siRNA沉默HLA-A2基因表达对人脐带间充质干细胞诱导成骨的影响

Effect of siRNA silent HLA-A2 gene on the induction of human umbilical cord mesenchymal stem cells into osteogenesis

目的探讨RNAi技术下调人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)HLA-A2基因表达后,对HLA-A2基因诱导成骨的影响。方法利用HLA-A2靶向小分子干扰RNA(small interfering RNA,siRNA)转染hUCMSCs,实验组为转染的细胞,对照组为未转染细胞。免疫细胞化学染色、Western Blot法检测两组细胞HLA-A2的表达;用诱导剂(0.1μmol/L地塞米松、10mmol/Lβ-甘油磷酸钠、50mg/L维生素C)行成骨诱导分化,茜素红矿化结节染色计数;碱性磷酸酶(ALP)染色以及比色法检测ALP活性。结果免疫细胞化学染色显示,实验组HLA-A2表达为弱阳性,对照组HLA-A2表达为阳性;Western Blot法检测HLA-A2蛋白的表达量对照组明显高于实验组。茜素红矿化结节染色显示:两组细胞均出现红色结节的阳性染色,但无差异(P>0.05);两组ALP染色细胞胞浆均呈蓝色阳性反应,ALP活性检测结果显示两组无差异(P>0.05)。结论应用RNAi技术下调HLA-A2基因表达后,不影响人脐带间充质干细胞的成骨诱导。

Objective To investigate the influence of the downregulation of HLA-A2 gene expression by RNAi on the osteogenic characteristics of umbilical cord mesenchymal stem cells(hUCMSCs).Methods hUCMSCs were transfected by HLA-A2 targeting small interfering RNA(siRNA),and immunohistochemical staining and Western blot were used to detect HLA-A2 expression in transfected and nontransfected hUCMSCs.hUCMSCs were cultured for the differentiation of osteoinduction with DMEM/F12(low sugar) medium(0.1μmol/L dexamethasone,10 mmol/Lβ-glycerol phosphate,50mg/L vitamin C).Alizarin red mineralized nodules staining and alkaline phosphatase(ALP) staining and colorimetric method were employed.Results Immunohistochemistry staining and Western blot showed that HLA-A2 expression level was much lower in transfected than in untransfected hUCMSCs.Alizarin red mineralized nodules staining and ALP staining showed that the positive products and ALP activity were similar in transfected and untransfected hUCMSCs(P>0.05).Conclusion The downregulation of the expression of HLA-A2 gene by RNAi exerts no influence on osteogenic characteristics of human umbilical cord mesenchymal stem cells.