作　　者： (陈凤英）; (吴雄灏）; (王念）; (梁恒瑞）; (陈俊邦）; (叶润杰）; (谢梓泓）; (杨伊霖）; (赵磊）;

机构地区： 511436,广州医科大学基础学院生理教研室

出　　处： 《中华生物医学工程杂志》

摘　　要： 目的 比较两种酶消化法原代培养小鼠气道平滑肌细胞(ASMC)的生长情况.方法取SPF级雄性昆明小鼠10只,处死后分离气管组织,分别采用Ⅰ型胶原酶消化法(单酶消化法)和链霉蛋白酶+Ⅰ型胶原酶消化法(双酶消化法)原代培养ASMC.倒置显微镜下观察细胞形态,免疫荧光鉴定细胞类型.结果单酶消化法培养2~3 d可见少量细胞贴壁伸展,15 d左右可进行传代.双酶消化法培养2~3d可见较多细胞贴壁伸展,7~9d可进行传代.免疫荧光检测显示,胞质中α-平滑肌肌动蛋白(α-SMA)呈红色阳性反应,两种方法所培养的细胞95%以上为ASMC.结论两种方法均可获得高纯度的ASMC,在细胞质量方面无明显差异.单酶消化法操作简便,但细胞培养周期较长,双酶消化法细胞生长较快但操作相对复杂. Objective To compare the growth of mice airway smooth muscle cells (ASMCs) cultivated by two enzymatic dispersion methods. Methods Ten healthy male SPF-grade Kunming mice were sacrificed and the tracheal tissue was separated. ASMCs were cultured from primary generation using enzymatic dispersion with collagenaseⅠ(single enzymatic dispersion) or pronase plus collagenaseⅠ(double enzymatic dispersion),respectively. The morphology of the obtained ASMCs was observed under the invert microscope,and immunofluorescence was used to identify the cell types. Results A few cells adhesion and spreading could be observed within 2 to 3 days by single enzymatic dispersion method,and be subcultured after about 15 days. More cells adhesion and spreading could be observed within 2 to 3 days by double enzymatic dispersion,and be subcultured around 7 to 9 days. The immunofluorescence showed that red positive reaction of α-smooth muscle actin in the cytoplasm,and over 95% of cultured cells were identified to be the ASMCs. Conclusion Highly purified ASMCs were obtained through both methods,and there was no significant difference in quality of cultivated cells between the two methods. Single enzymatic dispersion was simple to use but with longer culture cycle;in contrast,the double enzymatic dispersion resulted in quicker cell growth but required more complicated manipulation.

分 类 号： [Z1]