人脐带间充质干细胞向神经细胞分化过程中ILK的表达变化及意义

目的 研究在人脐带间充质干细胞(hUCMSC)向神经干细胞样细胞(hucNSC)、神经细胞(Nc)诱导分化中整合素连接激酶(ILK)的表达变化及意义.方法 将体外培养、传至第3代的hUCMSC,用无血清培养基加入bFGF、EGF和B27预诱导分化为hucNSC,继之将其用BHA、弗司扣林等向神经细胞定向诱导分化,并用AF488分别标记hUCMSC、hucNSC、NC细胞骨架变化过程加以证实,免疫组化及RT-PCR检测ILK在hUCMSC、hucNSC、NC中的表达,并用实时定量PCR加以定量分析.结果 hUCMSC被诱导后首先聚集成细胞球样悬浮生长,表达nestin、CD133,ILK表达明显增强;再被诱导后表达NSE、TH,GFAP,并持续表达ILK.hUCMSC细胞骨架呈丝状规律排列,hucNSC时则成巢状,定向诱导后的神经细胞骨架转变成多突起的树枝状.实时定量PCR检测ILK在hUCMSC中微量表达,hucNSC中表达明显上调,是hUCMSC表达水平的12倍;在NC中ILK表达较hucNSC降低,但仍高于hUCMSC 8.6倍(P＜0.05).结论 人脐带间充质干细胞向神经干细胞样细胞、神经细胞诱导分化及细胞形态重塑中ILK可能发挥重要作用.

Effect of ILK expression on differentiation of human umbilical cord mesenchymal stem cells into neural stem-like and neural cells

Objective To investigate effect of of ILK expression on differentiation of human umbilical cord mesenchymal stem cells(hUCMSC)into neural stem-like cells(hucNSC)and neural cells.Method After MSCs were obtained from human umbilical cord, induction of hucNSC processes were realized with EGF,bFGF and B27 supplement in the induction media,consequently induced to neural cells with neural induction medium contained BHA and Forskolin.Cystoskeleton changes of hUCMSC and neural cells were detected by AF488 immunostaining.RT-PCR analysis and Real-Time PCR to display changes of ILK expression in hUCMSC,hucNSC and neural cells.Results Induction with EGF,bFGF,B27 about 72h,hUCMSCs began to change into hucNSCs showing a neurosphere-like shape and expressed nestin and CD133.These hucNSCs could differentiated into the neural phenotypes expressed NSE.TH or GFAP when induced further by neural induction medium.Strong immunostaining of ILK was detected on hueNSCs and neural cells.ILK mRNA level in hucNSCs was 12 times than that in hUCMSCs.while ILK mRNA in neural cells was 8.6 times than that in hUCMSCs as analyzed by Real-Time PCR(P＜0.05). Cystoskeleton patterns varied from hUCMSCs to those of hucNSCs and neural cells.hUCMSCs revealed a silkiness pattern,hucNSCs displayed a nest shape, while neural cells appeared a branching pattern. Conclusions ILK expression was upregulated during the conversion of MSCs to neural cells,suggesting it may involve promoting difierentiation of hucNSCs and neural cells-derived from hUCMSCs.