流体剪切力抑制肿瘤坏死因子α诱导鼠成骨样细胞MC3T3-E1凋亡的实验研究

Fluid Shear Stress Inhibits Apoptosis Induced by TNF-α in Murine Osteoblastic MC3T3-E1 Cell

目的研究流体剪切力(fluid shear stress,FSS)对肿瘤坏死因子α(TNF-α)诱导鼠成骨样细胞MC3T3-E1凋亡的影响。方法将MC3T3-E1分为TNF-α干预组(实验组)和无TNF-α干预组(对照组),TNF-α(10ng/ml)×4h诱导MC3T3-E1凋亡后,四个实验组分别加载12dyn/cm2FSS作用0,15,30,60min。应用四甲基偶氮唑蓝(MTT)法、荧光显微镜和流式细胞技术(FΑCS)检测细胞的增殖能力和凋亡,免疫印迹法检测半胱氨酸蛋白激酶9(caspase9)和凋亡蛋白酶活化因子1(Αpaf-1)蛋白的表达。采用SPSS16.0软件包对数据进行单因素方差分析。结果TNF-α(10ng/ml)×4h能诱导明显的凋亡信号,FSS(12dyn/cm2)能明显抑制这种凋亡,而且随着刺激时间的增加,从0min逐渐延长至60min,细胞活性逐渐增加,凋亡细胞数逐渐减少,实验组与对照组单因素方差分析及各组间LSD两两比较有显著性差异(P<0.05),同时caspase9和Αpaf-1蛋白的表达也逐渐增加。结论生理范围的FSS能够抑制TNF-α诱导的MC3T3-E1细胞的凋亡,作为线粒体通路的关键蛋白,caspase9和Αpaf-1在凋亡时增加而FSS后表达减少,说明FSS抑制这种凋亡至少部分是减弱了凋亡的线粒体通路。

Objective To observe the effect of flow shear stress（FSS）on TNF-α-induced apoptosis in murine osteoblastic MC3T3-E1 cells.Methods MC3T3-E1 cells were divided into two groups：TNF-α intervention group（experimental group）and non-TNF-α intervention group（control group）.In the experimental group,cell apoptosis was induced by culturing with TNF-α（10 ngml）for 4 hours;and then,the MC3T3-E1 cells were stimulated by FSS for 0,15,30,or 60 minutes.Afterwards,MTT assay,fluorescence microscopy and flow cytometry were applied to measure the cellular proliferation and apoptosis,and Western blotting was used to detect the expressions of caspase 9 and Αpaf-1 protein.Statistical analysis was performed using SPSS16.0 for one way ANOVA.Results MC3T3-E1 cells showed significant signs of apoptosis within 4 hours of exposure to TNF-α（10 ngml）,while FSS（12 dyn/cm^2）attenuated this TNF-α-induced apoptosis significantly in a time-dependent manner;the percentage of survival osteoblasts increased with the time of FSS stimulation（P〈0.05）,meanwhile the expressions of caspase 9 and Αpaf-1 protein were raised as well.Conclusions FSS set at a physiological range can inhibit the TNF-α-induced apoptosis of MC3T3-E1 cells.The expressions of caspase 9 and Αpaf-1 protein,which are key proteins in mitochondrial pathway,increase at apoptosis,but decrease by FSS,indicating that FSS can attenuate,partly at least,the TNF-α-induced mitochondrial pathway of apoptosis.