原代胃癌相关成纤维细胞的分离培养与鉴定方法比较

Comparison of methods for isolation,culture and identification of primary gastric cancer-associated fibroblasts

目的 通过优化人原代胃癌相关成纤维细胞的分离培养方法来提高分离培养细胞的纯度和效率,以期为胃癌的治疗建立相关模型。方法 采用胰酶消化法和组织块消化法相结合,二次筛网过滤、差速贴壁法分离培养人原代胃癌相关成纤维细胞,观察胃癌相关成纤维细胞的形态和生长特点。HE染色和免疫组化鉴别胃癌相关成纤维细胞的表面标志物。结果 0.25%胰酶消化3~4h后,经过筛网过滤,可以得到较少的胃癌相关成纤维细胞,过滤后的组织块继续在培养瓶中培养可以得到较多的胃癌相关成纤维细胞。胰酶消化法得到的细胞在20d后可以铺满瓶底,组织块培养法得到的细胞可以在14d铺满瓶底。结论 胃癌相关成纤维细胞在第5代后的生长速度开始缓慢,并且随着传代次数的增多,细胞的形态发生相应的改变,细胞触角增多,胞体变短。所获得的细胞ɑ-平滑肌肌动蛋白和波形蛋白都是呈阳性表达,角蛋白CK-18呈阴性表达。

Objective To improve the purity and efficiency of isolating and culturing cells by optimizing the method for isolation and culture of cancer-associated fibroblasts in human primary gastric carcinoma,so as to establish a model for the treatment of gastric cancer.Methods The trypsin digestion and the tissue block digestion methods were combined in this study.Secondary screen filtration and differential adhesion method were employed to isolate and culture gastric cancer-associated fibroblasts.The morphology and growth characteristics of gastric cancer-associated fibroblasts were observed.HE staining and immunohistochemistry were used to identify the surface markers of gastric cancer-associated fibroblasts.Results After 3~4 hours of digestion with 0.25%trypsin,less gastric cancer-associated fibroblasts were obtained after sieve filtration.More gastric cancer-associated fibroblasts were obtained by further culture of the filtered tissue block in the culture flask.The cells obtained by trypsin digestion could fully cover the bottom of the flask after 20 days,and the cells obtained by tissue block culture could fully cover the bottom of the flask after 14 days.Conclusion The growth rate of gastric cancer-associated fibroblasts began to slow down after the 5th generation.And with the increase of passage times,the morphology of gastric cancer-associated fibroblasts changed correspondingly.Accordingly,the cell tentacles increased and the cell body became shorter.The obtained cellsɑ-smooth muscle actin and vimentin were positively expressed,while keratin CK-18 was negatively expressed.