应用改良的组织块酶消化法体外培养人大肠癌原代细胞

Application of improved tissue piece enzyme digestion method in primary culture of human colorectal cancer cells in vitro

目的探讨应用改良的组织块酶消化法体外培养人大肠癌原代细胞的方法.方法应用结合组织块法和Ⅳ胶原酶消化法的改良的组织块酶消化法体外原代培养人大肠癌细胞,通过对培养条件、促进贴壁、控制污染、细胞纯化等方面的探索得到了大肠癌细胞,并最终用形态学方法(瑞姬氏染色)、和免疫学方法(免疫细胞化学)鉴定所得细胞.结果经改良的组织块酶消化法体外培养的人大肠癌原代细胞,瑞姬氏染色进行细胞形态学鉴定,结果显示胞核呈紫红色,核质比明显增大,符合肿瘤细胞特征;细胞胃肠癌相关糖链抗原19-9(carbohydrate antigen 19-9,C A19-9)进行细胞免疫学鉴定,结果显示胞浆呈棕色,细胞CA19-9阳性.结论改良的组织块酶消化法在培养方法改良、促进贴壁、控制污染和去除杂细胞四方面进行了改进,既可快速获得游离细胞,又充分利用了未彻底消化的组织块.采用该改良的组织块酶消化法原代培养细胞成功率高,所培养的细胞进行初步鉴定证明为大肠癌细胞.

AIM To investigate methods of primary culture of human colorectal cancer cells in vitro by application of improved tissue piece enzyme digestion method.METHODS Human colorectal cancer cells were cultured by improved tissue piece enzyme digestion method, which combines the tissue explant method with IV collagenase digestion method. Colorectal cancer ceils were obtained by optimizing culture conditions, promoting adherence, controlling pollution, and purifying the cells. Wright Giemsa staining and immunocytochemistry were used to identify the cells. RESULTS Wright Giemsa staining of the cells showed karyomegaly and purple red-stained colorectal cancer cells. Immunocytochemistry staining showed that the cells were carbohydrate antigen 19-9 （CA19-9） positive and the cytoplasm was brown.CONCLUSION We have improved the tissue piece enzyme digestion method, which allows to obtain free cells quickly and make full use of the tissue pieces which have not been digested completely. The improved tissue piece enzyme digestion method has a high success rate, and cultured cells have been identified as colorectal cancer cells.