重组腺病毒rAd-E1A-CDglyTK对人卵巢癌SKOV3细胞的抑制作用  

Inhibitory effect of recombinant adenovirus rAd-E1A-CDglyTK on human ovarian carcinoma SKOV3 cells

作　　者：周丽君[1] 李秋香[1] 李冬田[1] 曹阳[2] 李光明[1] 佟惠春[1] 

机构地区：[1]天津医科大学基础医学院微生物学分子病毒学实验室,天津300070 [2]天津医科大学第二附属医院检验科,天津300211

出　　处：《中国肿瘤生物治疗杂志》2008年第3期223-227,共5页Chinese Journal of Cancer Biotherapy

基　　金：天津市卫生局资助项目(No.99KY047)~~

摘　　要：目的:探讨含腺病毒基因E1A、融合自杀基因CDglyTK及报告基因GFP的重组腺病毒rAd-E1A-CDglyTK与酶前体药物5-FC和(或)GCV联合应用对人卵巢癌SKOV3细胞的抑制作用。方法:将重组腺病毒质粒prAd-E1A-CDglyTK经脂质体介导转染293细胞,进行病毒的扩增、纯化与PCR鉴定,以紫外分光光度法与TCID50法测定纯化病毒的颗粒浓度与感染性滴度,计算病毒比活性;在SKOV3细胞中观察重组病毒的复制能力及感染效率,确定感染率达90%以上的最小MOI。用MTT法观察感染重组病毒的SKOV3细胞对5-FC或GCV的敏感性,并计算IC_(50);观察一定MOI的rAd-E1A-CDglyTK或重组复制缺陷型腺病毒rAd-CDglyTK与IC_(50)的5-FC和(或)GCV联合应用对SKOV3细胞生长的抑制作用。结果:经PCR和琼脂糖凝胶电泳证实,纯化重组腺病毒中存在E1A和CDglyTK两个基因片段,其病毒颗粒浓度和感染性滴度分别为9.47×10^(11) VP/ml和3.16×10^(10)IU/ml,病毒比活性为3.34%。重组病毒可在SKOV3细胞中复制,对SKOV3细胞的感染效率随MOI的增加而增加,可致细胞感染率达90%以上的最小MOI为50 IU/cell。5-FC或GCV明显抑制感染细胞的生长,且存在明显的剂量依赖效应。两种重组腺病毒/前药系统的抑瘤作用比较显示,rAd-E1A-CDglyTK组细胞生长抑制率[(16.57±1.59)%]显著高于rAd-CDglyTK组[(10.44±1.80)%,P<0.01];与IC_(50)的5-FC和GCV双药联合应用,其抑制率达(71.68±2.63)%,显著高于rAd-CDglyTK/5-FC+GCV组的(63.64±2.91)%(P<0.01)。结论:重组腺病毒rAd-E1A-CDglyTK与酶前体药物联合应用对卵巢癌SKOV3细胞具有显著的抑制作用,其效果优于重组复制缺陷型腺病毒rAd-CDglyTK。Objective： To investigate the inhibitory effect of recombinant adenovirus rAd-E1A-CDglyTK combined with enzyme prodrugs 5-flurocytosine （5-FC） and/or ganciclovir （GCV） on human epithelial ovarian carcinoma cell line SKOV3. Methods： The recombinant plasmid prAd-E1 A-CDglyTK was transfected into 293 cells by liposome. After amplification and purification, the virus was identified by PCR. The viral particle concentration was tested by ultraviolet spectrophotometry and its infectious titer was tested through TCID50 assay, rAd-E1A-CDglyTK was employed to infect SKOV3 cells for observation of the viral replication ability and the infection rate ; the minimum MOI leading to above 90% infection rate was determined. The drug sensitivity of the infected SKOV3 cells to 5-FC or GCV was observed by MTr assay and the IC50 values were determined. The growth inhibitory effects of rAd-E1A-CDglyTK or rAd-CDglyTK （at defined MOI） combined with 5-FC and GCV （ at IC50 ） were determined on SKOV3 cells. Results： PCR results confirmed the presence of EIA gene and CDglyTK fragments in recombinant adenovirus. The viral particle concentration was 9.47 ×10^11 VP/ml and its infectious titer was 3.16 × 10^10IU/ml. rAd-E1A-CDglyTK could replicate in SKOV3 cells; the infection rate of SKOV3 cells increased with the increase of MOI, the minimum MOI was 50 IU/cell. 5-FC or GCV inhibited the growth of infected SKOV3 cells in a dose-dependent manner. The inhibitory effect of rAd-CDglyTK group （ [ 10.44 ±1.80] % ） was significantly lower than that of rAd-E1A-CDglyTK group（ [ 16.57 ± 1.59 ] %, P 〈 0.01 ）. When combi- ning with 5-FC and GCV（ at IC50）, the growth inhibitory rate reached （71.68 ± 2.63）% in the rAd-E1A-CDglyTK/ 5-FC ＋ GCV group, significant higher than that in the rAd-CDglyTK/5-FC ＋ GCV （ [63.64 ± 2.91 ]%, P 〈 0.01 ）. Conclusion：Recombinant adenovirus rAd-E1A-CDglyTK combined with enzyme prodrugs can lead to a great cytotoxic effect on SKOV3 cells; the effect is superior to that o

关 键 词：卵巢癌 重组腺病毒 EIA基因 自杀基因 SKOV3细胞 

分 类 号：R737.31[医药卫生—肿瘤] R730.54[医药卫生—临床医学]