上调hMLH1基因表达对卵巢癌药物敏感性的影响

Up-regulation of hMLH1 Gene Expression on Cisplatin Sensitivity of Drug-resistant Ovarian Cancer Cells

构建携带错配修复基因hMLH1编码序列全长的真核表达质粒pCAN-hMLH1,并探讨其对卵巢癌细胞顺铂耐药的逆转作用。应用基因重组技术将pET28-hMLH1中的目的基因hMLH1定向克隆到真核表达载体pCAN,经酶切及测序鉴定;分别将pCAN-hMLH1和空质粒pCAN转染进卵巢癌耐药细胞SKOV3/DDP,同时以对顺铂敏感的SKOV3细胞和未转染的SKOV3/DDP细胞作为对照;应用RT-PCR和Western blot检测转染前后细胞内hMLH1 mRNA和蛋白的表达变化;四甲基偶氮唑蓝(MTT)比色法检测转染前后SKOV3/DDP细胞对顺铂敏感性的变化;Hoechst染色检测转染前后细胞的凋亡。结果提示:pCAN-hMLH1重组质粒经酶切及测序鉴定,表明真核表达质粒构建正确;采用脂质体法转染SKOV3/DDP细胞后,RT-PCR和Western blot检测到耐药细胞内hMLH1的表达增强;MTT结果显示转染重组质粒后SKOV3/DDP细胞对顺铂的敏感性显著增加;Hoechst染色观察到转染后耐药细胞的凋亡明显增强。该研究成功构建了pCAN-hMLH1重组质粒,在SKOV3/DDP细胞中进行表达,并能增强耐药细胞对顺铂的敏感性,促进耐药细胞的凋亡。

We constructed the eukaryotic expression plasmid pCAN-hMLH1 carrying the coding sequence and investigated its reversing effect on cisplatin-resistant ovarian cancer cells. With the recombinant DNA techniques, the hMLH1 gene in pET28-hMLH1 plasmid was subcloned into pCAN vector. The correctness of recombinant plasmid was evaluated by enzyme analysis and nucleotide sequencing. The SKOV3/DDP cells were transfected with the recombinant plasmid pCAN-hMLH1 and pCAN vectors by lipofectamine 2000. The expressions of hMLH1 were detected by RT-PCR and Westem blot. Chemosensitivity of SKOV3/DDP cells to cisplatin was measured by methyl thiazolyl tetrazolium （MTT）. Cell apoptosis was detected by Hoechst dyeing. The mRNA and protein levels of hMLH1 of SKOV3/DDP cells transient transfected with pCAN-hMLH1 were increased significantly. The chemosensitivity to cisplatin was enhanced after the hMLH1 gene being transfected into SKOV3/DDP cells. Hoechst dyeing showed that the apopototic rate of hMLH1 transfected cells was increased. The plasmid pCAN-hM-LH1 has been successfully constructed and expressed in SKOV3/DDP cells effectively, hMLH1 gene can increase the chemosensitivity to cisplatin and improve apoptosis.