新生小鼠心脏细胞体外共培养体系的建立及其在心肌细胞增殖研究中的应用  

The establishment of a co-culture system of cardiomyocytes and fibroblasts in neonatal C57 mice in vitro and its application in the study of cardiomyocytes proliferation

作　　者：胡金莲[1] 郑怡文[1] 谷晓蕾[1] 陆国才[1] 

机构地区：[1]第二军医大学热带医学与公共卫生学系卫生毒理学教研室,上海200433

出　　处：《北京医学》2015年第5期476-479,I0002,共5页Beijing Medical Journal

基　　金：国家自然科学基金(81473291;81402651);"重大新药创制"科技重大专项(2014ZX09J14106);上海市公共卫生重点实验室建设计划(12GWZX0501)

摘　　要：目的建立新生C57乳鼠心肌细胞和成纤维细胞体外原代培养和共培养体系,探讨不同日龄乳鼠心脏细胞的体外增殖特征。方法分离不同日龄C57新生小鼠的心肌细胞和成纤维细胞,分别进行原代培养和共培养,观察细胞的形态和活力变化,测定Cyclin D1、CDK4、GATA4、Nkx2.5以及FGF1的表达。结果差速贴壁90 min后成纤维细胞先贴壁,培养12 h后心肌细胞几乎全部贴壁;共培养心肌细胞多以细胞团的形式贴壁生长。出生5 d以内的乳鼠心肌细胞数量明显增加,共培养的心肌细胞活力更大、增殖速度快;出生5 d以后乳鼠的心肌细胞数量变化并不明显。出生5 d内乳鼠心肌细胞中Cyclin D1和CDK4的表达量较高,且共培养的心肌细胞表达量要高于单独培养的心肌细胞表达量。GATA4、Nkx2.5和FGF1的表达随日龄增大而增加。结论成功建立了新生小鼠心肌细胞和成纤维细胞的体外共培养体系;出生5 d内的乳鼠心肌细胞体外培养时具有较强的增殖能力,且成纤维细胞能促进心肌细胞的增殖,GATA4、Nkx2.5和FGF1有阻止心肌细胞增殖作用。出生5 d后乳鼠的心肌细胞增殖能力减弱甚至消失。Objective To establish a co-culture system of cardiomyocytes and fibroblasts in neonatal C57 mice, and to study the proliferation characteristics of neonatal mice cardiomyocytes at different periods. Methods Fibroblasts and cardiomyocytes of neonatal C57 mice at different day-age were isolated for in vitro culture and co-culture. The basic changes of chondrocytes and vitality of fibroblasts and cardiomyocytes were observed by inverted phase contrast micro- scope. The expression of CyclinD1, CDK4, GATA4, Nkx2.5 and FGF1 were detected via WB. Results In primary cell culture, fibroblasts were adhered 90 min later, and cardiomyocytes, 12 h later. In the co-culture system, several cardiomyocytes were gathered to form clusters. The cell number of cardiomyocytes within 5 day-age was significantly changed. Co-cultured cardiomyocytes grew faster and had relatively higher proliferation activity than the ones cultured alone. The expression of CyclinD1 and CDK4 of cardiomyocytes within 5 day-age was higher than those born 5 days later, the cocultured cardiomyocytes was higher than the cultured alone, and the expression of GATA4,Nkx2.5 and FGF1 presented an increasing trend with the increase of age. Conclusion The in vitro co-culture method of neonatal cardiomyocytes in mice is effective and feasible. Cardiomyocytes within 5 day-age have the ability of proliferation, while the ones born af- ter 5 days are weak or even disappeared. Cardiac fibroblasts can promote the growth and proliferation of cardiomyocytes while GATA4, Nkx2.5 and FGF1 have the opposite functions.

关 键 词：心肌细胞 成纤维细胞 新生小鼠 体外培养 共培养 细胞增殖 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]