热休克蛋白90抑制剂对间充质干细胞增殖、凋亡及成骨分化的影响  

Effects of heat shock protein 90 inhibitor on proliferation;apoptosis and osteogenic differentiation of mesenchymal stem cells

作　　者：余月明 周雷 张铁骑 王明海 Yu Yueming;Zhou Lei;Zhang Tieqi;Wang Minghai(Department of Orthopedics,the Fifth People’s Hospital of Fudan University,Shanghai 200240,Chin)

机构地区：[1]复旦大学附属上海市第五人民医院骨科,上海200240

出　　处：《中华实验外科杂志》2018年第8期1497-1499,共3页Chinese Journal of Experimental Surgery

摘　　要：目的观察热休克蛋白90抑制剂17-丙烯胺基-17-去甲氧基格尔德霉素（17-AAG）对间充质干细胞C3H10T1/2增殖、凋亡及成骨分化的影响。方法用0、0.001、0.010、0.100和1.000 μmol/L浓度17-AAG处理C3H10T1/2，细胞计数试剂盒（CCK-8）检测C3H10T1/2增殖；流式细胞术检测0、0.1和1.0 μmol/L浓度17-AAG作用下细胞的凋亡率；0、0.1、0.5、1.0 μmol/L 17-AAG处理后，Western blot检测凋亡相关蛋白的变化，半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3试剂盒检测Caspase-3酶活性的变化；0、0.01 μmol/L17-AAG处理后，碱性磷酸酶（ALP）染色和ALP活性测定检测两组成骨分化的强弱。结果0、0.001、0.010、0.100和1.000 μmol/L 17-AAG处理细胞后，随着浓度增加，C3H10T1/2的生长增殖明显受到抑制，呈时间浓度依耐性。流式细胞术检测，0、0.1、1.0 μmol/L 3组凋亡率分别为（1.36±0.43）%、（5.37±1.10）%、（13.30±1.94）%，0.1、1.0 μmol/L组较对照组差异均有统计学意义（P=0.014、0.011），0.1、1.0 μmol/L两组之间差异也有统计学意义（P=0.048）。Western blot结果显示随着17-AAG浓度的升高，抗凋亡蛋白B淋巴细胞/白血病-2（bcl-2）减少，促凋亡蛋白bcl-2相关X蛋白（bax）、BH3结构域凋亡诱导蛋白（bid）表达增加。Caspase-3活性测定17-AAG浓度0.1、0.5、1.0 μmol/L组与对照组比值分别为1.84±0.11、2.19±0.13、2.79±0.24，与对照组比较差异均有统计学意义（P=0.006，P=0.004，P=0.006）。成骨诱导14 d，0.01 μmol/L组ALP染色比未处理组颜色加深，0.01 μmol/L组ALP活力与对照组ALP活力比值为1.646±0.139，差异有统计学意义（P=0.015）。结论高浓度17-AAG抑制间充质干细胞C3H10T1/2的增殖，促进其凋亡，低浓度17-AAG能促进C3H10T1/2的成骨分化。ObjectiveTo observe Effects of Heat Shock Protein 90 Inhibitor 17-Allylamino-17-demethoxygeldanamycin （17-AAG） on Proliferation, Apoptosis and Osteogenic Differentiation of Mesenchymal Stem Cells C3H10T1/2.MethodsC3H10T1/2 cells were treated with 17-AAG （ 0, 0.001, 0.010, 0.100 and 1.000 μmol/L concentration）, the proliferation of cells were analyzed by cell counting kit-8 （CCK-8） assay. The apoptosis of cells treated with the concentrations of 17-AAG （0, 0.1 and 1.0 μmol/L） were detected by flow cytometry. Cells were treated with the concentrations of 17-AAG （0, 0.1, 0.5, 1.0 μmol/L）, the expressions of several apoptosis-related proteins were detected by western blotting. Caspase-3 Activity Assay Kit detected the activity of Caspase-3. Alkaline phosphatase staining and alkaline phosphatase activity assay detected the capability of osteogenic differentiation.ResultsC3H10T1/2 cells were treated with 17-AAG （ 0, 0.001, 0.010, 0.100 and 1.000 μmol/L concentration）, with the concentrations increased, the growth and proliferation of cells significantly inhibited and had a dose and time depent manner. The apoptotic rates of 0, 0.1, 1.0 μmol/L groups were （1.36±0.43）%, （5.37±1.10）% and （13.30±1.94）%, and the difference was statistically significant （P=0.014, P=0.011）. Differences between the 0.1 group and 1 μmol/L group were also statistically significant （P=0.048）. Western blotting showed that the expression of pre-apoptotic protein protein B cell lymphoma/lewkmia-2 （bcl-2）-Associated X （bax）and BH3 interacting domain death agonist （bid） were increased and the expression of anti-apoptotic bcl-2 was decreased with the increase of 17-AAG concentration. About the activity of Caspase-3, the ratio of 17-AAG concentration of 0.1, 0.5 and 1.0 μmol/L to the untreated control group were 1.84±0.11, 2.19±0.13 and 2.79±0.24, respectively. There were significant differences between the three groups and control group （P=0.006, 0.004, 0.006）. Osteogenic inductio

关 键 词：热休克蛋白90抑制剂 凋亡 成骨分化 间充质干细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]