【摘要】 背景:大多数研究通过使用小分子物质或病毒等方式诱导获取心肌样细胞,而关于重组蛋白用于细胞重编程获得心肌样细胞的报道较少。目的:探讨Nkx2.5重组蛋白对人骨髓间充质干细胞向心肌样细胞转分化的作用。方法:构建Nkx2.5原核表达载体,不同时间点诱导Nkx2.5重组蛋白表达和分离纯化。使用纳米转导试剂包裹Nkx2.5形成纳米-蛋白复合体,转染人骨髓间充质干细胞,观察蛋白转导效率;蛋白转导成功后,检测心肌细胞特异性标志MHC的表达,观察人骨髓间充质干细胞向心肌样细胞转化情况。结果与结论:(1)28℃ IPTG诱导培养4 h获取的蛋白表达量更高;(2)纳米-Nkx2.5复合体可成功转染人骨髓间充质干细胞并诱导其向心肌样细胞转化;(3)结果表明,重组蛋白能被高效转导到细胞内,并能促使人骨髓间充质干细胞重编程为心肌样细胞,但细胞转分化效率尚待提高。更多还原

【Abstract】 BACKGROUND:Most studies mainly focus on using small molecules or viruses to obtain cardiomyocytes, but reports about the use of recombinant proteins to reprogram cells to obtain cardiomyocytes are rare.OBJECTIVE:To explore the effect of recombinant Nkx2.5 protein on the transdifferentiation of human bone marrow mesenchymal stem cells into cardiomyocytes.METHODS:The prokaryotic expression vector of Nkx2.5 was constructed. Nkx2.5 protein was induced to express at different time points, and then was isolated and purified. Nkx2.5 was encapsulated with nano-transducers to form nano-protein complexes, which were transfected into human bone marrow mesenchymal stem cells. The protein transfection efficiency was then detected. After successful protein transduction, the expression of myosin heavy chain, a specific marker of cardiomyocytes, was detected and the transformation of human bone marrow mesenchymal stem cells into cardiomyocytes was observed.RESULTS AND CONCLUSION:The protein expression was higher when the cells were induced with isopropyl-β-d-thiogalactoside at 28 ℃ for 4 hours. To conclude, the nano-Nkx2.5 complexes can be efficiently transfected into human bone marrow mesenchymal stem cells and promote the reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes, but future studies are needed to improve the efficiency of the transdifferentiation.更多还原