血清诱导乳鼠骨髓间充质干细胞表达胰岛样细胞基因效果观察

Islet-like cell gene expression of bone marrow mesenchymal stem cells induced by serum in neonatal rats

目的观察血清诱导乳鼠骨髓间充质干细胞(MSC)表达胰岛样细胞基因的效果。方法 SD乳鼠10只,手术分离胫骨和股骨,分离、培养MSC,分别采用5%(对照组)、10%(低浓度组)和20%(高浓度组)的胎牛血清(FBS)刺激。纯化至第4代时以MTT法检测MSC增殖趋势;诱导14 d时用RT-PCR检测MSC细胞中的巢素蛋白(Nestin)、胰十二指肠同源性盒因子-1(PDX-1)、Insulin mRNA,用免疫荧光法检测Nestin、PDX-1、Insulin蛋白。结果随着血清浓度增高与刺激时间延长,MSC呈现明显的增殖趋势,细胞形态发生改变,形成胰岛β细胞轮廓。高浓度组MSC Nestin mRNA表达低于对照组,PDX-1、Insulin mRNA表达高于对照组(P均<0.05)。诱导14 d时Insulin、Nestin、PDX-1蛋白表达与7d时比较,P均<0.05。结论血清诱导下乳鼠MSC中胰岛相关基因PDX-1、Insulin表达上调,Nestin表达减少,显示MSC在一定条件下可被定向诱导分化成胰岛样细胞。

Objective To observe the effects of different serum concentrations on the islet-like cell gene expression of bone marrow mesenchymal stem cells （MSCs） in neonatal rats.Methods The tibia and femur were isolated from 10 neonatal SD rats and MSCs were separated and cultured .Then MSCs were induced by different serum concentrations of fetal bovine serum （FBS）, including 5%FBS （control group）, 10%FBS （low concentration group） and 20%FBS （high concentration group）.The proliferation of MSCs was examined by MTT way after being purified for the 4th generation.On the 14th day of induction, the nestin, pancreatic and duodenal homeobox gene-1（PDX-1） and insulin mRNA expression was detected by RT-PCR, and the nestin, PDX 1-and insulin proteins were examined by immunofluorescence .Results The increased concentrations of FBS and prolonged stimulation time enhanced the proliferation of MSCs , and the cell morphology changed and formed the islet-likeβcell contour .The nestin mRNA expression of the high concentration group was lower , but the PDX-1 and insulin mRNA expression was higher than that of the control group （all P〈0.05）.Significant differ-ence was found in the protein expression of nestin , PDX-1 and insulin between the 7th day and 14th day of induction （all P〈0.05）.Conclusion In the neonatal SD rats＆#39;MSCs induced by serum concentration , the insulin-related gene PDX-1and insulin expression is up-regulated and nestin expression is down-regulated, which shows that MSCs could be induced and differentiated into islet-like cells under certain conditions .