人肺癌细胞系NCI-H1650中肿瘤干细胞的分离及鉴定

摘要

Objective To isolate and identify the lung cancestem like cellfrom the cell line NCI-H1650 .MethodThe NCI-H1650 cellwere continuously cultured in serum-free medium with human insulin ,recombinanepidermal grow th facto(EGF) ,recombinanbasifibroblasgrowth facto(bFGF) and bovine serum albumin(BSA) ,and paclitaxel waadopted to induce the stem cell.Flow cytometry and immunofluorescenstaining were used to analyze the expression levelof lung cancestem cell markeCD133 and CD326 .The expressionof stem cell related markeOct4 ,Nanog and Sox-2 were detected by RT-Pcand West-ern blo.ResultThe NCI-H1650 cellcontinuously cultured by serum-free medium and induced by combining paclitaxel induction showed the spherical suspension growth .Compared with NCI-H1650 cellcultured by serum ,CD133 and CD326 had significantly high expression and the expression levelof Oct4 ,Nanog and Sox-2 were significantly increased (P＜0 .05) .Conclusion Adopting the serum-free culture in vitro combined with paclitaxel foinducing NCI-H1650 cellcan effectively isolate the stem cellamong them.%目的 在人肺癌细胞系NCI-H1650中分离出肺癌干细胞及鉴定.方法 将NCI-H1650细胞在含人胰岛素、重组人表皮生长因子(EGF)、重组人碱性成纤维细胞生长因子(bFGF)、牛血清清蛋白(BSA)的无血清培养基中连续培养 ,用紫杉醇刺激诱导干细胞 ,通过流式细胞术、免疫荧光的方法分析肺癌干细胞标志物CD133和CD326的表达;采用RT-PCR和Western blot的方法检测干细胞相关标记物Oct4、Nanog和Sox-2的表达.结果 经无血清连续培养结合紫杉醇诱导的NCI-H1650细胞呈球形悬浮生长 ,相比于有血清培养的NCI-H1650细胞 ,其干细胞标志物CD133和CD326高表达 ,干细胞相关标志物Oct4、Nanog和Sox-2表达水平提高(P＜0 .05).结论 采用体外无血清连续培养结合紫杉醇刺激诱导 NCI-H1650细胞能有效分离出其中干细胞.