摘要
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 study the reversing effect of hMLH1 gene on cisplatin resistance in ovarian cancer cell line SKOV3/DDP and its underlying mechanism. Methods SKOV3/DDP cells were transfected with recombinant eukaryotic expression plasmid pcDNA3.1-hMLH1 and plasmid pcDNA3.1, respectively, by using lipofectamine 2000. Expression of hMLH1 mRNA and protein was detected by RT-PCR and Western blotting, respectively. Chemosensitivity of SKOV3/DDP cells to cisplatin was assayed by MTT assay. KOV3/DDP cell apoptosis was detected by flow cytometry （FCM） and Western blotting, respectively, after treatment with cisplatin. Results MTT assay showed the expression level of hMLH1 mRNA and protein was significantly higher （P0.05） and the IC50 value（13.95±2.45）for cisplatin was obviously lower in SKOV3/DDP cells 24 h after transient transfection with hMLH1 gene than in controls（94.16±5.18）. FCM and Western blotting showed that the apoptotic rate of SKOV3/DDP cells transfected with the hMLH1 gene was higher （3.33%±2.31%） than that of controls and the expression level of Bcl-2 was lower in SKOV3/DDP cells transfected with the hMLH1 gene （1.35±0.39） than that in controls （P0.05）. Conclusion The hMLH1 gene can increase the chemosensitivity of human ovarian cancer cells to cisplatin, which may be related to the down-regulation of Bcl-2 expression and improvement of cell apoptosis.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2010年第23期2527-2529,共3页
Journal of Third Military Medical University
