猪心脏瓣膜间质细胞培养方法的改进

Improvement of culture method of porcine heart valve interstitial cells

目的探讨猪原代心脏瓣膜间质细胞分离培养的简便方法,并进行表型鉴定。方法取10头实验猪心脏,获取主动脉瓣,实验组采用改进后的Ⅱ型胶原酶一段15 min消化法分离培养间质细胞,对照组采用传统Ⅱ型胶原酶两段90min消化法分离培养间质细胞。观察同一时间2组间质细胞形态学差异,免疫细胞化学染色法分析间质细胞表型,CCK-8法检测培养1、3、5、7d吸光度值并绘制细胞生长曲线。结果实验组和对照组分离出的原代瓣膜间质细胞生长方式及形态特点无明显差异;细胞表型鉴定显示2组α-平滑肌肌动蛋白及波形蛋白均阳性;CCK-8检测结果及细胞生长曲线显示,实验组和对照组间质细胞生长趋势基本一致,1～3d为快速增长期,3～5d为平台期,5～7d为缓慢增长期。结论改进后的原代心脏瓣膜间质细胞分离培养方法操作时间短,为离体条件下行心脏瓣膜病的研究以及体外细胞模型的建立提供了可靠、快速的实验基础。

Objective To explore a simple and convenient method to culture valvular interstitial cells and identify the phenotypes.Methods The hearts from 10 experimental pigs were obtained to take the aortic valve.The interstitial cells were isolated and cultured with the modified type Ⅱ collagenase for 15 min digestion in experimental group and were cultured with conventional typeⅡcollagenase for 90 min digestion in control group.The morphological differences of two groups were observed at the same time.The phenotype of interstitial cells was analyzed by immunocytochemical staining,and the cell growth curve was drawn by CCK-8 method.Results There were no significant differences in the growth pattern and morphological characteristics of primary stromal cells between two groups.Cell phenotypic identification showed positiveα-smooth muscle actin and vimentin in two groups.The CCK-8 detection results showed that the growth trends of cells were basically the same in two groups,with the rapid growing phase from 1 to 3 d,platform phase from 3 to 5 d,and slow growing phase from 5 to 7 d.Conclusion The modified primary heart valve interstitial cell isolation and culture method has a short operation lasting time,which provides a reliable and rapid experimental basis for the study of valvular heart disease in vitro and the establishment of an in vitro cell model.