顺铂作用于人恶性睾丸生殖细胞瘤NT2细胞后对肿瘤干细胞标志物的影响

The Effects of Cisplatin on Cancer Stem Cell Markers in Human Malignant Testicular Teratoma NT2 Cells

为了探讨顺铂(cisplatin, CDDP)对人恶性睾丸生殖细胞瘤NT2细胞中干细胞标志物的影响,用20μmol/L CDDP作用于处于对数生长期的NT2细胞12 h、24 h、48 h,采用免疫荧光、实时定量PCR和Western-blot法检测NT2细胞中干细胞标志物Sox-2、Oct-4、Nanog的细胞定位以及mRNA和蛋白质的表达情况,同时对肿瘤干细胞耐药相关基因ABCG2的表达进行实时定量PCR检测。与对照组相比, CDDP处理组细胞变圆且漂浮细胞数量增多,存留活细胞数量随药物作用时长增加而减少;免疫荧光显示Sox-2、Oct-4、Nanog主要定位在细胞质中;实时定量PCR和Western-blot分析结果都显示Sox-2、Oct-4、Nanog在CDDP处理组中的表达较对照组上调;同时ABCG2 mRNA的相对表达量在CDDP处理组显著增加(P<0.05)。研究结果初步表明,顺铂导致人恶性睾丸生殖细胞瘤NT2细胞的肿瘤干细胞耐药机制加强。

To explore the effect of cisplatin （CDDP） on the expression of tumor stem cell markers in human malignant testicular germ cell line （NT2 cells）, 20 μmol/L CDDP was added to NT2 cells in the logarithmic growth phase for different times （12 h, 24 h, 48 h）. The cell localization, and changes in mRNA and protein expression of stem cell markers Sox-2, Oct-4 and Nanog were detected using immunofluorescence, real-time PCR and Western-blot. The molecular marker ABCG2 related to tumor stem cell resistance was detected by realtime PCR. Micrographs showed that the cells in the cisplatin group became rounder and more floating compared with the control group, and that the number of viable cells decreased with the increase of the drug concentration. Immunofluorescence showed that Sox-2, Oct-4 and Nanog were mainly located in the cytoplasm. The results of both real-time PCR and Western-blot showed that the expressions of three stem cell markers （Sox-2, Oct-4 and Nanog） in the cisplatin group were increased, compared with those in the control group. The relative expression of A BCG2 mRNA was statistically significantly higher （P〈0.05） in cisplatin group than that in the control group. The results showed that drug resistance of cancer stem cells in human malignant testicular teratoma NT2 cells was enhanced by cisplatin.