骨髓间充质干细胞对体外损伤心肌细胞凋亡的影响

Anti-apoptotic effects on damaged cardiomyocytes co-cultured with mesenchymal stem cells in vitro

目的:探讨骨髓间充质干细胞(mesenchymal stem cells,MSCs)在体外共培养条件下对多柔比星(doxorubi-cin,DOX)损伤心肌细胞凋亡的影响及可能机制。方法:贴壁法分离、培养骨髓间充质干细胞,第3代MSCs用于实验。原代培养3天的SD乳鼠心肌细胞,1.0 mg/L浓度的DOX处理4 h,建立心肌细胞损伤模型。将心肌细胞分为正常对照组、DOX损伤组、DOX损伤+MSCs共培养组。ELISA法检测细胞条件培养基内胰岛素样生长因子-1(insulin-like growth factor 1,IGF-1)的浓度。MTT检测MSCs条件培养基对DOX损伤心肌细胞活性的影响。并检测各组心肌细胞caspase-9,caspase-3活性及各组心肌细胞凋亡率。结果:第3代MSCs及原代心肌细胞均有IGF-1分泌,但MSCs条件培养基内IGF-1的浓度明显高于心肌细胞。MSCs共培养组心肌细胞活性高于DOX损伤组,但低于正常对照组(P<0.05)。DOX损伤+MSCs共培养组caspase-9,caspase-3活性及心肌细胞凋亡率低于DOX损伤组,但高于正常对照组(P<0.05)。结论:MSCs对多柔比星诱导的心肌细胞凋亡有保护作用,这种保护作用与旁分泌机制有关。

Objective： To investigate the effects of mesenchymal stem cells （MSCs） on doxorubicin （DOX）-injured cardiomyocytes apoptosis in vitro on the condition of co-culture and the possible mechanism. Methods： Mesenchymal stem cells（MSCs） were isolated and purified by adherentscreening method, then expanded in vitro. The third passage was used for study. The primary cultured neonatal SD rat cardiomyocytes were exposed to 1.0 mg/L Doxorubicin for 4 h to establish experimental model of toxic cardiomyocytes after having been cultured for 3 days. The cardiomyocytes were divided into 3 groups： cultured alone （control group）, DOX-injured, DOX-injured and co-cultured with the MSCs. The level of insulin-like growth factor 1 （IGF-1） in the conditioned medium was measured with ELISA kit. The effects of MSCs-condition medium on cardiomyocytes viability was determined by MTT assay. The activity of caspase-9 and caspase-3 were measured by caspase kits. The cardiomyocyte apoptosis was detected by Annexin V-FITC. Results： IGF-1 was significantly higher in the medium from cultured MSCs than in the medium from cultured cardiomyocytes. The viable cell number was larger in MSCs-conditioned medium group than that of DOX-injured group, but both decreased compared to control group （P 〈 0.05）. The activity of caspase-9, caspase-3 and the apoptosis rate decreased significantly in co-cultured group compared with the DOX-injured group, but increased compared to control group （ P 〈 0.05 ）. Conclusion ： It was concluded that paracrine mediators secreted by MSCs were involved in preventing DOX-induced cardiomyocytes apoptosis.