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目的探讨错配修复基因hMLH1对人卵巢癌耐药细胞株SKOV3/DDP顺铂耐药的逆转作用及其机制。方法分别用重组质粒pcDNA3.1-hMLH1和空质粒pcDNA3.1转染SKOV3/DDP细胞。RT-PCR和Western blot检测hMLH1的表达。MTT检测转染前后SKOV3/DDP细胞对顺铂敏感性的变化。经顺铂DDP作用后,流式细胞术和Westernblot法检测细胞凋亡情况。结果与对照组相比,瞬时转染24h后,SKOV3/DDP细胞中hMLH1 mRNA和蛋白的表达水平均显著升高(P<0.05)。MTT结果显示,转染细胞的IC50值(13.95±2.45)较未转染的细胞(94.16±5.18)明显减小。流式细胞仪和Westernblot检测结果表明,顺铂作用24h后,转染了重组质粒的耐药细胞的凋亡率提高到(33.33±2.31)%,同时凋亡抑制蛋白Bcl-2的表达受到抑制(1.35±0.39),与对照组相比,差异均有统计学意义(P<0.05)。结论 hMLH1基因能增强卵巢癌耐药细胞对顺铂的敏感性,其机制可能与降低该耐药细胞内Bcl-2的表达水平,促进细胞凋亡有关。
Objective To investigate the reversal effect of mismatch repair gene hMLH1 on cisplatin-resistant human ovarian cancer cell line SKOV3 / DDP and its mechanism. Methods SKOV3 / DDP cells were transfected with recombinant plasmid pcDNA3.1-hMLH1 and empty plasmid pcDNA3.1 respectively. The expression of hMLH1 was detected by RT-PCR and Western blot. Sensitivity of cisplatin to SKOV3 / DDP cells before and after transfection by MTT assay. After cisplatin DDP effect, apoptosis was detected by flow cytometry and Western blot. Results Compared with the control group, the expression of hMLH1 mRNA and protein in SKOV3 / DDP cells was significantly increased after transfection 24h (P <0.05). The results of MTT showed that the IC50 of transfected cells (13.95 ± 2.45) was significantly lower than that of untransfected cells (94.16 ± 5.18). The result of flow cytometry and Western blot showed that the apoptosis rate of drug-resistant cells transfected with recombinant plasmids increased to (33.33 ± 2.31)% and the expression of Bcl-2 was inhibited (1.35 ± 0.39), respectively, which were significantly different from the control group (P <0.05). Conclusion hMLH1 gene can enhance the sensitivity of cisplatin-resistant ovarian cancer cells, and its mechanism may be related to the decrease of Bcl-2 expression and the promotion of cell apoptosis.