兔骨髓间充质干细胞体外向心肌样细胞诱导分化：缝隙连接蛋白43的

背景：骨髓间充质干细胞经过诱导因素刺激后，能够分化为心肌细胞，同时表达缝隙连接蛋白43，移植后可改善心功能或修复受损的心脏起搏传导系统。而缝隙连接蛋白43在维持心脏正常功能中发挥着重要作用。目的：观察兔骨髓间充质干细胞体外诱导分化为心肌样细胞过程中缝隙连接蛋白43的表达变化，及诱导后骨髓间充质干细胞间隙连接通讯功能的改变。方法：采用Percoll非密度梯度离心法与贴壁法分离培养兔骨髓间充质干细胞，正常组不进行任何干预，诱导组加入5-氮胞苷向心肌样细胞诱导，免疫荧光及流式细胞仪检验细胞诱导前后缝隙连接蛋白43的表达。将原代培养1 d的乳鼠心肌细胞分别接种于上述两组细胞爬片上，免疫荧光观察兔骨髓间充质干细胞与心肌细胞形成间隙连接的情况。划痕试验设立正常组、诱导组以及添加甘草次酸的阻滞剂组，观察兔骨髓间充质干细胞诱导前后细胞间隙连接的功能变化。结果与结论：正常组骨髓间充质干细胞缝隙连接蛋白43呈弱表达，5-氮胞苷诱导2，4周后缝隙连接蛋白43的表达均显著增加(P < 0.01)，细胞间隙连接通讯功能明显增强(P < 0.001)，且随诱导时间的延长呈依赖性增强(P < 0.05)。骨髓间充质干细胞与心肌细胞共培养后，诱导组缝隙连接蛋白43明显呈线状表达在两个相邻细胞的接触面。与诱导4周时比较，阻滞剂组细胞间隙连接通讯功能明显受到抑制(P < 0.001)。提示骨髓间充质干细胞能够自发表达缝隙连接蛋白43，在移植后早期能与心肌细胞形成间隙连接，从而有利于移植后心肌电传导，并发挥骨髓间充质干细胞的旁分泌效应。关键词：缝隙连接蛋白43；间隙连接；5-氮胞苷；诱导；心肌细胞；骨髓间充质干细胞

Differentiation of rabbit bone marrow mesenchymal stem cells into cardiomyocytes in vitro following induction: Changes in connexin 43 expression

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) could differentiate to cardiomyocytes-like cells and express connexin 43 (CX43) after induction by inducing factors. Cell transplantation could improve cardiac function or repair damaged cardiac pacing conduction system. While CX43 plays an important role in maintaining normal function of the heart.OBJECTIVE: To observe CX43 expression in the cardiomyocytes-like cells which were induced from the rabbit BMSCs in vitro, and study the gap junction intercellular communication between BMSCs and cardionmyoctes following induction. METHODS: Rabbit BMSCs were derived and cultured by Percoll discontinuous density gradient centrifugation and adherent culture method. Cells in the normal group did not receive any intervention. Cells in the induction group were induced by 5-azacitidine to differentiate into cardiomyocytes-like cells. Immunofluorescence and flow cytometry were used to determine CX43 expression in BMSCs before and after induction. Neonatal rat cardiomyocytes following primary culture of 1 day were seeded on above-mentioned two groups of cell plates. Immunofluorescence was used to study the gap junction between BMSCs and cardiomyocytes. Normal, induction and additional enoxolone blocker groups were established in cell scratch test to investigate the GJIC transformation before and after BMSC induction. RESULTS AND CONCLUSION: CX43 had weak expression in the normal group, but the CX43 expression was significantly increased at 2 and 4 weeks following 5-azacitidine induction (P < 0.01). GJIC was significantly enhanced (P < 0.001), and showed dependent increase with prolonged induction time (P < 0.05). Following coculture of BMSCs and cardiomyocytes, CX43 expression showed significantly linear expression on contact surface of two neighboring cells in the induction group. Compared with that at 4 weeks following induction, GJIC was significantly suppressed in the blocker group (P < 0.001). Above-mentioned results indicated that BMSCs could express CX43 spontaneously, which showed gap junction with cardiomyocytes in the early transplantation, resulting in cardiac electrical conduction following transplantation and the paracrine effect of BMSCs.