大鼠骨髓间充质干细胞体外诱导失巢凋亡过程中半胱氨酸蛋白酶-3的作用

Significance of casepase-3 in induced anoikis of mesenchymal stem cells(MSCs)in vitro

[目的]探讨在体外诱导骨髓间充质干细胞失巢凋亡过程中半胱氨酸蛋白酶-3(Caspase-3)的作用。[方法]将体外培养的骨髓间充质干细胞随机分为3组:对照组、诱导凋亡组、抑制凋亡组。应用Caspase-3活性检测试剂盒,荧光比色法和W estern b lotting法检测失巢凋亡中Caspase-3活性的改变;借助流式细胞仪分析骨髓间充质干细胞凋亡的变化。[结果]流式细胞术显示诱导凋亡组出现了明显的凋亡峰,凋亡率随诱导时间的延长而明显增加;其它两组的凋亡率较低且较稳定。W estern b lotting检测和Caspase-3活性荧光检测显示,诱导凋亡组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,以后者更灵敏,其余两组活性无明显变化。[结论]Caspase-3蛋白在诱导骨髓间充质干细胞失巢凋亡的过程中发挥着重要作用;抑制Caspase-3的活性可以有效降低细胞失巢凋亡率。

[ Objective] To investigate the significance of Caspase-3 in anoikis induced by preventing mesenchymal stem cells （MSCs） from adherence in vitro. [ Method ] 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 blotting. [ Result ] The flow cytometry 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 blotting and fluorometric assay showed the caspase-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 blotting. [ Conclusion ] Caspase-3 plays a vital role in suspended culture induced anoikis of MSCs in vitro. Decreasing the activity of caspase-3 by Caspase-3 Inhibitor can reduce the anoikis rate.