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目的:体外研究miR-21对脐带血间充质干细胞(umbilical cord blood derived mesenchymal stem cells,UCBMSCs)血管向分化的促进作用。方法:培养UCBMSCs(来源于同济大学干细胞库);构建Lenti-Lac Z-Luciferase和LentimiR-21-Luciferase慢病毒载体;目的基因转染UCBMSCs后0、1、4、7及14d,分别采用实时荧光定量PCR及Western印迹法检测关键成血管因子的表达。用第3代UCBMSCs分别转染Lenti-Lac Z-Luciferase和Lenti-miR-21-Luciferase后,接种于Matrigel上培养,显微镜下观察其管样结构形成情况。采用SPSS13.0软件包对数据进行统计学分析。结果:成功培养脐带血骨髓间充质干细胞并成功转染目的基因;q PCR以及Western印迹法结果表明,miR-21组HIF-1α和VEGF在第4天开始显著高表达,并持续到7 d和14 d;Matrigel结果显示,Lenti-miR-21-Luciferase组管状结构最多,长度最长。结论:Mi R-21具有促进UCBMSCs血管向分化的作用。
AIM: To investigate the role of miR-21 in promoting the differentiation of umbilical cord blood derived mesenchymal stem cells (UCBMSCs). Methods: Lenti-Lac Z-Luciferase and LentimiR-21-Luciferase lentiviral vector were constructed by culturing UCBMSCs (derived from stem cell bank of Tongji University). The target gene was transfected into UCBMSCs at 0,1,4,7 and 14 days, Quantitative PCR and Western blotting were used to detect the expression of key angiogenic factors. Lenti-Lac Z-Luciferase and Lenti-miR-21-Luciferase were transfected with the third generation of UCBMSCs respectively, and then seeded on Matrigel and observed under a microscope for tube-like structure formation. SPSS13.0 software package for statistical analysis of the data. Results: The cord blood MSCs were successfully cultured and successfully transfected into the target gene. The results of q PCR and Western blotting showed that the expression of HIF-1α and VEGF in miR-21 group were significantly increased from the 4th day to the 7th day And 14 days respectively. The results of Matrigel showed that Lenti-miR-21-Luciferase group had the most tubular structure and the longest length. Conclusion: Mi R-21 can promote the differentiation of UCBMSCs into blood vessels.