miR-210促进小鼠BMMSCs血管内皮生长因子的表达和分泌

目的 通过体内与体外的手段验证miR-210能够有效的促进骨髓间充质干细胞(BMMSCs)血管内皮生长因子(VEGF)的表达和分泌.方法 构建慢病毒载体,分别转染BMMSCs和人脐静脉内皮细胞(HUVEC),Lenti-miR-210/BMMSCs和Lenti-LacZ/BMMSCs培养第7天RT-PCR法检测VEGF表达,第14天Western blot法检测VEGF表达,PKH26荧光染色Lenti-miR-210/HUVEC和Lenti-LacZ/HUVEC,Matrigel成管实验24 h,荧光显微镜观察成管效果;agomir-210和agomir-NC分别与BMMSCs复合HyStem-HP凝胶缓释系统裸鼠皮下注射7d后取标本CD31免疫荧光染色.结果 miR-210慢病毒载体转染BMMSCs,第7天RT-PCR和第14天Western blot法检测VEGF表达,明显高于Lenti-LacZ/BMMSCs(P ＜0.05);Matrigel成管实验24 h结果显示:LentimiR-210/HUVEC组与Lenti-LacZ/HUVEC组相比,端端接触明显且管状结构完整;agomir-210和agomir-NC与BMMSCs复合HyStem-HP凝胶缓释系统裸鼠皮下注射7d后标本CD31免疫荧光染色结果:agomir-210组CD31阳性细胞含量明显高于agomir-NC组.结论 通过体内与体外的检测,miR-210过表达能够有效促进BMMSCs血管内皮生长因子的表达和分泌.

miR-210 promotes the expression and secretion of vascular endothelial growth factor in mouse BMMSCs

Objective To verify miR-210 effectively promoting the expression and secretion of vascular endothelial growth factor in BMMSCs by means of in vivo and in vitro.Methods To construct the lentiviral vector,we respectively transfected BMMSCs and HUVEC and cultured Lenti-miR-210/BMMSCs and Lenti-LacZ/BMMSCs.The expression of VEGF was deceted by RT-PCR on day 7 and Western blot on day 14.After fluorescent stained by PKH26 red kit,Lenti-miR-210/HUVEC and Lenti-LacZ/HUVEC were cultured on matrigel for 24 h,then observed by fluorescence microscope.Two hydrogel systems(agomir-210,BMMSCs,and hyetem-HP;agomir-NC,BMMSCs,and hyetem-HP) were injected subcutaneously into nude mice.On the seventh day,the nude mice were killed and the sample was immunofluorescence stained by CD31.Results The expression of VEGF in Lenti-miR210/BMMSCs group deceted by RT-PCR on day 7 and Western blot on day 14 was much higher than that of the control group(P ＜ 0.05).After cultured in matrigel for 24 h,cells on Lenti-miR-210/HUVEC group developed much more vessel-like structures and junction size than those on Lenti-LacZ/HUVEC group.And CD31 positive cells in agomir-210 group were significantly higher than that of agomir-NC group by immunofluorescence stain.Conclusion Through in vivo and in vitro detection,miR-210 overexpression could effectively promote the expression and secretion of vascular endothelial growth factor BMMSCs.