共表达胞嘧啶脱氨酶和胸苷激酶双自杀基因杀伤大鼠血管平滑肌细胞的机制研究  

Killing Machanism of Coexpressing Cytosine Deaminase/Thymidine Kinase Double Suicide Gene in Rat Vascular Smooth Muscle Cells

作　　者：曹慧青[1] 孟宪敏[1] 刘冬青[1] 赵秀文[1] 丁金凤[1] 

机构地区：[1]中国医学科学院中国协和医科大学心血管病研究所阜外心血管病医院分子医学中心,北京市100037

出　　处：《中国循环杂志》2004年第4期309-312,共4页Chinese Circulation Journal

基　　金：国家自然科学基金项目 (NO .39970 2 97)经费资助

摘　　要：目的 :研究共表达胞嘧啶脱氨酶 (CD)和胸苷激酶 (TK)双自杀基因对大鼠血管平滑肌细胞的杀伤机制。方法 :将表达CD和 (或 )TK的单、双自杀基因重组腺病毒载体分为 4组即Ad EF1α CD组、Ad CMV TK组、Ad EF1α CD CMV TK组和Ad lacZ组。感染原代培养的大鼠主动脉血管平滑肌细胞 ,感染复数为 2 0。给予相应的前体药物 5 氟胞嘧啶和脱氧鸟苷后 ,用四甲基偶氮挫盐微量酶反应比色法 (MTT)和流式细胞术分析细胞死亡率。荧光染料Hoechst 3 3 3 42核染色观察凋亡细胞。琼脂糖凝胶电泳检测凋亡细胞基因组脱氧核糖核酸 (DNA)的降解。结果 :血管平滑肌细胞可被重组腺病毒有效感染 ( 70 % )。MTT检测发现Ad EF1α CD CMV TK组双自杀基因及其前体药物 [5 氟胞嘧啶 ( 3 0 μg/ml)和脱氧鸟苷 ( 0 3 μg/ml) ]的细胞毒效应可达 91% ,高于Ad CMV TK组 ( 68% )和Ad EF1α CD组 ( 63 % )单自杀基因组。流式细胞仪分析Ad CMV TK组和Ad EF1α CD CMV TK组的杀伤作用是以细胞毒性坏死和凋亡为主。凋亡细胞荧光染色显示Ad EF1α CD CMV TK组和Ad CMV TK组细胞的凋亡率分别为 11 0 %和 12 0 % ,高于Ad CMV LacZ组 ( 1 2 % )和Ad EF1α CD组 ( 5 0 % )。琼脂糖凝胶电泳发现在Ad CMV TK组和Ad EF1α CD CMV TK组的基因组DNA可?Objective:To investigate the killing machanism of rat vascular smooth muscle cells mediated by combined expression of E. coli cytosine deaminase (CD) and herpes simplex virus type-1 thymidine kinase (TK) double suicide genes with exposure to their matching prodrugs 5-fluorocytosine and ganciclovir.Methods:The primary cultured vascular smooth muscle cells were infected by recombinant adenovirus carrying double or single suicide genes including Ad-EF1α-CD-CMV-TK,Ad-CMV-TK and Ad-EF1α-CD at a multiplicity of infection (MOI) of 20. Cell mortality rates were tested by MTT assay and flow cytometry. Apoptotic cells were analyzed using Hoechst 33342 fluorescence dye as a DNA probe. Genomic DNA-cleavage of apoptotic was detected by agarose gel electrophoresis.Results:Vascular smooth muscle cells were effectively infected by the recombinant adenovirus. Cytotoxic effects of Ad-EF1α-CD-CMV-TK double suicide genes combined with 5-fluorocytosine (30μg/ml) and ganciclovir (0.3μg/ml) reached 91%,which were superior to those of Ad-CMV-TK (68%) and Ad-EF1α-CD (63%) single gene by MTT method. Flow cytometry analysis indicated that the killing machanism among groups was different. The double gene group was via cytotoxic and apoptosis approach. Based on the DNA stainability with Hoechst 33342,the apoptotic rates of vascular smooth muscle cells in Ad-EF1α-CD-CMV-TK (11.0%) and Ad-CMV-TK group (12.0%) were higher than those of Ad-CMV-LacZ (1.2%) and Ad-EF1α-CD (5.0%) group. DNA ladders were observed in both Ad-CMV-TK and Ad-EF1α-CD-CMV-TK group after electrophorosis.Conclusions:Killing machanism of recombinant adenovirus coexpressing CD/TK double suicide genes was mainly due to cytotoxic necrosis and apoptosis.

关 键 词：双自杀基因 CMV 血管平滑肌细胞 大鼠 胸苷激酶 胞嘧啶脱氨酶 凋亡细胞 共表达 鸟苷 脱氧核糖核酸 

分 类 号：R735.7[医药卫生—肿瘤] R392[医药卫生—临床医学]