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_临床医学论文.docVIP

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_临床医学论文 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_临床医学论文 作者：冯建军，杨述华，孙立，唐欣 【关键词】 骨髓间充质干细胞；,,Caspase 　　摘 要：［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（Caspase3）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Caspase3活性检测试剂盒，荧光比色法和Western blotting法检测失巢凋亡中Caspase3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。Western blotting检测和Caspase3活性荧光检测显示，诱导凋亡组Caspase3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］Caspase3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Caspase3的活性可以有效降低细胞失巢凋亡率。 关键词：骨髓间充质干细胞； Caspase3； 失巢凋亡 Abstract：［Objective］To investigate the significance of Caspase3 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 Caspase3 inhibitor groupMSCs anoikis was detected by flow cytometryThe caspase3 activity was evaluated by Caspase3 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 onThe apoptosis rate of the others were lower and stableWestern blotting and fluorometric assay showed the caspase3 expressive level or activity in induced group was the highest and was related with its apoptosis rateThe fluorometric assay technique was sensitive to the western blotting［Conclusion］Caspase3 plays a vital role in suspended culture induced anoikis of MSCs in vitroDecreasing the activity of caspase3 by Caspase3 Inhibitor can reduce the anoikis rate Key words：Mesenchymal stem cells; Caspase3; Anoikis 骨髓间充质干细胞作为组织工程最有希望的种子细胞之一，越来越受到重视。体外培养的骨髓间充质干细胞呈贴壁生长特性，如果将它改变为悬浮状态，由胞外输入的存活信号通路就会中断，从而诱发失巢凋亡〔1、2〕。Caspase3（半胱氨酸蛋白酶3）是涉及细胞凋亡的重要蛋白酶，它的激活意味着细胞不可避免的发生凋亡〔3、4〕。本实验选用琼脂糖预先铺被培养皿的方法来阻止骨髓间充质干细胞贴壁〔5〕，诱导失巢凋亡，并应用Caspase3抑制剂（DEVDCHO）以探讨Caspase3骨髓间充质干细胞失巢凋亡中的作用。 1 材料与方法 11 材 料 低糖DMEM培养基、特优级胎牛血清（fetal bovine serum，FBS）购自美国GIBCO公司；Caspase3抑制剂DEVDCHO：美