表达白细胞介素18的条件增殖腺病毒在肾癌Ketr-3细胞中的生物活性及抗肿瘤作用

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.Westem blot检测结果 发现ZD55-IL-18能在肿瘤细胞内表达E1 A并有效介导IL-18表达,在病毒感染Ketr-3细胞48 h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±2.90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4 d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Kerr-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.

The bioactivity and antitumor effects of conditionally replicating adenovirus vector expressing IL-18 gene in human renal carcinoma ketr-3 cells

Objective To study the bioaetivity and antitumor effects of conditionally replicating adenovirus vector expressing IL-18 gene(ZD55-IL-18)in human Fenal carcinoma ketr-3 cells.Methods The infection and proliferation of replicating adenovirus vector(ZD55-EGFP)in ketr-3 ceils were ohserved by fluorescence microscopy.ZD55-IL-18 were used to infect ketr-3cells.The expression of IL-18 and E1 A protein was detected by Western blot.The expression of IL-18 antigen was also detected by immunecytochemical technique.The apoptosis of eels 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 rephcative 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 hiher than that of Ad-IL-18 treatment group(118.7±2.9).IL-18 iminunohistochemical 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 tramfection.respectively.MTT assay confirmed ZD55-IL-18 had significantly inhibitory effects as compared withAd-IL-18.Cell viabilityrate in ZD55-IL-18and Ad-IL-18 treatment groupswas(32.6±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.