Ad-生长抑制因子4-polyA-promoter-白细胞介素-24对NCI-H460肺癌细胞抑癌增效作用的体内实验研究

Enhanced anticancer effect by Ad-inhibitor of growth 4-polyA-promoter-interleukin-24 for lung adenocarcinoma(NCI-H460)in vivo

目的探讨腺病毒介导的生长抑制因子4(ING4)和白细胞介素(IL)-24双基因共表达对NCI-H460肺癌细胞裸鼠移植瘤的抑癌增效作用及分子机制。方法根据腺病毒携带基因类型,把实验分5组,PBS、Ad、Ad-IL-24、Ad-ING4、Ad-ING4-IL-24组。以50的感染复数(MOI)各重组腺病毒分别感染NCI-H460细胞(苏州大学基础医学院细胞和分子生物学教研室提供),蛋白质印迹法(Western blot)鉴定ING4和/或IL-24基因在NCI-H460细胞中的表达;噻唑蓝(MTT)法检测各重组腺病毒对NCI-H460细胞的生长抑制作用;各重组腺病毒注入裸鼠瘤体内进行抗肿瘤实验,观察各组瘤体体积变化;治疗15 d后处死裸鼠、摘取瘤体并称瘤体质量;免疫组织化学检测瘤体组织中B细胞淋巴瘤/白血病-2相关X蛋白(bax)、半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3、B细胞淋巴瘤/白血病-2(bcl-2)、生存素(Survivin)等相关因子的表达。统计学采用单因素方差分析。结果动物实验结果表明,腺病毒介导白细胞介素-24(Ad-IL-24)、腺病毒介导生长抑制因子4(Ad-ING4)、腺病毒介导白细胞介素-24及生长抑制因子4(Ad-ING4-IL-24)组的肿瘤平均体积明显低于腺病毒(Ad)组[(559.880±43.704)mm3,t=12.811,P<0.01;(573.720±42.019)mm3,t=13.654,P<0.01;(834.700±44.948)mm3,t=18.570,P<0.01]和磷酸盐缓冲液(PBS)组[(-710.210±32.608)mm3,t=21.780,P<0.01;(724.050±30.313)mm3,t=23.886,P<0.01;(-985.030±34.257)mm3,t=28.754,P<0.01],平均瘤体重量明显低于Ad组[(0.547±0.080)g,t=6.812,P<0.01;(0.607±0.082)g,t=7.433,P<0.01;(0.919±0.082)g,t=11.221,P<0.01]和PBS组[(0.572±0.067)g,t=8.597,P<0.01;(0.632±0.068)g,t=9.260,P<0.01;(0.944±0.069)g,t=13.775,P<0.01],差异均有统计学意义;Ad-ING4-IL-24组对裸鼠NCI-H460肺癌移植瘤的抑癌作用明显优于Ad-IL-24、Ad-ING4单基因组(0.302±0.048,t=6.213,P<0.01;0.250±0.053,t=4.685,P<0.01),差异有统计学意义,呈现抑癌增效相加作用(Q=0.940)。ING4和/或IL-24能够上调bax、Caspase-3等因子的表达,下调bcl-2、Survivin等因子的表达,且Ad-ING4-IL-24组优于Ad-IL-24、Ad-ING4单基因组(0.263±0.055,t=4.286,P<0.01),差异有统计学意义。结论Ad-ING4-IL-24双基因共表达在体内对NCI-H460细胞裸鼠移植瘤均具有抑癌增效作用,其分子机制可能与上调bax、Caspase-3等促凋亡因子的表达,下调bcl-2、Survivin等凋亡抑制因子的表达有关。

Objective To study the enhanced anticancer effect and its mechanism by Ad-inhibitor of growth 4(ING4)-polyA-promoer-interleukin(IL)-24 for lung adenocarcinoma(NCI-H460).Methods NCI-H460 cells were trasnfected with the different recombinant adenoviruses by 50 MOI.The expression of ING4 and/or IL-24 was identified by Western blotting.The influence of cell growth was tested by methyl thiazolyl tetrazolium(MTT)essay.Anti-tumor experiment was conducted on these mice bearing lung adenocarcinoma transplantation of NCI-H460 cells by intratumorally injecting with the recombinant adenovirus.And tumor growth was recorded by volume.All tumors were taken out and weighed at 15th day after treatment.The expression levels of cytokines including B cell lymphoma/leukemia-2 associated X protein(bax),cysteinyl aspartate-specific protease(Caspase)-3,B cell lymphoma/leukemia-2(bcl-2),Survivin were detected by immunohistochemisty.Results Animal experimental results showed that the average volumes in Ad-IL-24,Ad-ING4,Ad-ING4-IL-24 groups were significantly reduced as compared with those in Ad group[(559.880±43.704)mm3,t=12.811,P<0.01;(573.720±42.019)mm3,t=13.654,P<0.01;(834.700±44.948)mm3,t=18.570,P<0.01]and PBS group[(710.210±32.608)mm3,t=21.780,P<0.01;(724.050±30.313)mm3,t=23.886,P<0.01;(985.030±34.257)mm3,t=28.754,P<0.01];and average tumor weight was significantly lower than in Ad group[(0.547±0.080)g,t=6.812,P<0.01;(0.607±0.082)g,t=7.433,P<0.01;(0.919±0.082)g,t=11.221,P<0.01],PBS group[(0.572±0.067)g,t=8.597,P<0.01;(0.632±0.068)g,t=9.260,P<0.01;(0.944±0.069)g,t=13.775,P<0.01].Ad-ING4-IL-24 had significantly better antitumor effect than Ad-IL-24 and Ad-ING4 single genome(0.302±0.048,t=6.213,P<0.01;0.250±0.053,t=4.685,P<0.01),showing a synergistic effect(Q=0.940).ING4 and/or IL-24 could up-regulate the expression of bax,Caspase-3,and down-regulate the expression of bcl-2 and Survivin,and Ad-ING4-IL-24 was better than Ad-IL-24 and AdING4 single genome(t=4.286,P<0.01).Conclusion It is proved that there is enhanced anticancer effect by Ad-ING4-polyA-promoter-IL-24.The mechanism may be related to the up-regulations of bax and Caspase-3 and the down-regulations of bcl-2 and Survivin.