利用c-kit^+骨髓间充质干细胞与心脏脱细胞骨架共培育心肌组织

Cultivation of myocardial tissue by using c-kit^+ bone marrow mesenchymal stem cells and decellularized heart matrix

背景:研究发现c-kit阳性骨髓间充质干细胞可特异分化为心肌细胞,可能成为理想的种子细胞。目的:验证利用c-kit+骨髓间充质干细胞及心脏脱细胞骨架培育心肌组织的可行性。方法:取成年大鼠心脏脱细胞后备用。取大鼠骨髓间充质干细胞,培养至第8代后行c-kit富集,采用5μmol/L的5氮杂胞苷诱导2周后行二氢吡啶受体α2富集,然后继续培养行心肌分化鉴定或种植于心脏脱细胞骨架内行心肌组织培养。6周后,利用心肌细胞特异性蛋白表达及动作电位鉴定心肌细胞分化,利用免疫荧光染色分析新生心肌组织结构。结果与结论:第2次富集6周后,约60%的骨髓间充质干细胞表达心肌肌钙蛋白T、GATA结合蛋白4、连接蛋白43,电生理分析显示这些细胞可产生心肌细胞样动作电位,证实c-kit+骨髓间充质干细胞可特异分化为心肌细胞,种植于脱细胞心脏骨架后可形成排列有序的心肌组织。

BACKGROUND：Studies have found that c-kit^＋ bone marrow mesenchymal stem cels can differentiate into myocardial cels specificaly, which may be the ideal seed cels. OBJECTIVE：To investigate the feasibility of cultivation of myocardial tissue by using c-kit^＋ bone marrow mesenchymal stem cels and decelularized heart matrix. METHODS：Heart tissues harvested from adult rats were decelularized for the folowing experiments. Primary rat bone marrow-derived mesenchymal stem cels were culturedin vitro. Until passage 8, bone marrow mesenchymal stem cels were enriched for c-kit and induced by 5 μmol/L 5-azacytidine for 2 weeks, and a second enrichment for the dihydropyridine receptor subunit α2δ1 was performed before analysis of cardiac differentiation or implantation into decelularized heart matrix for cultivation of myocardial tissue. Six weeks later, myocardial differentiation was identified by specific cardiac protein and action potential. Immunofluorescence staining was used to analysis neonatal myocardial tissue. RESULTS AND CONCLUSION：Six weeks after the second enrichment, 60% bone marrow mesenchymal stem cels expressed cardiac troponin T, GATA binding protein 4, and connexin 43, and these cels could be induced to yield cardiac action potential, which was identified as cardiac differentiation. And when implanted into decelularized heart matrix, these cels could form myocardial tissue arranged regularly.