携带IL24基因复制缺陷型腺病毒对膀胱癌生长的抑制作用

Study of suppressive effect of replication-deficient recombinant adenovirus expressing IL24 gene on bladder carcinoma in vitro and in vivo

目的研究携带人IL24基因的复制缺陷型腺病毒(Ad-IL24)对膀胱癌的杀伤效应。方法聚合酶链反应(PCR)鉴定Ad-IL24,并扩增、纯化和滴度测定。Ad-IL24感染人膀胱癌Biu87细胞系,通过结晶紫染色及四甲基偶氮唑盐(MTT)法检测细胞杀伤作用;Western blot法检测腺病毒IL24蛋白的表达情况;并进一步建立裸鼠膀胱癌模型,应用Ad-IL24瘤内注射体内观察抗肿瘤效果。结果PCR鉴定说明Ad-IL24包含目的基因且为复制缺陷型腺病毒;Western blot结果表明Ad-IL24能有效介导IL24基因在Biu87细胞中的表达;结晶紫染色结果表明Ad-IL24对Biu87细胞有明显的杀伤作用,MTT表明用MOI=10的Ad-IL24和Ad-EGFP感染Biu87细胞,6天后存活率分别为(47.6±2.6)%、(84.7±4.5)%,比较差异有统计学意义(P<0.05)。成功建立膀胱癌动物模型后,瘤内注射病毒Ad-IL24、Ad-EGFP和PBS后5周,AdIL-24、Ad-EGFP和PBS各组肿瘤大小分别为(420.3±49.0)mm3、(639.8±52.9)mm3和(766.1±75.8)mm3。结论成功获得纯化的Ad-IL24,并进一步证明Ad-IL24能在体内外有效抑制膀胱癌的生长,显示出良好的抗肿瘤效果,为进一步应用于膀胱癌基因治疗奠定基础。

Objective To investigate the antitumor effects of replication - deficient recombinant adenovirus expressing IL - 24 gene （ Ad - IL24） on human bladder carcinoma in vitro and in vivo. Methods Replication - deficient recombinant adenovirus Ad -IL24 were verified by polymerase chain reaction （PCR）. These viruses were further propagated and purified and their functional PFU titers were determined by plaque assay on 293 cells. Ad - IL24 were used to infect Biu87 cells, and the inhibition of Biu87 cell proliferation by Ad - IL24 was determined by MTT assay and crystal violet staining for different MOI values. The expressions of Ad - IL24 protein was determined by Western blot. Bal B/C nude mice were inoculated subcutaneously with Biu87 cells to establish the nude mice bladder carcinoma model. The tumor - bearing nude mice were injected intratumorally with Ad - IL24 for measurement of the tumor size at different intervals to observe the antitumor effect. Results The PCR analysis indicated the recombinant adenovirus Ad- IL24 contained IL24 gene instead of E1A gene. Functional PFU titers of Ad - IL24 were 2.0 × 10^10 PFU/ml. Western blot analysis confirmed that Ad - IL24 can express IL24 protein in Biu87 ceils with high efficiency. Crystal violet staining and MTF assay showed Ad - IL24 could obviously induce cytotoxic effects and growth inhibition of Biu87 cells. Cells viability rate were （47.6 ± 2.6） % and （ 84.7 ± 4.5 ） %, respectively after tumor cells were infected with Ad - IL24, Ad - EGFP at MOI = 10 on the 6th postinfective day. 5 weeks later, the mean tumor size of the Ad - IL24 treatment group was 420.3 ± 49.0 mm^3, much smaller than that of the Ad -EGFP treatment group [ （639.8 ± 52.9） mm^3,P 〈 0.05 ] and PBS treatment group [ （766.1 ± 75.8） mm3,P 〈 0.05 ]. Conclusion Replication - deficient recombinant adenovirus Ad - IL24, which was successfully obtained and purified, could exert potential antitumor activity on human bladder cancer in vitro and in vivo, and benefit further research on gene therapy of bladder carcinoma.