成年小鼠心脏成纤维细胞的分离、纯化和原代培养  

Isolation,purification and primary culture of adult mouse cardiac fibroblasts

作　　者：李如君[1] 龚开政[1] 张振刚[1] 

机构地区：[1]扬州大学第二临床医学院心血管内科,江苏扬州225012

出　　处：《细胞与分子免疫学杂志》2017年第1期67-71,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81270197)

摘　　要：目的探讨并建立成年小鼠心脏成纤维细胞原代培养方法。方法采用0.8 g/LⅣ型胶原蛋白酶和1 g/L胰蛋白酶为消化液,以多次短时间水浴振荡的方式消化成年小鼠心脏组织,差速贴壁法分离纯化心脏成纤维细胞。波形蛋白免疫荧光细胞化学染色法鉴定细胞类型及纯度;倒置相差显微镜观察细胞生长情况,绘制细胞生长曲线;CCK-8法测定细胞增殖能力、Western blot法检测转化生长因子β1(TGF-β1)刺激后细胞SMAD家族成员2/3(Smad2/3)蛋白及磷酸化水平。结果差速贴壁后在相差显微镜下可见大量球形贴壁细胞;3 d后细胞逐渐由圆形伸展为长梭形并迅速增殖,分裂象多见;5 d后细胞数量显著增多,完全汇合无空隙;细胞生长曲线近似"S"形。波形蛋白阳性率可达95%。CCK-8法显示在第3~5天细胞增殖能力最显著,给予第2代心脏成纤维细胞TGF-β1刺激显示Smad2/3蛋白磷酸化水平明显增加。结论建立了一种快速、经济、有效获取成年小鼠心脏成纤维细胞的方法。Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0. 8 g/L collagenase Ⅳ by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion,adherent fibroblasts formed spherical cell mass and after 3 days,cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly＂S＂shape. The positive expression rate of vimentin was95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.

关 键 词：成年小鼠 心脏成纤维细胞 原代培养 细胞鉴定 

分 类 号：R542.2[医药卫生—心血管疾病]