摘要
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肺癌细胞抑癌增效作用及其机制。方法以50感染复数(MOI)各重组腺病毒分别感染NCI-H460细胞,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;经膜联蛋白V-藻红蛋白(Annexin V-PE)/7-氨基放线菌素D(7-AAD)染色后流式细胞术(FCM)检测细胞凋亡率变化;反转录-聚合酶链反应(RT-PCR)检测NCI-H460细胞中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)等因子的表达,Western blot法检测其中裂解的Caspase-3(Cleaved Caspase-3)因子的表达。结果 ING4和/或IL-24基因在NCI-H460细胞中能够有效表达;96 h时相点生长抑制率Ad-IL-24、Ad-ING4、Ad-ING4-IL-24组对NCI-H460细胞的生长抑制率明显高于Ad组(27.800±4.271,t=6.508,P<0.01)、(27.130±3.341,t=8.121,P<0.01)、(50.767±2.671,t=19.005,P<0.01),差异有统计学意义;Ad-ING4-IL-24组对NCI-H460细胞的生长抑制率明显优于Ad-IL-24(22.967±4.933,t=4.655,P<0.01)、Ad-ING4(23.633±4.154,t=5.689,P<0.01)单基因组,差异有统计学意义,并呈现抑癌增效相加作用(Q=0.93)。FCM检测结果表明,Ad-IL-24、Ad-ING4、Ad-ING4-IL-24组作用于NCI-H460细胞72 h的诱导细胞凋亡率明显高于磷酸盐缓冲液(PBS)组(16.440±0.896,t=18.340,P<0.01)、(14.000±1.562,t=13.255,P<0.01)、(29.660±1.519,t=19.522,P<0.01)和Ad组(14.450±1.058,t=13.660,P<0.01)、(12.010±1.196,t=1.040,P<0.01)、(27.670±1.620,t=17.082,P<0.01),差异有统计学意义,其中Ad-ING4-IL-24组中NCI-H460细胞的细胞凋亡率明显高于Ad-IL-24(15.660±1.773,t=8.834,P<0.01)、Ad-ING4(13.220±1.628,t=7.858,P<0.01)单基因组,差异有统计学意义;Ad-ING4-IL-24组可明显上调bax、Caspase-3等因子的表达,下调bcl-2、Survivin等因子的表达。结论 Ad-ING4-IL-24双基因共表达在体外对NCI-H460细胞具有抑癌增效作用,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:The NCI-H460 cells were transfected with the different recombinant adenovirus by 50 multiplicity of infection(MOI).The expression of ING4 and/or IL-24 was identified by Western blotting.The influence of cell growth was tested by MTT assay.The cell apoptotic effect was evaluated by flow cytometry(FCM).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)and Survivin were detected by reverse transcriptase-polymerase chain reaction(RT-PCR).The expression of cleaved Caspase-3 was detected by Western blotting.Anti-tumor experiment was conducted on the mice bearing lung adenocarcinoma transplantation of NCI-H460 cells by intratumorally injecting with the recombinant adenovirus.The tumor growth was recorded by volume.All tumors were taken out and weighed at 15th day after treatment.The expression levels of cytokines including bax,Caspase-3,bcl-2 and Survivin were detected by immunohistochemisty.Results:Western blotting proved that ING4 and/or IL-24 were/was effectively transcribed in NCI-H460 cells.At 96 h,the growth inhibition rate in Ad-IL-24 group,Ad-ING4 group and Ad-ING4-IL-24 group on NCI-H460 cells was significantly higher than that in Ad group(27.800±4.271,t=6.508,P<0.01;27.130±3.341,t=8.121,P<0.01;50.767±2.671,t=19.005,P<0.01).The cell growth was obviously inhibited by the recombinant adenovirus.The effect of Ad-ING4-IL-24 group was better than that of Ad-IL-24(22.967±4.933,t=4.655,P<0.01)and Ad-ING4 group(23.633±4.154,t=5.689,P<0.01),which showed an enhanced anticancer effect and an additive effect(Q=0.93).FCM results showed that the apoptosis rate of NCI-H460 cells treated with Ad-IL-24,Ad-ING4 and Ad-ING4-IL-24 for 72 h was significantly higher than that in phosphate buffer(PBS)group(16.440±0.896,t=18.340,P<0.01;14.000±1.562,t=13.255,P<0.01;29.660±1.519,t=19.522,P<0.01)and Ad group(14.450±1.058,t=13.660,P<0.01;12.010±1.196,t=1.040,P<0.01;27.670±1.620,t=17.082,P<0.01),and the difference was statistically significant.The apoptosis rate of NCI-H460 cells in Ad-ING4-IL-24 group was significantly higher than that in Ad-IL-24 group(15.660±1.773,t=8.834,P<0.01)and Ad-ING4 group(13.220±1.628,t=7.858,P<0.01).RT-PCR proved Ad-ING4-IL-24 could obviously up-regulate the expression of bax and Caspase-3,and down-regulate the expression of bcl-2 and Survivin.Experiments showed the anti-tumor effects in Ad-IL-24,Ad-ING4,and Ad-ING4-IL-24 groups were significantly better than those in PBS group or Ad group.Ad-ING4-IL-24 could obviously regulate the expression of bax,Caspase-3,bcl-2 and Survivin.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,Caspase-3 and the down-regulations of bcl-2 and Survivin.
作者
梁光辉
丁志丹
袁东风
李振轩
杨吉成
赵军
邢文群
Liang Guanghui;Ding Zhidan;Yuan Dongfeng;Li Zhenxuan;Yang Jicheng;Zhao Jun;Xing Wenqun(Department,of Thoracic Surgery,Affiliated Cancer Hospital of Zhengzhou University,Zhengzhou 450008,China;Department of Thoracic Surgery,the First Affiliated Hospital of Zhengzhou Universityy Zhengzhou 450052,China;Department of Cell and Molecular Biology,School of Basic Medicine,Suzhou University,,Suzhou 215123,China;Department of Cardiothoracic Surgery,the First Affiliated Hospital of Soochow University,Suzhou 215000,China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2020年第7期1267-1271,共5页
Chinese Journal of Experimental Surgery
