摘要
目的观察表达白细胞介素18（IL-18）基因的条件增殖腺病毒在肾癌Ketr-3细胞中的生物活性及其对Ketr-3细胞的杀伤作用。方法通过荧光显微镜观察表达绿色荧光蛋白的条件增殖腺病毒（ZD55-EGFP）在肾癌Ketr-3细胞中的感染和增殖情况。分别将表达IL-18的条件增殖腺病毒（ZD55-IL-18）及表达IL-18的普通腺病毒（Ad-IL-18）感染人肾癌Ketr-3细胞系，通过Western blot法检测病毒E1A和IL-18蛋白的表达；免疫细胞化学染色检测IL-18抗原表达；TUNEL法检测Ketr-3细胞的凋亡情况；噻唑蓝（MTT）比色法检测Ketr-3细胞存活情况。结果ZD55-EGFP能有效感染肾癌Ketr-3细胞并在其中大量增殖。Western blot检测结果发现ZD55-IL-18能在肿瘤细胞内表达E1A并有效介导IL-18表达，在病毒感染Ketr-3细胞48h后，ZD55-IL-18、Ad-IL-18处理组的IL-18蛋白表达量分别为255．6±3．1、118．7±2．9。免疫组织化学检测显示ZD55-IL-18、Ad-IL-18处理组的IL-18抗原阳性率分别为（82．4±3．2）％和（23．4±1．9）％。TNUEL检测结果显示ZD55-IL-18、Ad-IL-18处理组的细胞凋亡率分别为（52．2±3．5）％和（25．5±1．9）％。病毒感染4d后，MTT检测结果显示ZD55-IL-18、Ad-IL-18处理组细胞存活率分别为（32．6±2．3）％和（73．3±2．5）％，表明ZD55-IL-18对Ketr-3细胞有显著的杀伤作用。结论ZD55-IL-18能在Ketr-3细胞中高效特异性表达IL-18基因并显示出良好的抗肿瘤作用。
Objective To study the bioactivity and antitumor effects of conditionally replicating adenovirus vector expressing IL-18 gene （ ZD55-IL-18 ） in human renal carcinoma ketr-3 cells. Methods The infection and proliferation of replicating adenovirus vector （ZD55-EGFP） in ketr-3 cells were observed by fluorescence microscopy. ZD55-IL-18 were used to infect ketr-3cells. The expression of IL-18 and E1A protein was detected by Western blot. The expression of IL-18 antigen was also detected by immunocytochemical technique. The apoptosis of cells was tested by TUNEL. The inhibition of ketr-3 cell proliferation by ZD55-IL-18 was determined by MTT assay. Results Fluorescence microscopy indicated ZD55-EGFP showed much stronger specific replicative ability than Ad-EGFP. Western blot analyses revealed ZD55-IL-18 could effectively express IL-18 protein and could express E1A in adenovirus-infected ketr-3 cells in vitro. IL-18 protein expression in the ZD55-IL-18 treatment group was 255.6 ± 3.1 at 48 h post transfection,much higher than that of Ad-IL-18 treatment group （118.7 ± 2.9 ）. IL-18 immunohistochemical analysis verified IL-18 staining positivity rate in the ZD55-IL-18 treatment group was （82.4 ± 3.2） % ,significantly higher than that of the Ad-IL-18 treatment groups （25.5 ± 1.9）%. The average apoptosis rate in the ZD55-IL-18 and Ad-IL-18 treatment group was （52.2 ± 3.5 ） % and （25.5 ± 1.9 ） % after the transfection, respectively. MTT assay confirmed ZD55-IL-18 had significantly inhibitory effects as compared with Ad - IL - 18. Cell viability rate in ZD 5 5 - IL - 1 8 and Ad - IL - 18 treatment groups was （ 3 2.6 ± 2.3） % and （73.3 ± 2.5 ） % respectively at MOI = 10 4 days after infection. Conclusion ZD55-IL-18 can replicate selectively and effectively express IL-18 gene in ketr-3 cells, and produce powerful antitumor effects.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2008年第9期1181-1183,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（30570385、30700999）
关键词
肾癌
腺病毒
IL-18
Renal cell carcinoma
Adenovirus
IL-18 gene
