过表达IGF-1的小鼠骨髓间充质干细胞分泌物对缺氧心肌细胞的抗凋亡作用

Effect of bone marrow mesenchymal stem cells overexpressing IGF-1 on cardiomyocyte apoptosis during hypoxia

目的探讨IGF-1过表达的小鼠骨髓间充质干细胞(BMSCs)对缺氧时小鼠心肌细胞凋亡的影响。方法以小鼠BMSCs为研究对象,利用慢病毒转染技术,构建过表达IGF-1的BMSCs(IGF-1-BMSCs)及其对照细胞(NC-BMSCs)。与正常培养相比,1%氧浓度构建低氧诱导的细胞凋亡模型。流式细胞术观察低氧诱导对BMSCs及心肌细胞凋亡率的影响;流式细胞术观察加入IGF-1-BMSCs上清液时低氧诱导对心肌细胞凋亡率的影响;Western blotting检测低氧模型细胞内Bcl-2家族促凋亡蛋白(Bax)及抗凋亡蛋白(Bcl-2)、Caspase家族蛋白(caspase-3、8、9)及细胞色素C(Cyto C)的表达。结果与NC-BMSCs细胞对比,低氧诱导时IGF-1-BMSCs细胞的凋亡率明显下降[(36.85±1.02)%vs(15.66±4.36)%,P<0.05],抗凋亡蛋白Bcl-2明显增加,而促凋亡蛋白Bax、caspase-3、caspase-8及细胞色素C的释放明显减少(P<0.05)。与正常培养相比,低氧处理显著诱导心肌细胞凋亡[(10.89±1.62)%vs(42.30±2.53)%,P<0.05]。与NC-BMSCs细胞对比,IGF-1-BMSCs细胞上清液培养的心肌细胞凋亡率显著降低[(45.78±3.26)%vs(21.45±5.46)%,P<0.05],且抑制凋亡的效应呈时间及浓度依赖性。与NC-BMSCs细胞对比,IGF-1-BMSCs细胞上清液使低氧诱导的心肌细胞内凋亡蛋白Bcl-2明显增加,而促凋亡蛋白Bax、caspase-3、caspase-8及细胞色素C的释放明显减少(P<0.05)。结论过表达IGF-1可增强小鼠骨髓间充质干细胞自身抗凋亡能力,其细胞培养上清液可抑制低氧诱导时心肌细胞凋亡。

Objective To investigate the effect of mouse bone marrow mesenchymal stem cells(BMSCs)overexpressing IGF-1 on the apoptosis of cardiomyocytes under hypoxia.Methods BMSCs were transfected with IGF-1 or NC lentivirus to construct IGF-1-BMSCs and control cells(NC-BMSCs).Flow cytometry was used to observe the hypoxia-induced apoptosis of BMSCs and cardiomyocytes.Flow cytometry was applied to observe the hypoxia-induced apoptosis of cardiomyocytes after treatment with IGF-1-BMSCs supernatant.Western blotting was performed to detect the expression of intracellular Bcl-2 family pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2,caspase family proteins(caspase-3,8,9)and cytochrome C(Cyto C)in BMSCs and cardiomyocytes under hypoxia.Results Compared with NC-BMSCs,the apoptosis rate of IGF-1-BMSCs was decreased significantly under hypoxia(36.85%±1.02%vs 15.66%±4.36%,P<0.05),the anti-apoptotic protein Bcl-2 in IGF-1-BMSCs was significantly increased,while the expression levels of pro-apoptotic proteins Bax,caspase-3,caspase-8 and cytochrome C were significantly reduced(P<0.05).Compared with normal culture,hypoxia treatment significantly induced the apoptosis of cardiomyocytes(10.89%±1.62%vs 42.30%±2.53%,P<0.05).Comparing to NC-BMSCs supernatant,IGF-1-BMSCs supernatant significantly reduced the apoptosis rate of cardiomyocytes(45.78%±3.26%vs 21.45%±5.46%,P<0.05),and this inhibitory effect was in a time-and concentration-dependent manner.Comparing to NC-BMSCs cell supernatant,Bcl-2 protein in cardiomyocytes under hypoxia was increased significantly after adding IGF-1-BMSCs cell supernatant,while the expression of pro-apoptotic proteins Bax,caspase-3,caspase-8 and cytochrome C was decreased significantly(P<0.05).Conclusion Overexpression of IGF-1 can enhance the anti-apoptotic ability of mouse bone marrow mesenchymal stem cells,and its cell culture supernatant can inhibit the apoptosis of cardiomyocyte induced by hypoxia.