用极量稀释法从HeLa细胞中筛选多药耐药的亚克隆细胞株

目的探讨肿瘤细胞亚克隆筛选在多药耐药研究中的应用.方法运用极量稀释法从亲代人宫颈癌HeLa细胞中筛选单个细胞的亚克隆细胞株,并采用Western Blot和MTT法检测亲代HeLa细胞株和亚克隆细胞多药耐药基因1（mdr^-1）的基因产物P-糖蛋白（P-gp）的表达及细胞的多药耐药性.结果共筛选了10个亚克隆细胞株,其中4个亚克隆细胞株有P-gp的表达,而有6个亚克隆株没有P-gp的表达.表达P-gp的克隆细胞株和亲代HeLa细胞株对柔红霉素、环磷酰胺及长春新碱具有多药耐药性,其IC50明显高于不表达P-gp的细胞株（P＜0.05）.结论HeLa细胞株有原发性耐药倾向,而且P-gp表达上具有异质性,亚克隆细胞的筛选既可以用于原发性耐药的研究,也可以筛选出细胞生物学特性一致的敏感细胞株进行继发性耐药细胞模型的建立及相关的研究.

The subcloned cell line of multidrug resistance derived from parental HeLa cell using the method of maximum dose dilution

Objective To investigate the application of screening subcloned cell line from parental tumor cells in study on multidrug resistance. Methods The subcloned cell line derived from single cell clone of parental HeLa cells was screened using the method of maximum dose dilution. Western Blotting and MTT assay were used to test the expression of the multidrug resistance gene, cytoplasm membrane P-glycoprotein(P-gp) , and the activity of the multidrug resistance. Results In 10 subcloned cell lines, the expression of P-gp was identified only in four of them, and loss of P-gp expression in the other six. The multidrug resistance to daunorubicin, cyclophosphamide and vinblastine was emerged in both the cloned cell lines and the parental HeLa cell lines, which expressed P-gp, and the IC50 of them were obviously higher than those of P-gp rare or absent cell lines( F <0. 05). Conclusion HeLa cell line was shown to have the trend of primary drug-resistance, and it also appeared that the expression of cytoplasm membrane P-gp takes the forms of heterogeneity. The screening subcloned cell line further extends the application not only to investigate the primary multidrug resistance,but also to screen the sensitive cell lines, which have the coherent bionomics to construct some models for study secondary multidrug resistance and do some, related researches.