靶向性双自杀基因系统对脐静脉内皮细胞的杀伤作用

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (02): 139-143.

靶向性双自杀基因系统对脐静脉内皮细胞的杀伤作用

黄宗海¹, 杨文宇¹, 龚小卫², 钱勇³, 车小燕⁴

1. 第一军医大学珠江医院普外科, 广东, 广州, 510282;
2. 第一军医大学病理生理教研室, 广东, 广州, 510515;
3. 广州军区广州总医院放射科, 广东, 广州, 510010;
4. 第一军医大学珠江医院中心实验室, 广东, 广州, 510282

出版日期:2004-02-20 发布日期:2004-02-20
基金资助:
收稿日期:2003-8-16。
基金项目:国家863计划项目(2001AA₂17171);广东省自然科学基金重点资助项目(013072)
作者简介:黄宗海(1954-),男,第一军医大学珠江医院普通外科主任,教授,博士生导师,电话:020-61643213

Killing effect of suicide gene system under control by KDR promotor on human umbilical vein endothelial cells

HUANG Zong-hai¹, YANG Wen-yu¹, GONG Xiao-wei², QIAN Yong³, CHE Xiao-yan⁴

1. 第一军医大学珠江医院普外科, 广东, 广州, 510282;
2. 第一军医大学病理生理教研室, 广东, 广州, 510515;
3. 广州军区广州总医院放射科, 广东, 广州, 510010;
4. 第一军医大学珠江医院中心实验室, 广东, 广州, 510282

Online:2004-02-20 Published:2004-02-20

摘要/Abstract

摘要： 目的研究腺病毒介导的前药/血管内皮生长因子受体(KDR)启动子-双自杀基因系统对血管内皮细胞的杀伤作用。方法应用PCR扩增出KDR启动子、CD基因、TK基因序列,构建pKDR-CDglyTK质粒;以Adeasy-1系统为载体,采用细菌内同源重组“两步转化法”构建携带KDR启动子调控下的CD与TK的融合基因腺病毒重组质粒pAdKDR-CDglyTK,重组质粒在293细胞中包装成病毒,并进一步扩增、纯化,以不同的感染复数(MOI)体外感染脐静脉血管内皮细胞(HUVEC),利用重组病毒携带的绿色荧光蛋白(GFP)基因,荧光显微镜下观察重组腺病毒的感染效率,并给予不同浓度的GCV(ganciclovir)和/或5-FC(5-fluorocytosine),比较GCV(ganciclovir)和/或5-FC联合对转基因HUVEC细胞的杀伤作用。结果成功构建了AdKDR-CDglyTK重组病毒,并高效地转染了HUVEC,其转染效率随重组病毒的MOI增加而升高。被转染的HUVEC对GCV和5-FC高度敏感。另外,MOI一定时,细胞存活率随GCV和5-FC浓度增加递减;GCV和5-FC浓度一定时,细胞存活率随MOI升高而降低。不同前药组细胞存活率结果显示:联合应用GCV+5-FC较单用GCV或5-FC对感染细胞的杀伤作用更强(P<0.05)。结论 KDR启动子-双自杀基因系统对脐静脉内皮细胞具有强烈的杀伤作用,其杀伤效率与GCV和5-FC的浓度及重组腺病毒的MOI相关,联?

Abstract: Objective To study the killing effect of adenovirus-mediated double suicide gene under the regulation of kinase domain-containing receptor (KDR) promoter on human umbilical vein endothelial cells (HUVECs). Methods The sequences of human KDR promoter gene, CD gene and TK gene were amplified by PCR, and the plasmid pKDR-CDglyTK was constructed. A two-step transformation protocol was employed for the construction of a recombinant adenoviral plasmid pAdKDR-CDglyTK that was transfected into 293 packaging cells to further multiply and purify the adenovirus. HUVECs were infected by the resultant recombinant adenovirus of different multiplicities of infection (MOI), and the infection rate was measured by observing the expression of green fluorescence protein (GFP). The infected cells were cultured in the culture media containing ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the killing effects were evaluated. Results Recombinant adenovirus AdKDR-CDglyTK were successfully constructed, which could efficiently infect HUVEC cells, with the infection rate associated with the MOI of the recombinant adenovirus. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of the recombinant adenovirus. The killing effect of the two produrgs used in combination was much stronger than that of exclusive use of GCV or 5-FC. Conclusions Prodrug/KDR-CdglyTK system is effective in killing HUVEC cells, and its killing effect is correlated with the concentration of the prodrugs and the MOI of the recombinant adenovirus. Combination of the two prodrugs produces stronger killing effect on the cells.

中图分类号:

R394.1

黄宗海¹, 杨文宇¹, 龚小卫², 钱勇³, 车小燕⁴. 靶向性双自杀基因系统对脐静脉内皮细胞的杀伤作用[J]. 南方医科大学学报, 2004, 24(02): 139-143.

HUANG Zong-hai¹, YANG Wen-yu¹, GONG Xiao-wei², QIAN Yong³, CHE Xiao-yan⁴. Killing effect of suicide gene system under control by KDR promotor on human umbilical vein endothelial cells[J]. Journal of Southern Medical University, 2004, 24(02): 139-143.

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