【摘要】 背景:干细胞已成为组织工程中理想的种子细胞,为细胞移植治疗心肌梗死提供了研究方向。目的:探讨骨形态发生蛋白2联合5-氮胞苷体外诱导骨髓间充质干细胞向心肌样细胞分化的可行性。方法:采用全骨髓贴壁法培养骨髓间充质干细胞,将第3代骨髓间充质干细胞进行分组诱导,分别为对照组(普通IMDM培养基)、骨形态发生蛋白2组(200 ng/L)、5-氮胞苷组(10μmol/L)、5-氮胞苷+骨形态发生蛋白2组。诱导3 d后更换正常培养基继续培养4周,免疫细胞化学法检测cTnI的表达,免疫荧光化学法检测Desmin、cTnT的表达,Western blot检测cTnT和cTnI的表达;诱导3 d后更换正常培养基继续培养1,2,4周,采用RT-qPCR检测心肌早期转录因子GATA-4、Nkx2.5 mRNA的表达。结果与结论:(1)Western blot检测诱导组均表达cTnT、cTnI,联合诱导组cTnT、cTnI的表达高于单独诱导组(P <0.01);(2)免疫细胞化学法检测诱导组cTnI均呈阳性表达,联合诱导组高于单独诱导组(P <0.05);免疫荧光细胞化学法检测诱导组Desmin、cTnT均呈阳性表达,联合诱导组高于单独诱导组(P <0.05);(3)RT-qPCR检测诱导组均有GATA-4、Nkx2.5 mRNA的表达,联合诱导组高于单独诱导组(P <0.05);(4)结果表明,骨髓间充质干细胞经5-氮胞苷和骨形态发生蛋白2联合诱导后可以提高心肌样细胞的分化效率。更多还原

【Abstract】 BACKGROUND: Stem cells have become ideal seed cells in tissue engineering, which provides a research direction for cell transplantation in the treatment of myocardial infarction.OBJECTIVE: To investigate the feasibility of bone morphogenetic protein 2 combined with 5-azacytidine on the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro.METHODS: Bone marrow mesenchymal stem cells were cultured by whole bone marrow culture adherent method. The third generation of bone marrow mesenchymal stem cells was divided into four groups: control group(ordinary Iscove’s modified Dulbecco’s medium), bone morphogenetic protein 2 group(200 ng/L), 5-azacytidine group(10 μmol/L), 5-azacytidine and bone morphogenetic protein 2 group. After induction for 3 days, the normal medium was changed and cultured for 4 weeks. The expression of cTnI was detected by immunocytochemistry. The expression of Desmin and cTnT was detected by immunofluorescence. The expression of cTnT and cTnI was detected by western blot assay. After 3 days of induction, the normal medium was changed and cultured for 1, 2 and 4 weeks. The mRNA expression of GATA-4 and Nkx2.5 was detected by RT-qPCR. RESULTS AND CONCLUSION:(1) Western blot assay showed that cTnT and cTnI were expressed in the induction group. The expression levels of cTnT and cTnI in the combined group were higher than those in other groups(P < 0.01).(2) Immunocytochemistry showed positive expression of cardiac troponin(cTnI) in the induction group; the expression was higher in the combined group than that in the induction group(P < 0.05). Immunofluorescence cytochemistry assay showed the positive expression of Desmin and cTnT; the expression in the combined group was higher than that in the induction group(P < 0.05).(3) RT-qPCR showed the mRNA expression of GATA-4 and Nkx2.5 in the induction group; the expression was higher in the combined group than that in the induction group(P < 0.05).(4) The results showed that bone marrow mesenchymal stem cells induced by 5-azacytidine and bone morphogenetic protein 2 could improve the differentiation efficiency of cardiomyocyte-like cells.更多还原