RNAi干扰MHCⅡ基因表达后的骨髓间充质干细胞向血管内皮样细胞分化的研究

Sdudy on Rats Bone Marrow Mesenchymal Stem Cells of RNA Inhibiting Effect to MHCⅡ Gene Expression in Differentiate into Vascular Endothelial Cells

目的 慢病毒沉默BMSCs中的MHCⅡ基因表达,再向血管内皮样细胞诱导分化;检测慢病毒感染后的BMSCs的增殖活性、分化效应及分化后的免疫原性变化。方法 从大鼠骨髓中分离、培养BMSCs,用慢病毒(LV)沉默MHCⅡ基因的表达,观察其增殖情况。使用血管内皮生长因子(VEGF)诱导感染后的BMSCs向血管内皮样细胞(VEC)分化,观察分化后细胞的形态、蛋白及mRNA是否有变化,电镜下观察细胞质是否出现Weibel-Palade小体。结果 慢病毒感染后的BMSCs生长曲线与未感染的BMSCs无明显差别,增殖活性不受影响。LV-CIITA-BMSCs、LV-NC-BMSCs及BMSCs经过诱导后细胞形态发生变化,细胞质中出现Weibel-Palade小体,诱导后BMSCs的表面标志物CD34、Ⅷ因子及Flt-1 mRNA明显高于未诱导组,各个诱导组之间无统计学意义;LV-CIITA-BMSCs的MHCⅡ蛋白及mRNA表达明显低于LV-NC-BMSCs及诱导后的BMSCs。结论 BMSCs经过慢病毒感染后,不影响其增殖活性及向VEC分化。BMSCs经过分化后,其MHCⅡ蛋白及mRNA表达升高;但是LV-CIITA-BMSCs诱导后MHCⅡ蛋白及mRNA的上升幅度较LV-NC-BMSCs及BMSCs低。

Objective To study the MHCⅡ gene expression of BMSCs inhibited by the Lentivirus, LV-CIITA-BMSCs induced into VECs, and infected BMSCs of proliferation activity, differentiation effect and the immunogenicity changes after differentiation.Methods BMSCs was isolated and cultured from rat bone marrow.The MHCⅡ gene expression was inhibited by the Lentivirus, observe the activity of proliferation.The infected BMSCs were induced by VEGF.Then, dBMSCs were obseved in the shape、mRNA、protein and found the WP body in the cytoplasm with electron microscope.Results There was no evident difference between infected BMSCs and uninfected BMSCs in the growth curve.It was changed that LV-CIITA-BMSCs、LV-NC-BMSCs and BMSCs were induced in the shape, and the WPB were found in the cytoplasm after being induced.The BMSCs of being induced were higher than that of uninduced in the expression of CD34、Ⅷ and Flt-1 mRNA,and there was no difference in the groups of being induced.The LV-CIITA-BMSCs was lower than LV-NC-BMSCs and BMSCs after being induced in the expression of MHCⅡ protein and mRNA.Conclusion The lentivirus to infect does not decrease the BMSCs of proliferation and differentiation to VECs.The MHCⅡ protein and mRNA expression are increased after being induced, but that of the LV-CIITA-BMSCs is lower than that of LV-NC-BMSCs and BMSCs.