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目的研究转染BIG-3基因对人骨髓基质干细胞(hBMSCs)向软骨纠胞诱导分化的影响。方法将构建好的pMSCVpuro-BIG-3转染至建系的hBMSCs,5μg/mL嘌罗霉素筛选12 d后,扩增并收集细胞,电泳证实特定条带无误。实验分三组：hBMSCs-BIG-3组、hBMSCs-EV组和hBMSCs组,每组重复6次,将三组细胞制成微粒模拟三维培养模式,分别加入浓度为400 ng/mL的重组人骨形态发生蛋白-2(rhBMP一2)作用14 d后,检测甲苯胺蓝及Ⅱ型胶原mRNA,利用MPS60病理图象分析系统采集图像,并进行灰度分析,半定量比较基质表达量。结果灰度值：hBMSCs-BIG-3组为684 481 822,hBMSCs-EV组为439 101 780,hBMSCs组为441 082 183,hBMSCs-BIG-3组灰度值明显高于后两组(P＜0．05),hBMSCs-EV组和hBMSCs组之间差异无显著性意义(P=0．862)。结论BIG-3基因有助于hBMSCs向软骨细胞诱导分化。
Objective To investigate the effect of BIG-3 gene transfection on the differentiation of human bone marrow stromal cells (hBMSCs) into cartilage. Methods The constructed pMSCVpuro-BIG-3 cells were transfected into hBMSCs with established hBMSCs. After 5μg / mL puromycin was screened for 12 days, the cells were expanded and collected. The specific bands were confirmed by electrophoresis. The experiment was divided into three groups: hBMSCs-BIG-3 group, hBMSCs-EV group and hBMSCs group. Each group was repeated 6 times, and the three groups of cells were made into micro-particle simulation three-dimensional culture model. After exposure to rhBMP-2 for 14 days, the mRNA of toluidine blue and type II collagen was detected. The images were collected by MPS60 pathological image analysis system and analyzed by grayscale. The expression of matrix was semi-quantitatively compared. RESULTS: The gray value of hBMSCs-BIG-3 group was 684 481 822, that of hBMSCs-EV group was 439 101 780, and that of hBMSCs group was 441 082 183. The gray value of hBMSCs-BIG-3 group was significantly higher than that of the latter two groups (P < 0.05). There was no significant difference between hBMSCs-EV group and hBMSCs group (P = 0.862). Conclusion BIG-3 gene contributes to hBMSCs differentiation into chondrocytes.