骨髓干细胞向心肌细胞分化的基因芯片分析

摘要

Objective To investigate gene expression changes in BMSCs differentiation into myocardial cells under special micro-environment. Methods BMSCs were purified from bone marrow of clinical patients by density gradient centrifugation. They were cocultured with neonatal rat ventricular myocytes hy semipermeahle membrane. After cocultured for 2 weeks , gene expression profile of BMSCs was analyzed by 22K Human Genome Array. And the level of cardiac myogenesis related genes were presented by cluster analysis. The time course expression of some BMSCs differentiation related genes were confirmed with RT-PCR. Results Microarray analysis revealed that the expression of 511 genes were upregulated (ratio ＞ 5) , while 336 genes were downregulated (ratio ＜0.2) in BMSCs co-cultured for 2 weeks. And most of the upregulated genes are related to development , myogenesis and anti-apoptosis. RT-PCR analysis confirmed that the expression of HOP , GATA4, NKx2.5, β-MHC and ANF mRNA were upregulated in co-cultured BMSCs, and reached to their peak after for 2 weeks. Conclusions Upregulated expression of differentiation related genes such as IGF and FGF may play a role in the cardiac:diff'erentiation. And the differentiation of BMSCs in micro-environment may also be regulated by several transcription factors such as HOP, Nkx2.5 and GATA4. However, the mechanisms of BMSCs differentiation into cardiomyocytes is very complicated , and further experiment is needed to reveal the functional interaction among different transcription factors in this process.%目的:观察骨髓间充质干细胞(BMSCs)在特殊微环境下向心肌细胞分化的过程中基因表达的差异变化.方法:用密度梯度离心法培养BMSCs,原代培养乳鼠心室肌细胞,与BMSCs一起用半透膜共培养.共培养后2周提取细胞总RNA,进行22K Human Genome Array芯片检测.用RT-PCR检测心肌分化相关的基因的时序表达.结果:基因芯片检测BMSCs诱导前后差异基因的表达水平,发现诱导2周后表达明显升高(ratio>5)的基因有572个,表达下调(ratio<0.2)的基因有336个.基因的时序性表达发现HOP,Nkx2.5和GATA4基因在诱导前几乎没有检测到阳性表达,诱导后0.5周表达增强,1～2周表达达到高峰,维持一定水平后到4～8周表达呈下降趋势.心肌特异性基因β-MHC和ANF在0.5周开始出现,表达随时间逐渐增加,2周达到高峰.结论:分化诱导相关基因如IGF、FGF等的表达与心肌发育分化相关,GATA4和Nkx2.5等转录因子在微环境诱导心肌分化过程中起重要作用.然而心肌的分化实际上极为复杂,转录因子间相互作用的机制尚需进一步探讨.