自噬在肿瘤坏死因子-α诱导成骨细胞凋亡中的作用

The role of autophagy in tumor necrosis factor-α induced apoptosis in osteoblasts

目的探讨自噬在肿瘤坏死因子-α(TNF-α)诱导成骨细胞凋亡中的作用。方法将培养液按处理方式的不同分为对照组(不做处理)、TNF-α观察组(加入40μg/L TNF-α)和雷帕霉素(RAP)观察组(加入100nmol/L RAP作用1 h后再加入40μg/L TNF-α)。采用四甲基偶氮噻唑蓝法检测MC3T3-E1成骨细胞增殖活力,流式细胞仪检测MC3T3-E1成骨细胞的凋亡,免疫印迹法检测蛋白表达。结果 (1) MC3T3-E1成骨细胞增殖抑制率、细胞凋亡率:3组比较差异均有统计学意义(P <0.001);RAP观察组、TNF-α观察组均明显高于对照组(P <0.01);TNF-α观察组明显高于RAP观察组(P <0.01)。(2) MC3T3-E1成骨细胞自噬蛋白LC3-Ⅱ/LC3-Ⅰ比值:RAP观察组显著高于对照组和TNF-α观察组(P <0.01);TNF-α观察组显著低于对照组(P <0.01)。结论自噬在TNF-α诱导MC3T3-E1成骨细胞凋亡中发挥一定作用,增强自噬可以显著降低TNF-α对成骨细胞的损伤。

Objective To investigate the role of autophagy in tumor necrosis factor-α( TNF-α) induced apoptosis in osteoblasts. Methods The MC3 T3-E1 osteoblasts culture fluid were divided into control group( non-management),TNF-α observation group( adding 40 μg/L TNF-α) and rapamycin( RAP) observation group( adding 100 nmol/L RAP for 1 h,and then adding 40 μg/L TNF-α),according to different treatment methods. The methyl thiazolyl tetrazolium( MTT) assay was used to detect MC3 T3-E1 cell proliferation,the apoptosis of cells was detected by floecytometry,the protein expression was detected by Western Blot. Results(1) The proliferation inhibition rate and apoptotic rate of MC3 T3-E1 osteoblasts: there were significant differences among three groups( P < 0. 001);RAP observation group,TNF-α observation group were significantly higher than those in the control group( P < 0. 01);and TNF-α observation group was significantly higher than RAP observation group( P < 0. 01) .(2) The ratio of LC3-Ⅱ/LC3-Ⅰ of autophagy protein in MC3 T3-E1 osteoblasts: RAP observation group was significantly higher than the control group and TNF-α observation group( P < 0. 01);TNF-α observation group was significantly lower the control group( P < 0. 01).Conclusions Autophagy plays a role in TNF-α induced apoptosis of MC3 T3-E1 osteoblasts,and enhancing autophagy can significantly reduce TNF-α induced damage to osteoblasts.