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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。
To construct the eukaryotic expression plasmid pCAN-hMLH1 carrying the full-length coding sequence of mismatch repair gene hMLH1 and investigate its reversal effect on cisplatin-resistant ovarian cancer cells. The hMLH1 gene of pET28-hMLH1 was cloned into the eukaryotic expression vector pCAN by gene recombination technology and identified by restriction enzyme digestion and sequencing. PCAN-hMLH1 and empty plasmid pCAN were transfected into SKOV3 / DDP cells, At the same time, cisplatin-sensitive SKOV3 cells and untransfected SKOV3 / DDP cells were used as a control. The expression of hMLH1 mRNA and protein was detected by RT-PCR and Western blot. MTT MTT) colorimetric assay before and after transfection SKOV3 / DDP cells sensitivity to cisplatin; Hoechst staining before and after transfection of cells apoptosis. The results showed that the recombinant plasmid pCAN-hMLH1 was identified by restriction enzyme digestion and sequencing, which indicated that the eukaryotic expression plasmid was constructed correctly. After transfected SKOV3 / DDP cells by liposome, the hMLH1 The results of MTT assay showed that the sensitivity of SKOV3 / DDP cells to cisplatin was significantly increased after transfection of recombinant plasmids. The apoptosis of drug-resistant cells was obviously enhanced by Hoechst staining. This study successfully constructed pCAN-hMLH1 recombinant plasmid, expressed in SKOV3 / DDP cells, and can enhance the resistance of cisplatin-resistant cells and promote apoptosis of drug-resistant cells.