上调hMLH1基因表达对卵巢癌药物敏感性的影响

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—hMLHl，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-hMLHl中的目的基因hMLHl定向克隆到真核表达载体pCAN，经酶切及测序鉴定：分别将pCAN—hMLHl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Westemblo凇测转染前后细胞内hMLHlmRNA和蛋白的表达变4Jc；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—hMLHl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Westernblot检测到耐药细胞内hMLHl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．hMLHl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。

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

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.