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目的:研究5-氮胞苷(5-Aza)对培养人骨髓间充质干细胞(MSC)的作用,并对分化后的心肌样细胞进行鉴定。方法:采用密度梯度离心法分离到骨髓单个核细胞(MB-MNC),用含200 ml/L胎牛血清的低糖型DMEM培养液进行培养。采用差速贴壁法纯化MSC,用流式细胞仪检测细胞表面抗原。以5-Aza诱导第3代MSC 24 h后继续培养。培养4周,用免疫细胞化学染色法检测肌系标记抗原:α-肌动蛋白(α-actin)及心肌细胞特异性标记抗原:肌钙蛋白T(cTnT);在透射电镜下观察细胞的超微结构。结果:MSC经5-Aza诱导分化后,可表达α-actin和cTnT,未经诱导的同培养天数的MSC中均未见表达。透射电镜可观察到肌丝等心肌细胞的特异性结构。结论:5-Aza可诱导MSC分化为心肌样细胞。
AIM: To investigate the effect of 5-azacytidine (5-Aza) on cultured human bone marrow mesenchymal stem cells (MSCs) and to identify differentiated cardiomyocyte-like cells. Methods: Bone marrow mononuclear cells (MB-MNC) were isolated by density gradient centrifugation and cultured in low glucose DMEM containing 200 ml / L fetal calf serum. MSC were purified by differential adherence and cell surface antigens were detected by flow cytometry. The third generation of MSCs was induced by 5-Aza for 24 h and then cultured. After cultured for 4 weeks, the myogenic marker antigen (α-actin and cardiomyocyte-specific marker antigen: troponin T (cTnT)) was detected by immunocytochemical staining. microstructure. Results: After induced by 5-Aza, MSC could express α-actin and cTnT, and no expression was found in MSC without induction for the same number of culture days. Transmission electron microscopy can be observed myofilament and other specific structure of myocardial cells. Conclusion: 5-Aza can induce MSCs to differentiate into cardiomyocytes.