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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 effect of RNAi on HLA-A2 gene-induced osteogenesis after down-regulating HLA-A2 gene expression in human umbilical cord mesenchymal stem cells (hUCMSCs). Methods Human umbilical vein endothelial cells (hUCMSCs) were transfected with HLA-A2 targeting small interfering RNA (siRNA). The experimental group was transfected cells and the control group was untransfected cells. Immunocytochemical staining and Western Blot were used to detect the expression of HLA-A2 in both groups. Osteogenic differentiation was induced by inducer (0.1μmol / L dexamethasone, 10mmol / L sodium β-glycerophosphate and 50mg / L vitamin C) Alizarin red mineralized nodule staining count; alkaline phosphatase (ALP) staining and colorimetric assay ALP activity. Results Immunocytochemical staining showed that the expression of HLA-A2 in the experimental group was weakly positive and the expression of HLA-A2 in the control group was positive. The expression of HLA-A2 protein in the control group was significantly higher than that in the experimental group by Western Blot. Alizarin red mineralization of nodular staining showed: the two groups of cells were positive for staining of red nodules, but no difference (P> 0.05); two groups of ALP-stained cytoplasm showed a blue positive reaction, ALP activity test results show There was no difference between the two groups (P> 0.05). Conclusion Down-regulation of HLA-A2 gene expression using RNAi technology does not affect osteogenic induction of human umbilical cord mesenchymal stem cells.