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目的分析hBMSCs经5-氮杂胞苷(5-azacytidine,5-aza)诱导向心肌样细胞分化过程中基因表达谱的改变。方法取胸外科非血液病患者手术中废弃的肋骨骨髓分离培养hBMSCs,5-aza诱导第2代hBMSCs。取诱导前及诱导后的细胞,免疫细胞化学法检测α-actin、肌钙蛋白T(cardiac troponin T,cTnT)和连接蛋白43(connexin 43)表达,流式细胞仪检测cTnT阳性细胞百分率;利用人表达谱基因芯片技术筛选分化过程中的差异表达基因,对部分基因进行功能分类和分层聚类分析。结果 hBMSCs经5-aza诱导后,部分呈肌细胞样形态。免疫细胞化学染色检测示诱导前细胞α-actin、cTnT染色呈弱阳性,connexin 43染色呈阴性;诱导后3周细胞α-actin、cTnT、connexin 43染色均呈阳性。流式细胞仪检测示诱导前cTnT阳性细胞百分率为7.43%±0.02%,诱导后3周为49.64%±0.05%。分化过程中共检测到1 814个显著差异表达基因,对其中647个基因分层聚类,聚为5类,生物功能包括信号传导、细胞代谢、增殖分化、发育以及形态发生等。结论 hBMSCs经5-aza诱导向心肌样细胞分化过程受信号传导通路、转录基因、生长因子等多种因素在不同时间点上的共同调控。
OBJECTIVE: To analyze the gene expression profiles of hBMSCs differentiated into cardiomyocyte-like cells induced by 5-azacytidine (5-aza). Methods hBMSCs were isolated and cultured from ablated rib bone marrow in patients undergoing thoracic surgery and non-hematological diseases. The second passage of hBMSCs was induced by 5-aza. The expressions of α-actin, cTnT and connexin 43 were detected by immunocytochemistry before and after induction. The percentage of cTnT positive cells was detected by flow cytometry. Gene expression profiling of differentially expressed genes was screened by cDNA microarray technology and functional classification and hierarchical cluster analysis of some genes were performed. Results After hBMSCs were induced by 5-aza, some of the hBMSCs were myoblast-like. Immunocytochemical staining showed weak positive staining of α-actin and cTnT cells and negative connexin 43 staining before induction, and positive staining of α-actin, cTnT and connexin 43 cells 3 weeks after induction. Flow cytometry showed that the percentage of cTnT positive cells before induction was 7.43% ± 0.02% and that after 3 weeks was 49.64% ± 0.05%. A total of 1 814 differentially expressed genes were detected during the differentiation process, of which 647 genes were clustered and clustered in 5 categories. Biological functions include signal transduction, cell metabolism, proliferation and differentiation, development and morphogenesis. Conclusion The differentiation of hBMSCs into cardiomyocyte-like cells induced by 5-aza is regulated by signal transduction pathway, transcriptional gene, growth factor and other factors at different time points.