in vitr. Methods eDNA microarray technique with 34,000 genes was used to analyze the gene expression profiles during entochondrostosis in the limbs of mice embryo from E10 to E14. Pathway analysis of Casepase-3 was performed with GCOS1.2 software. The cultured MSCs were randomly divided into 3 groups ： control group, induced group and Caspase-3 inhibitor group. MSCs anoikis was detected by flow cytometry. The Caspase-3 activity was evaluated by Caspase-3 Fluorometric Assay Kit and Western blot. Results There were obvious down regulation of Caspase-3 during the critical period of entoehondrostosis （E12） in microarray. The flow eytometry analysis showd that apoptosis peak was appeared significantly in the induced groupand increased dramaticly along with the apoptosis conditions going on. The apoptosis rate of the others were lower and stable. Western blot and fluorometric assay showed the easpas e-3 expressive level or activity in induced group Was the highest and Was related with its apoptosis rate. The fluorometric assay technique Was sensitive to the western blot. Conclusion This finding revealed Caspase-3 plays a vital role in apoptosis of MSCs in vivo and in vitro. Down regulation of Caspase-3 during the critical period of entoehondrostosis （E12） promote chondrogenesis in mice embryonal limb bud. Decreasing the activity of Caspase-3 by Caspase-3 Inhibitor can reduce the anoikis rat in vitro.

关 键 词：基因芯片 半胱天冬酶3 间质干细胞 脱噬作用 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]