腺病毒介导的HSV-TK/GCV系统在Cfos启动子调控下对胶质瘤细胞的杀伤作用  

Adenoviral-mediated delivery of herpes simplex virus thymidine kinase gene controlled by Cfos promoter confers cytotoxic effect on human glioma cells

作　　者：王浩[1] 潘建青[1] 胡继良 冯诣 宋伟健[1] 罗杰[1] 刘欣民[1] 魏强国[1] 洪全球[1] 翟宝进[1] 

机构地区：[1]深圳市南山区人民医院神经外科,518052 [2]深圳市人民医院神经外科,518020

出　　处：《中华神经医学杂志》2015年第6期558-562,共5页Chinese Journal of Neuromedicine

基　　金：深圳市南山区科技计划项目（2012017）

摘　　要：目的 构建人Cfos启动子调控的单纯疱疹病毒（HSV）-胸苷激酶（TK）自杀基因重组腺病毒载体,观察其介导的HSV-TK/更昔洛韦（GCV）系统对胶质瘤细胞的杀伤作用. 方法 构建重组腺病毒载体Ad-Cfos-TK-IRES-hr绿色荧光蛋白（GFP）、Ad-CMV-TK-IRES-hrGFP,转染至HEK293细胞获得重组腺病毒Ad-Cfos-TK-IRES-hrGFP、Ad-CMV-TK-IRES-hrGFP,荧光计数法检测病毒滴度;将病毒感染U251、U87细胞,荧光显微镜下观察GFP的表达.将病毒感染U251、U87细胞,24 h后加入1000、100、10、1、0.1、0.01、0μmol/L GCV作用48 h,CCK-8法检测各组细胞抑制率. 结果 成功构建重组腺病毒载体pDC315-CMV-TK-IRES-hrGFP、pDC315-CFOS-TK-IRES-hrGFP.荧光计数法检测显示2种腺病毒滴度均达1＆#215;1010 PFU/mL.在病毒感染复数（MOI）为100、GCV浓度为1、10、100、1000 μmol/L时,转染Ad-Cfos-TK-IRES-hrGFP组U251细胞抑制率分别为（24.18±6.01）％、（30.39±9.67）％、（57.07±9.29）％、（94.50±3.48）％,转染Ad-Cfos-TK-IRES-hrGFP组U87细胞细胞抑制率分别为（9.78±2.24）％、（86.33±5.06）％、（98.48±0.79）％、（98.76±0.93）％. 结论 腺病毒介导的HSV-TK/GCV系统在Cfos启动子调控下在体外可以有效杀伤胶质瘤细胞.Objective To construct recombinant adenovirus vectors containing herpes simple virus thymidine kinase （HSV-TK） controlled by human Cfos promoter and cytomegalovirus （CMV） promoter and observe the expression of adenovirus in human glioma U251 cells and their specific cytotoxic effect on U251 cells in combination with ganciclovir （GCV） in vitro.Methods Two recombinant adenovirus vectors containing herpes simplex virus thymidine kinase gene controlled by human Cfos promoter and CMV promoter were constructed （Ad-Cfos-TK-IRES-hr green fluorescent protein [GFP] and Ad-CMV-TK-IRES-hrGFP）,respectively.For packaging virus,two adenovirus vectors were transfected into HEK293 cells.Ad-CMV-TK-IRES-hrGFP and Ad-Cfos-TK-IRES-hrGFP were collected,purified and transfected into U251 cells.The expression of EGFP was observed under a fluorescence microscope.Recombinant adenovirus infected U251 cells were cultured with 1,10,100 and 1000 μmol/L GCV to observe specific cytotoxic effect on glioma.Results The recombinant plasmid vectors Ad-CMV-TK-IRES-hrGFP and Ad-cfos-TK-IRES-hrGFP were verified by restriction endonuclease digestion digestion and sequencing.Ad-CMV-TK-IRES-hrGFP and Ad-Cfos-TK-IRES-hrGFP adenovirus were collected and purified,successfully.The titers of adenovirus reached 1 ＆#215;1010 IU/ml.In company with the rising of GCV concentration gradient （1,10,100 and 1000 μmol/L）,the inhibition rate of U251 cells gradually increased （24.18%±6.01%,30.39%±9.67%,57.07%±9.29% and 94.50%±3.48%） at mutiplicity of infection （MOI） 100.In company with the rising of GCV concentration gradient （1,10,100 and 1000 μmol/L）,the inhibition rate of U87 cells gradually increased （9.78%± 2.24%,86.33% ±5.06%,98.48% ±0.79% and 98.76% ±0.93） at MOI 100.Conclusion Adenoviral-mediated delivery of herpes simplex virus thymidine kinase gene controlled by Cfos promoter can confer cytotoxic effect on human glioma cells in vitro.

关 键 词：神经胶质瘤 基因治疗 腺病毒 Cfos启动子 

分 类 号：R739.41[医药卫生—肿瘤]