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目的 探讨以抗Ⅰ型粘蛋白单链抗体 (anti MUC1)为靶向的慢病毒高效转染自杀基因后联合更昔洛韦 (GCV)对Ⅰ型粘蛋白阳性 (MUC1+ )卵巢上皮性癌细胞的靶向性基因治疗。方法 包装以anti MUC1为靶向的慢病毒 ,介导单纯疱疹病毒胸苷激酶 (HSV tk)基因转染卵巢上皮性癌细胞株SKOV3(MUC1+ )及正常成纤维细胞GF(MUC1-) ,荧光显微镜下观察转染效果。聚合酶链反应 (PCR)、逆转录聚合酶链反应 (RT PCR)技术鉴定HSV tk基因在靶细胞 (SKOV3及GF)中的整合转录结果。光学显微镜观察GCV作用后细胞形态变化 ;用四甲基偶氮唑蓝 (MTT)法测定GCV的体外杀伤效应。结果 荧光显微镜下观察到以anti MUC1为靶向的慢病毒可有效转染SKOV3细胞 ,PCR、RT PCR均证明HSV tk基因已在靶细胞SKOV3中整合且转录表达 ;而在GF细胞中未见HSV tk基因的整合及转录表达。光学显微镜下观察到HSV tk基因转录后的细胞在GCV作用后出现明显形态变化 ;MTT法显示GCV对转染HSV tk基因后的SKOV3细胞有显著杀伤作用 ,其半数抑制浓度为 0 10mg/L。结论 以anti MUC1为靶向的慢病毒可将HSV tk基因高效、靶向性转染MUC1+ 卵巢上皮性癌细胞 ,联合应用GCV可高效、靶向性杀伤MUC1+ 卵巢上皮性癌细胞
Objective To investigate the effect of ganciclovir (GCV) on the expression of type Ⅰ mucin positive (MUC1 +) epithelial ovarian cancer cells after efficient transfection of lentivirus with lentivirus targeting anti-type Ⅰ mucin scFv (anti MUC1) Targeted gene therapy. Methods Lentivirus targeting MUC1 was transfected into ovarian epithelial carcinoma cell line SKOV3 (MUC1 +) and normal fibroblasts (MUC1-) mediated by herpes simplex virus thymidine kinase (HSV tk) gene. Fluorescence microscopy Under the observation of transfection. Polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT PCR) were used to identify the integrated transcriptional activity of HSV tk gene in target cells (SKOV3 and GF). The morphological changes of cells were observed under optical microscope after GCV treatment. The killing effect of GCV in vitro was determined by MTT assay. Results The lentivirus targeting anti MUC1 was successfully transfected into SKOV3 cells under fluorescence microscope. Both HSV tk gene and HSV tk gene were confirmed by PCR and RT PCR. HSV tk gene was integrated and transcribed in target cell SKOV3. No HSV was found in GF cells tk gene integration and transcriptional expression. After transfection of HSV tk gene under optical microscope, obvious morphological changes were found after the cells were treated with GCV. MTT assay showed that GCV had a significant killing effect on SKOV3 cells transfected with HSV tk gene, with a median inhibitory concentration of 10 mg / L. Conclusion The lentivirus targeting anti MUC1 can transfect the HSV tk gene into MUC1 + epithelial ovarian cancer cells efficiently and in combination with GCV, which can kill MUC1 + epithelial ovarian cancer cells