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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 primary culture and co-culture system of neonatal C57 neonatal rat cardiomyocytes and fibroblasts in vitro and to explore the in vitro proliferation characteristics of heart cells of different age-age neonatal rats. Methods Cardiomyocytes and fibroblasts from C57 neonatal mice of different ages were isolated and cultured. The primary culture and co-culture were performed. The changes of cell morphology and viability were observed. The expressions of Cyclin D1, CDK4, GATA4, Nkx2.5 and FGF1 were determined. RESULTS: After 90 min of differential adherence, fibroblasts adhered first, and almost all of cardiomyocytes adhered to each other after cultured for 12 h. Most of the co-cultured cardiomyocytes grew in the form of cell clusters. The number of cardiomyocytes in neonatal rats within 5 d after birth was significantly increased, and the co-cultured cardiomyocytes were more viable and proliferated rapidly. The number of cardiomyocytes in neonatal rats after 5 days of birth was not significantly changed. The expression of Cyclin D1 and CDK4 in neonatal rat cardiomyocytes was higher than that in neonatal rat hearts within 5 days of birth, and the expression level of co-cultured cardiomyocytes was higher than that of cultured cardiomyocytes. GATA4, Nkx2.5 and FGF1 expression increased with age. Conclusion The in vitro co-culture system of neonatal mouse cardiomyocytes and fibroblasts has been established successfully. The neonatal rat cardiomyocytes cultured in vitro for 5 days have a strong proliferative capacity and fibroblasts can promote the proliferation of cardiomyocytes. GATA4 , Nkx2.5 and FGF1 prevent cardiomyocyte proliferation. After 5 days of birth, the proliferative ability of neonatal cardiomyocytes attenuated or even disappeared.