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目的构建含融合自杀基因Fcy::Fur重组逆转录病毒,用自杀基因治疗系统Fcy::Fur/5-氟胞嘧啶(5-FC)对裸鼠胶质瘤进行体内抑瘤作用的实验研究。方法扩增Fcy::Fur基因并构建Fcy::Fur基因重组逆转录病毒载体;载体转染包装细胞获得高滴度病毒并转染鼠胶质瘤细胞C6,筛选并鉴定阳性转基因克隆;构建裸鼠荷胶质瘤动物模型,腹腔注射5-FC,观察裸鼠肿瘤重量变化及电镜、流式细胞仪(FCM)检测肿瘤的凋亡。结果PCR法扩增出全长Fcy::Fur基因,经测序证实序列正确;构建了PLXSN-Fcy::Fur逆转录病毒载体,载体转染包装细胞PT67,获得滴度为3.5×106CFU/mL的逆转录病毒;转染C6获转基因阳性克隆C6-Fcy::Fur,检测C6-Fcy::Fur有Fcy::Fur基因的mRNA表达;裸鼠前肢背部接种转基因细胞,成瘤后腹腔注射5-FC,转基因肿瘤的生长较对照组明显抑制。FCM法检测到凋亡峰,电镜观察到转基因肿瘤细胞有凋亡小体。结论AdE1CMVCD与5-FC联合在体内对胶质瘤有明显的抑制作用,为临床胶质瘤基因治疗提供了可靠的理论及应用依据。
OBJECTIVE: To construct a recombinant retrovirus containing Fcy :: Fur fusion gene and to study the antitumor effect of Fcy :: Fur / 5-fluorocytosine (5-FC) in nude mice. Methods Fcy :: Fur gene was amplified and Fcy :: Fur gene recombinant retroviral vector was constructed. The vector was transfected into packaging cells to obtain high titer virus and transfected into C6 glioma cells. The positive transgenic clones were screened and identified. The rat model of glioma was induced by intraperitoneal injection of 5-FC. The changes of tumor weight in nude mice were observed. The apoptosis of tumor was detected by electron microscopy and flow cytometry (FCM). Results The full-length Fcy :: Fur gene was amplified by PCR and confirmed by sequencing. The PLXSN-Fcy :: Fur retroviral vector was constructed and the vector was transfected into the packaging cell PT67 to obtain a titer of 3.5 × 106 CFU / mL Retrovirus; transfection positive C6 clone transfected with C6-Fcy :: Fur, C6-Fcy :: Fur Fcy :: Fur gene expression was detected; nude mice were inoculated with transgenic cells back of the forelimb, FC, transgenic tumor growth was significantly inhibited compared with the control group. The apoptotic peak was detected by FCM, and apoptotic bodies were observed by electron microscopy in transgenic tumor cells. Conclusion The combination of AdE1CMVCD and 5-FC significantly inhibits glioma in vivo and provides a reliable theory and application basis for clinical glioma gene therapy.