小鼠心肌祖细胞的永生化及筛选

Immortalization and screening of cardiac progenitor cells in mice

背景:移植外源干细胞治疗功能性心肌细胞死亡或凋亡,易导致移植并发症,因此心脏本身存在的心肌祖细胞成为理想的种子细胞。目的:建立稳定来源于小鼠心脏的心肌祖细胞株,为研究影响心肌祖细胞在成体心肌细胞受损时的增殖分化因素提供理想的细胞模型。方法:①分离培养胚胎15.5 d的小鼠心肌细胞。②感染反转录病毒SSR69使心肌细胞永生化,通过抗生素筛选和无限稀释法获得永生化的单克隆心肌祖细胞。③筛选出具有心肌祖细胞特性的单克隆细胞株。结果与结论:①成功分离培养出心肌细胞,部分细胞可见明显搏动。②感染病毒后,通过潮霉素筛选成功选出76个单克隆,并将其进行命名为CP15-#。③对第1-20号克隆,根据心肌细胞标志基因筛选出具有心肌祖细胞特性的克隆。表明通过可逆SV40 T抗原介导的途径成功建立永生化心肌祖细胞。

BACKGROUND：Transplantation of exogenous stem cells for functional cardiac celldeath or apoptosis easily induced complications after transplantation. Therefore, cardiac progenitor cells of heart itself have been ideal seed cells. OBJECTIVE：To establish stable celllines of cardiac progenitor cells from mouse heart, and to provide ideal cellmodels for studying proliferation and differentiation factors affecting cardiac progenitor cells during adult myocardial cells were damaged. METHODS：（1） Myocardiocytes were isolated from embryonic 15.5 days mice. （2） The cultured myocardiocytes were immortalized using retrovirus SSR69. Immortalized monoclonal myocardial cells were obtained using antibiotic selection and infinite dilution. （3） Monoclonal cellline with the property of cardiac progenitor cells was screened out. RESULTS AND CONCLUSION：（1） Myocardiocytes were successful y isolated and cultured. Partial cells showed obvious beating. （2） Myocardiocytes infected with retrovirus SSR69 were cloned and got 76 clones, then were named as CP15-#, （3） Screening the first 20 clones, the reasonable clones with the characteristics of cardiac progenitor cells were obtained according to myocardial cellmarker genes. The results suggested that immortalized cardiac progenitor cells were established mediated by reversible SV40 T antigen.