慢病毒介导GDNF基因对食蟹猴骨髓间充质干细胞生物学特性的影响

Effects of lentivirus-mediated GDNF on biologic characteristics of cynomolgus monkey mesenchymal stem cells

目的探讨慢病毒介导胶质源性神经营养因子(GDNF)对食蟹猴骨髓间充质干细胞(cMSCs)生物学特性的影响。方法无菌条件下取食蟹猴骨髓,梯度密度离心法分离培养cMSCs,以含有GDNF基因的慢病毒感染cMSCs。利用ELISA、Real-time PCR、流式细胞术(FCM)、BrdU掺入法等实验方法,观察慢病毒感染后GDNF基因的表达,以及慢病毒感染前后的cMSCs的表面抗原表达、体外增殖和分化能力。结果感染慢病毒后,体外培养的cMSCs高表达GDNF。慢病毒感染后cMSCs的细胞形态无明显改变,细胞表面标志CD34、CD90和Stro-1阳性细胞比例增高。BrdU掺入结果显示,与对照组(0.61±0.11)比较,GDNF慢病毒感染组(0.50±0.13)明显降低(P<0.05)。但GDNF慢病毒感染并没有影响cMSCs体外脂肪诱导分化能力。结论慢病毒感染影响cMSCs的表面抗原表达和体外增殖能力,但对cMSCs体外脂肪分化能力无影响。

Objective To explore the effects of lentivirus-mediated GDNF on biologic characteristics of cynomolgus monkey mesenchymal stem cells.Methods The bone marrow mononuclear cells of cynomolgus monkey were isolated by density-gradient centrifugation and MSCs were cultured in vitro.GDNF lentivirus were used to infect cynomolgus monkey MSCs（cMSCs）.Using ELISA,Real-time PCR,flow cytometry（FCM）,BrdU incorporation and other experimental methods,GDNF gene expression,cell surface markers,proliferation and differentiation of cynomolgus monkey MSCs were observed before and after the infection of lentivirus.Results After the infection of GDNF lentivirus,cMSCs highly expressed GDNF in vitro,and moreover,cell morphology and adipocyte differentiation of cMSCs did not change significantly,but cell surface markers CD34,CD90 and Stro-1 positive cells increased in vitro,and compared with control（0.61±0.11）,the proliferative potential of GDNF group（0.50±0.13） showed a significant decrease.Conclusions The cell surface markers and the proliferative potential of cMSCs changed after the infection of GDNF lentivirus,but the adipocyte differentiation ability of MSCs had no effect.