新生大鼠心肌细胞体外培养及其与心肌成纤维细胞形态比较

Cardiac myocyte of neonatal rats cultured in vitro and compared with cardiac fibroblast on morphology

目的探索一种快速分离和培养新生大鼠心肌细胞的方法,比较心肌细胞和成纤维细胞的形态差异。方法胰蛋白酶消化法分离新生大鼠心肌细胞进行体外培养,差速贴壁法纯化心肌细胞,倒置显微镜跟踪观察心肌细胞的生长及搏动情况,台盼兰负染法检测心肌细胞活力,通过HE染色观察心肌细胞和成纤维细胞的形态学差异。结果接种24 h后细胞全部贴壁,部分细胞开始自发性搏动,48 h后,细胞体积增大,并伸出伪足,形状以三角形和梭形为主,3 d后细胞聚集成片并同步搏动且细胞活力均在90%以上。心肌细胞为梭形或三角形,胞质致密,多为单核细胞;心肌成纤维细胞呈泡状,双核和三核细胞较多,培养过程中不发生搏动。结论胰蛋白酶消化法培养新生大鼠心肌细胞快速有效,差速贴壁法纯化的心肌细胞可用于实验研究。

Objective To introduce a rapid method of isolateing and culturing cardiac myocyte of neonatal rats and to compare the difference with cardiac fibroblast.Methods Cardiac myocytes were isolated with enzyme digestion and purified by means of differential attachment technique.Morphological changes and beating condition of cardiac myocytes were observed dynamically with inverted microscope.The vitality of cardiac myocytes was evaluated with trypan blue negative staining.The difference between cardiac myocyte and cardiac fibroblast was differentiated by HE staining.Results 24 h after inoculating all cardiac myocytes had attached and partly of them began to beat spontaneously.48 h after inoculating,volume of cardiac myocytes enlarged and pseudopodia stretched out,the main shape were triangle or clostridial form.Cardiac myocytes got together and beat synchronously in clusters after 3 days.The ratio of survival cells was above 90%.Most cardiac myocytes were clostridial form or triangle and had single nucleu.Cardiac fibroblasts were circle shaped and had two or three nucleus,and they could not beat during culturing.Conclusion The method of isolating and culturing cardiac myocyte of neonatal rats is a rapid and effective method and purified cardiac myocytes by means of differential attachment technique can be used for experiment.