慢病毒转染自杀基因后联合更昔洛韦治疗卵巢上皮性癌的实验研究

Targeted killing effect of mucin-1 single chain antibody targeting, lentivirus-mediated suicide gene therapy and ganciclovir in mucin-1^+ ovarian epithelial carcinoma cells

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+

Objective To investigate the targeted killing effect of mucin 1 single chain antibody targeting, lentivirus mediated suicide gene therapy and ganciclovir(GCV) in mucin 1 + ovarian epithelial carcinoma cells Methods Mucin 1 single chain antibody targeting lentivirus produced by packaging cell line 293T transduced herpes simplex virus thymidine kinase (HSV tk) gene into the ovarian epithelial cancer cell line SKOV3 (MUC1 +) The infection effect was observed through fluorescence microscopy Polymerase chain reaction (PCR) and reverse transcription PCR (RT PCR) were resorted to demonstrate the successful transduction and transcription of the HSV tk gene After administration of GCV, changes of those cells were observed through optical microscopy The cytotoxicity efficacy of HSV tk/GCV system was evaluated by methyl thiazolyl tetrazolium method Results It was observed through fluorescence microscopy that anti MUC1 directed lentivirus can specificly infect MUC1 + ovarian cancer cells A fragment of 600 bp was generated through PCR and RT PCR which indicated successful transduction and transcription of the HSV tk gene in SKOV3 cells Changes of cells followed by administration of GCV were observed with optical microscopy Significant cytotoxicity efficacy of GCV to SKOV3 was observed Conclusions The HSV tk gene can be targetedly transducted into MUC1 + ovarian cancer cell line under the mediation of anti MUC1 directed lentivirus, and such HSV tk/GCV system has targetingly killing effect on MUC1 + cancer cells