慢病毒载体介导的自杀基因系统YCD/5-FC对人急性淋巴细胞白血病Jurkat细胞杀伤效应的研究  

The killing effect of YCD/5-FC suicide gene system mediated by lentiviral vector on the human Jurkat cells

作　　者：侯军[1,2] 陈洁[1] 陈萍萍[1] 冯曹波[1] 周虹[1] 章卫平[1] 王健民[1] 

机构地区：[1]第二军医大学附属长海医院血液科,上海200433 [2]同济大学附属上海第十人民医院血液科,上海200072

出　　处：《中国癌症杂志》2011年第1期30-35,共6页China Oncology

摘　　要：背景与目的:酵母菌胞嘧啶脱氨酶(yeast cytosine deaminase,YCD)可将无毒性的抗真菌药5-氟胞嘧啶(5-fluorocytosine,5-FC)转化为细胞毒性产物氟尿嘧啶(5-fluorouracil,5-FU),进而阻抑DNA、RNA和蛋白质合成,引起细胞死亡。本研究中构建含YCD基因的慢病毒载体质粒,体内外观察YCD/5-FC自杀基因系统的杀伤效应。方法:应用亚克隆技术将YCD和绿色荧光蛋白基因(green fluorescence protein,GFP)连接至慢病毒空载体pFUW,构建获得pGC-FU-YCD-GFP。将3质粒系统(含包装质粒、包膜蛋白质粒和转移质粒)脂质体法共转染包装细胞293T,获得的慢病毒转染人Jurkat细胞,流式细胞仪(FACS)和Western blot检测Jurkat/YCD-GFP表达水平;加入前体药物5-FC,观察对靶细胞的杀伤情况和旁观者效应。转基因细胞接种至裸鼠前肢皮下,观察体内肿瘤杀伤情况。结果:慢病毒载体3质粒系统感染293T细胞后,获得的慢病毒滴度为1.2×108 TU/mL。该病毒可高效转染人Jurkat细胞,感染率达96.93%,Western blot显示转基因细胞可分泌YCD蛋白。100μmol/L 5-FC作用96 h可杀伤(89.37±6.57)%的Jurkat/YCD-GFP细胞。混合培养提示存在明显的旁观者效应。转基因细胞可在小鼠体内致瘤,5-FC可明显抑制肿瘤生长。结论:成功构建慢病毒载体pGC-FU-YCD-GFP,YCD/5-FC自杀基因系统在体内外均有显著的特异性杀伤效应。Background and purpose： Yeast cytosine deaminase （YCD） can change the nonpoisonous drug 5-fluorocytosine （5-FC） into poisonous 5-fluorouracil （5-FU）, which inhibits cells from producing DNA, RNA and proteins, therefore killing the cell The purpose of this study was to construct the plasmid of lentiviral vectors carrying the YCD gene and to investigate the killing effect of the YCD/5-FC suicide gene system on the Jurkat cells in mice with tumors. Methods： The YCD gene and the green fluorescent （GFP） gene were ligated to plasmid pFUW using sub-cloning technology to construct plasmid pGC-FU-YCD-GFE Using LipofectamineTM2000, human kidney 293T cells were co-transfeeted with the three-plasmid system which contains packaging plasmid pHelper 1.0, plasmid pGC- FU-YCD-GFP and envelope plasmid pHelper 2.0. The plasmid particles were collected and then to transferred into the Jurkat cells. The transferring efficacy and expression of YCD protein in Jurkat cells were tested using flow cytometry （FACS） and Western blot, respectively. The killing capacity and bystander effects were measured after added 5-FC into the cultures. The Jurkat/YCD-GFP cells were subsequently injected into the forelimb of node mice and established human leukemia tumors in situ. The tumor bearing mice were then treated with 5-FC. Results： Lentivirus containing the YCD gene was obtained and the titer of the virus was 1.2×10^8 TU/mL. Of the Jurkat cells, 96.93% could be transfered efficiently using virus particles without the need for fluorescence sorting. Western blot showed that transgenic cells could excrete YCD protein. Of the Jurkat/YCD-GFP cells, （89.37±6.57）% cells were killed by 5-FC in 100 μmol/L at the end of 96 h. The bystander effect appeared in a mixed culture oftransgene cells and Jurkat cells. YCD/5-FC could also in vivo efficiently kill the Jurkat cells that were carrying the YCD gene. Conclusion： The lentiviral vector pGC- FU-YCD-GFP was successfully constructed. Besides, the lentiviral vector-mediated Y

关 键 词：自杀基因 慢病毒载体 酵母菌胞嘧啶脱氨酶 JURKAT细胞 

分 类 号：R733.7[医药卫生—肿瘤]