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目的:探讨ERCC1 Ⅷ外显子剪接变异对卵巢癌细胞顺铂耐药的影响。方法:分别构建缺失Ⅷ外显子和野生型的ERCC1表达载体pD-DE(含突变型ERCC1基因)和pD-WE(野生型ERCC1),脂质体转染法转染A2780细胞,Western blot检测ERCC1表达水平研究该剪接变异对ERCC1基因表达水平的影响。MTT法检测肿瘤细胞的顺铂耐药性,彗星实验检测转染后细胞DNA的损伤修复情况。结果:成功构建重组质粒pD-DE和pD-WE;Western blot结果显示,该剪接变异未显著影响ERCC1表达;转染pD-DE的细胞与转染pD-WE的细胞相比,明显降低了顺铂的耐受性与细胞DNA损伤修复的能力。结论:ERCC1 Ⅷ外显子剪接变异降低卵巢癌细胞DNA损伤修复能力和对顺铂的耐药性。
Objective: To investigate the effect of ERCC1 Ⅷ exon splicing on cisplatin-resistant ovarian cancer cells. Methods: The ERCC1 expression vector pD-DE (containing mutant ERCC1 gene) and pD-WE (wild-type ERCC1) with exon and wild-type deletion were constructed and transfected into A2780 cells by lipofectamine. ERCC1 expression levels of the splicing variation ERCC1 gene expression levels. MTT assay of cisplatin resistance of tumor cells, comet assay DNA damage detection after transfection cells. Results: The recombinant plasmids pD-DE and pD-WE were successfully constructed. The results of Western blotting showed that the splicing variant did not significantly affect the expression of ERCC1. Compared with the cells transfected with pD-DE, the expression of pD-DE significantly decreased Platinum tolerance and cellular DNA damage repair capabilities. Conclusion: Exon splicing variants of ERCC1 Ⅷ reduce DNA damage repair ability and resistance to cisplatin in ovarian cancer cells.