鹿茸多肽联合GDNF基因修饰的雪旺细胞对人骨髓间质干细胞体外增殖的影响

Effect of velvet antler polypeptides combined with Schwann cells modified with GDNF gene on proliferation of human bone marrow mesenchymal stem cells in vitro

目的:探讨鹿茸多肽(PAP)联合胶质细胞源性神经营养因子(GDNF)基因修饰的雪旺细胞对人骨髓间质干细胞(BMSCs)体外增殖的影响。方法:按常规方法抽取10mL健康志愿者骨髓,接种于培养瓶中进行培养,原代培养细胞完全融合前进行传代,传至第3代时收获BMSCs,调整细胞浓度为5×106 mL-1备用。加入4μL GDNF基因修饰的雪旺细胞为GDNF组,加入4μL(10mg·L-1)PAP联合GDNF基因修饰的雪旺细胞作为联合组,对照组未加入任何药物仅加入等量的培养基。采用酶联免疫检测法检测各组细胞细胞增殖活力,ELISA法检测各组BMSCs中增殖细胞核抗原(PCNA)水平,AnnexinⅤ-FIFC/PI细胞凋亡检测试剂盒检测细胞凋亡率。结果:原代培养48h后大部分细胞贴壁,形态转变为多角形,少数呈梭形。传代细胞几乎呈梭形,为BMSCs,且均为非造血干细胞。与对照组比较,GDNF组和联合组细胞增殖活力和BMSCs中PCNA水平升高(P<0.05),细胞凋亡率降低(P<0.05);与GDNF组比较,联合组细胞增殖活力和BMSCs中PCNA水平升高(P<0.05),细胞凋亡率降低(P<0.05)。结论:PAP联合GDNF基因修饰的雪旺细胞对人BMSCs体外增殖具有明显的促进作用。

Objective:To investigate the effect of pilose antler polypeptide combined with Schwann cells modified by glial cell line derived neurotrophic factor(GDNF)gene on the proliferation of human bone marrow mesenchymal stem cells(BMSCs)in vitro.Methods:According to the conventional method,the bone marrow(10mL)was extracted from the healthy volunteers and was inoculated into the culture flask.The primary cultured cells were completely fused.The BMSCs were harvested at the3rd generation and the cells were adjusted to5×106mL-1.4μL GDNF gene modified Schwann cells was added into GDNF group,4μL(10mg·L-1)PAP combined with GDNF gene modified Schwann cells was added into combination group,and only same amount of medium was added into control group.The proliferative activities,cell nuclear antigen(PCNA)levels and apoptotic rates of BMSCs in various groups were detected by enzyme linked immunosorbent assay,ELISA method and AnnexinⅤFIFC/PI cell apoptosis detection kit,respectively.Results:After primary culture for48h,most of the cells adhered to the wall,and the morphology of the cells changed into polygonal shape and few of them showed spindle.The passaged cells showed spindle spindle,the cells were confirmed as BMSCs,and all of them were non hematopoietic stem cells.Compared with control group,the proliferative activities and the PCNA level of the BMSCs in GDNF group and combination group were increased(P<0.05)and the apoptotic rates were decreased(P<0.05);compared with GDNF group,the proliferative activity and the PCNA level of the BMSCs in combination group were increased(P<0.05)and the apoptotic rate was decreased(P<0.05).Conclusion:PAP combined with Schwann cells modified by GDNF gene can promote the proliferation of human BMSCs in vitro.