目的: 探讨通过封闭ERCC1基因表达干扰DNA损伤修复路径及其对卵巢癌细胞对铂类药物耐药性的影响。方法: 构建ERCC1基因反义表达载体，转染人卵巢癌细胞A2780及A2780/CP70，采用Northern杂交及Western Blot分析人卵巢癌细胞内ERCC1基因RNA及蛋白表达水平变化；利用荧光素酶报告基因系统观察宿主细胞对DNA损伤的修复作用；采用MTT实验研究细胞对铂类药物耐药性的影响。结果: 卵巢癌细胞转染反义ERCC1基因后，ERCC1基因转录水平、蛋白表达水平明显下降。荧光素酶报告基因系统结果证明，宿主细胞的DNA修复活性下降。MTT实验表明，伴随ERCC1基因表达水平下降，细胞对顺铂的耐药性降低。结论: 转染ERCC1反义基因显著影响宿主细胞的核苷酸切除修复，影响细胞对顺铂的耐药性。

Objective: To study the effect on cytotoxicity and drug resistance by blocking ERCC1 gene expression in ovarian cancer cell lines. Methods: ERCC1 antisense expression vector was constructed and transfected into human ovarian cancer cell lines. Northern blotting and Western blotting were used to detect the RNA and protein expression level in the cells. Luciferase assay system was used to reveal the host cell reactivation function. MTT assay was used to study the effect on cytotoxicity and drug resistance in the cell lines.Results: After transfection of the antisense ERCC1, Northern and Western blotting indicated that the RNA and protein expression level of ERCC1 was obviously decreased. Luciferease assay showed reduced DNA-damage repair capacity as assessed by host cell reactivation. MTT assay also showed decreased drug resistance to cisplatin in the lines. Conclusion: Transfection of antisense ERCC1 may enhance the cisplatin cytotoxicity by disturbing the NER pathway in cisplatin-resistant cell lines.

本课题受美国“The National Center for Research on Minority Health and Health Disparity”资助