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目的研究PARP-1基因在卵巢癌化疗耐药发生机制中的作用,探讨以该基因为靶向的小干扰RNA(siR-NA)能否有效逆转C13*细胞对顺铂(CDDP)的耐药性。方法采用实时荧光定量PCR(RFQ-PCR)和蛋白印迹法(Western blotting)检测C13*、OV2008细胞中PARP-1 mRNA和蛋白表达差异;转染PARP1-siRNA后,通过RFQ-PCR和Western法验证干扰效果;应用四甲基偶氮唑蓝(MTT)法观察转染后细胞对CDDP敏感性变化及对细胞增殖活性的影响。结果 C13*细胞中PARP-1 mRNA和蛋白的表达量分别是OV2008细胞表达量的(2.12±0.97)和(1.89±0.23)倍;特异性PARP1-siRNA能有效降低C13*细胞中PARP-1 mRNA、蛋白水平及其对顺铂的半数抑制浓度(IC50),分别下降(71.23±5.41)%、(73.84±3.78)%和(89.77±10.06)%(P<0.05),且C13*细胞的生长活力明显下降(P<0.05)。结论 PARP-1蛋白在卵巢上皮性癌细胞中的表达与顺铂敏感性相关;靶向PARP-1基因的siRNA可在转录和翻译水平抑制PARP-1基因表达,并能有效逆转C13*细胞对CDDP的耐药性。
Objective To investigate the role of PARP-1 gene in the mechanism of chemoresistance in ovarian cancer and to explore whether siRNA targeting siR-NA can effectively reverse the drug resistance of cisplatin (CDDP) in C13 * cells Sex. Methods The mRNA and protein expressions of PARP-1 in C13 * and OV2008 cells were detected by real-time quantitative PCR (RFQ-PCR) and Western blotting. PARP1-siRNA was transfected into PARP1-siRNA and confirmed by RFQ- Interfering effect was observed. MTT assay was used to observe the changes of sensitivity to CDDP and the effect on cell proliferation after transfection. Results The expression of PARP-1 mRNA and protein in C13 * cells was (2.12 ± 0.97) and (1.89 ± 0.23) times higher than that in OV2008 cells respectively. The specific PARP1-siRNA could effectively decrease the PARP-1 mRNA (71.23 ± 5.41)%, (73.84 ± 3.78)% and (89.77 ± 10.06)% (P <0.05), respectively, and the half-maximal inhibitory concentration (IC50) Vitality decreased significantly (P <0.05). Conclusions The expression of PARP-1 protein in epithelial ovarian cancer cells is related to cisplatin sensitivity. SiRNA targeting PARP-1 gene can inhibit the expression of PARP-1 gene at the transcriptional and translational level and effectively reverse C13 * cell pair CDDP resistance.