炎症环境下线粒体自噬与大鼠骨髓间充质干细胞成骨分化相关性研究

The correlation between mitophagy and osteogenic differentiation of rat bone marrow mesenchymal stem cells in inflammatory environment

目的:探索高浓度肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)诱导的炎症环境下线粒体自噬与大鼠骨髓间充质干细胞(Rat Bone marrow mesenchymal stem cells,rBMSCs)成骨分化能力的相关性。方法:利用100ng/ml TNF-α构建体外炎症微环境。探讨在炎症微环境下,线粒体自噬与成骨分化的相关性,将实验分为T组(TNF-α100ng/ml组)、T+促进剂组(TNF-α100ng/ml+雷帕霉素(Rapamycin)组)和T+抑制剂组(TNF-α100ng/ml+3-甲基腺嘌呤(3-MA)组)。使用免疫荧光观察线粒体自噬变化,通过CCK-8,碱性磷酸酶(alkaline phosphatase,ALP)活性测定、茜素红染色法检测线粒体自噬的变化对细胞增殖与成骨分化的影响,利用RT-PCR、Western Blot分别检测成骨与线粒体自噬相关基因和蛋白的表达情况,以期探索炎症环境下线粒体自噬对成骨分化能力的影响。结果:TNF-α100ng/ml成功构建体外炎症微环境。Mito-tracker与LC3共定位PCC结果显示:T+促进剂组优于T+抑制剂组(P<0.05)。CCK-8、ALP活性测定与茜素红染色结果显示:与T组相比,T+促进剂组rBMSCs增殖能力、ALP活性、钙结节矿化程度增强,而T+抑制剂组rBMSCs增殖能力、ALP活性、钙结节矿化程度减弱(P<0.05)。RT-PCR和Western Blot结果显示,与T组相比,T+抑制剂组成骨相关蛋白与基因ALP和OPN表达量显著下降,线粒体自噬相关蛋白LC3II/LC3I、PINK1、Parkin显著下降,P62/SQSTM1显著升高(P<0.05)。T+促进剂组与T组相比,成骨相关蛋白与基因ALP和OPN表达显著上升,线粒体自噬相关蛋白LC3II/LC3I、PINK1、Parkin表达显著上升,P62/SQSTM1表达显著下降(P<0.05)。结论:在TNF-α100ng/ml构建的体外炎症微环境下,随着rBMSCs线粒体自噬能力下降,成骨分化能力受到抑制。

Objective:To investigate the correlation between mitophagy and osteogenic differentiation of rat bone marrow mesenchymal stem cells(rBMSCs)in an inflammatory environment induced by high concentrations of tumor necrosis factor-α(TNF-α).Methods:An in vitro inflammatory microenvironment was constructed using 100 ng/ml TNF-α.To investigate the correlation between mitophagy and osteogenic differentiation in the inflammatory microenvironment,the experiment was divided into T group(TNF-α100ng/ml group),T+promoter group(TNF-α100ng/ml+Rapamycin group)and T+inhibitor group(TNF-α100ng/ml+3-MA group).The mitophagy promoter Rapamycin and the inhibitor 3-methyladenine(3-MA)were used to observe the changes in mitophagy by immunofluorescence co-localization,and the changes in mitophagy were detected by CCK-8,alkaline phosphatase(ALP)activity assay and alizarin red staining on cell proliferation.The expression of genes and proteins related to osteogenesis and mitophagy were examined by RT-PCR and Western Blot respectively,in order to explore the effect of mitophagy on osteogenic differentiation under inflammatory environment.Results:TNF-α100ng/ml was successfully constructed in an in vitro inflammatory microenvironment.Mito-tracker and LC3 colocalization results showed that the T+promoter group outperformed the T+inhibitor group(P<0.05).The results of CCK-8 and ALP activity assay with alizarin red staining showed that the proliferative capacity,ALP activity and calcium nodule mineralization of rBMSCs were enhanced in the T+promoter group compared with the T group,whereas the proliferative capacity,ALP activity and calcium nodule mineralization of rBMSCs were diminished in the T+inhibitor group(P<0.05).RT-PCR and Western Blot results showed that Compared with the T group,the T+inhibitor group showed a significant decrease in the expression of bone-related proteins and genes ALP and OPN,a significant decrease in mitophagy-related proteins LC3II/LC3I,PINK1 and Parkin,and a significant increase in P62/SQSTM1(P<0.05).the T+promoter group showed a significant increase in the expression of bone-related proteins and genes ALP and OPN compared with the T group,mitophagy-related proteins LC3II/LC3I,PINK1,Parkin expression was significantly increased and P62/SQSTM1 expression was significantly decreased(P<0.05).Conclusion:In the in vitro inflammatory microenvironment constructed with TNF-α100ng/ml,the osteogenic differentiation ability was inhibited as the mitophagy capacity of rBMSCs decreased.