逆转录病毒介导Fcy::Fur融合基因联合5-FC治疗胶质瘤的体内试验研究

Anticancer effects of fusion gene Fcy::Fur/5-fluorocytosine therapies to glioma in vivo

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 evaluate the killing effects of Fcy： ：Fur/5-FC suicide gene therapy system on the growth of rat glioma xenograft in nude mice. Methods Fcy： ： Fur gene was cloned and Fcy： ：Fur expression retroviral vector was constructed, the vector was transferred into packag- ing cell line PT67 and high-titer virus was abtained. Then the tumorigenic glioma cell line C6 was infected, selected and evaluated. Then nude mice were implanted subcutaneously with approximate 0.1 mL gene transfected cells C6 at concentration （1 × 10^6/20 μL） compared with implanted non- trasfected cells C6. The tumors grew with diameter of approximate 3 mm, These animals were administered Fcy：： Fur at doses of 400 mg/kg daily by intratumoral injection from day 1 to day 7. meanwhile normal saline was used in control group with the same volume, then tumor volumes were measured in following days, and Finally, the killing efficacy of 5-FC on gene-transfered tumors ob- served by electron-microscope and FCM assays. Results A Fcy： ：Fur expression retroviral vector PLXSN- Fcy： ： Fur was constructed. Transfering it into packaging cell line PT67, high-titer （3.5 × 10^6 CFU/mL） virus was obtained. A gene-transfered cell clone, C6-Fcy： ：Fur, was selected, and Fcy ： ： Fur gene mRNA expression was detected in vivo. Compared with control group , the growth of tumors in test group was significantly suppressed. The survival days of nude mice in test group were prolonged distinctly. Electron microscope examination showed that there were apoptotic bodies in gene-transfered C6 cells control group and the percentage of apoptosis calculated by the flow cytometry was much higher than that of the contrast. Conclusion This research has shown the significant effects of Fcy： ：Fur/5-FC on glioma in nude mice mode.