MSCs旁分泌对多柔比星诱导的H9C2细胞凋亡的影响

Effect of hepatocyte growth factor on doxorubicin-induced H9C2 cells apoptosis

目的:探讨骨髓间充质干细胞(mesenchymal stem cells,MSCs)旁分泌因子肝细胞生长因子(hepatocyte growth factor,HGF)对多柔比星诱导的H9C2细胞凋亡的影响及其机制。方法:将SD大鼠MSCs传代至第3代后分为未转染组、转染HGF-siRNA组及转染阴性对照(NC)-siRNA组,分别进行相应处理。通过ELISA、蛋白质印迹法检测各组MSCs细胞上清中及细胞中HGF、转化生长因子β1(transforming growth factorβ1,TGF-β1)含量和蛋白表达量。用浓度为1μmol/L的多柔比星处理H9C2细胞4 h后分为4组:单独培养组、MSCs共培养组、与HGF-siRNA-MSCs共培养组及与NC-siRNA-MSCs共培养组。培养24 h后通过流式细胞术AnnexinⅤ/PI双染法检测H9C2细胞凋亡率。结果:HGF-siRNA组MSCs上清中TGF-β1浓度为(519.23±24.34)pg/mL,明显高于未转染组[(459.65±11.78)pg/mL]及NC-siRNA组[(459.33±11.78)pg/mL](P均<0.05);转染HGF-siRNA组MSCs中TGF-β1表达量亦明显高于其他两组。HGF-siRNA-MSCs与H9C2细胞共培养组中H9C2细胞凋亡率为(18.54±0.64)%,较MSCs共培养组[(6.65±0.49)%]及NC-siRNA-MSCs共培养组[(9.70±1.62)%]明显增加(P均<0.05)。结论:MSCs旁分泌因子HGF通过抑制TGF-β1的表达对多柔比星诱导的H9C2细胞凋亡起保护作用。

Objective:To investigate the effect of hepatocyte growth factor (HGF) on doxorubicin (DOX)-induced H9C2 cells apoptosis.Methods:The third generation of mesenchymal stem cells(MSCs)were divided into three groups:cultured alone,HGF-siRNA-MSCs and NC-siRNA-MSCs.The concentration of HGF and transforming growth factor β1 (TGF-β1) were measured with both ELISA and Western-blot kit.H9C2 cells were exposed to 1.0 μmol/L doxorubicin for 4 hours.Then they were divided into four groups:cultured alone,co-cultured with MSCs,co-cultured with HGF-siRNA-MSCs and co-cultured with NC-siRNA-MSCs.After 24 hours,the apoptosis rate was determined by flow cytometry (FCM).Results:Compared with other groups,the concentration of TGF-β1 in MSCs were significantly increased in the HGF-siRNA-MSCs [(519.23 ± 24.34) pg/mL] than cultured alone [(459.65 ± 11.78) pg/mL] and NC-siRNA-MSCs [(459.33 ± 11.78) pg/mL,P ＜ 0.05].The apoptosis rate of H9C2 cells was increased significantly in cocultured with HGF-siRNA-MSCs(18.54 ± 0.64) ％ than co-cultured with MSCs(6.65 ± 0.49) ％ and NCsiRNA-MSCs [(9.70 ± 1.62) ％,P ＜0.05].Conclusion:HGF prevented DOX-induced H9C2 cells apoptosis by inhibition of TGF-β1.