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目的 探讨切割修复交叉互补基因 2 (excisionrepaircross complementingrodentrepairdeficiencygene 2 ,ERCC2 )在人脑胶质瘤耐药中的作用。方法 采用来自p2E ER2的 2 4kbEcoRⅠ片段和pcDNA3载体组建成pcDNA3ERCC2质粒。再用脂质体转染技术 ,将pcDNA3ERCC2质粒转入SKMG 4细胞。转染ERCC2的细胞用G41 8连续筛选 2周。再从存活的细胞集落挑取单细胞进行克隆。克隆细胞进一步增殖并经RT PCR和Westernblot证实为稳定转染细胞用于下一步试验。转染不带ERCC2的pcDNA3载体作为对照。然后 ,采用改良的SulforhodamineB(SRB)抗癌药筛选法 ,对稳定转染和亲代SKMG 4细胞进行细胞毒试验。结果 细胞毒性试验结果显示转染ERCC2的SKMG 4胶质瘤细胞对顺铂和麻法林的耐药性明显增加。顺铂的IC90 ( μmol/L)在亲代SKMG 4、转染pcDNA3载体的对照SKMG 4和转染ERCC2的SKMG 4细胞分别为 1 1 75、1和 1 75 ;麻法林的IC90 ( μmol/L)分别为 5、5 6和 9。转染ERCC2的SKMG 4细胞与转染空载体的对照细胞相比较 ,顺铂和麻法林的IC90 分别增加了 75 %和 61 %。结论 转染ERCC2基因的胶质瘤细胞对顺铂和麻法林的耐药性明显增加 ,证明ERCC2在烷化剂类抗癌药物耐药中起重要作用。
Objective To investigate the role of excision repair complementing human depairdeficiency gene 2 (ERCC2) in human glioma drug resistance. Methods The pcDNA3ERCC2 plasmid was constructed by using the 2 4kb EcoRI fragment from p2E ER2 and pcDNA3 vector. The plasmid pcDNA3ERCC2 was transfected into SKMG 4 cells by lipofectamine. Cells transfected with ERCC2 were screened continuously with G41 8 for 2 weeks. Single colonies from surviving colonies were picked for cloning. Clone cells were further propagated and confirmed by RT PCR and Westernblot as stably transfected cells for use in the next assay. The pcDNA3 vector without ERCC2 was transfected as a control. Then, the stably transfected and parental SKMG4 cells were cytotoxic tested using a modified SulforhodamineB (SRB) anti-cancer drug screening method. Results Cytotoxicity test results showed that SKMG 4 glioma cells transfected with ERCC2 significantly increased resistance to cisplatin and terfaulin. IC50 (μmol / L) of cisplatin was 11 75,1 and 1 75 in parental SKMG 4, control SKMG 4 transfected with pcDNA3 vector, and SKMG 4 cells transfected with ERCC2, respectively; IC90 (μmol / L) ) Are 5, 5, 6 and 9, respectively. The IC90 of cisplatin and melphalin increased by 75% and 61%, respectively, in SKCC4 cells transfected with ERCC2 compared to control cells transfected with empty vector. Conclusion The ERCC2 gene transfected glioma cells significantly increased the resistance to cisplatin and mafarin, indicating that ERCC2 plays an important role in the chemoresistance of alkylating agents.