腺病毒介导IL-24基因诱导皮肤鳞状细胞癌细胞COLO16的凋亡  

Adenovirus-mediated IL-24 ge ne expression induces apoptosis in a human squamous cell carcinoma cell line COLO 16

作　　者：马欣欣[1] 张孟丽[1] 李玲君[1] 曹玉萍[1] 吴秋菊[1] 马鹏程[1] 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所,南京210042

出　　处：《中华皮肤科杂志》2015年第9期611-615,共5页Chinese Journal of Dermatology

摘　　要：目的探讨腺病毒介导IL-24基因表达载体（Ad—IL-24）对皮肤鳞状细胞癌（鳞癌）细胞COLO16的凋亡的影响，并探讨其作用机制。方法将构建的Ad—IL-24腺病毒感染COLO16细胞，qPCR法检测IL-24基因的表达，MTr法检测IL_24过表达对细胞生长的影响，流式细胞仪检测IL-24过表达对COLO16细胞凋亡的影响，激光扫描共聚焦显微镜观察在IL-24过表达后COLO16细胞凋亡的形态变化，Western印迹检测Bax，Bcl-2以及活化的caspase3蛋白水平，qPCR法检测Bax、Bcl-2基因mRNA的表达，免疫荧光方法，检测IL-24过表达后Bax，Bcl-2的表达情况。结果MTT结果显示，Ad—IL-24组的细胞从第4天开始出现明显抑制，第6天差异最明显（P〈0．05），并呈现时间依赖性，而Ad—GFP组与对照组比较，差异无统计学意义（P〉0．05）。流式细胞仪显示。Ad—IL-24组的COLO16细胞凋亡率（13．10±0．92）％，显著高于对照组（3．69±0．36）％（P〈0．05）和空载体组（3．39±1．06）％（P〈0．05），后两组间比较，差异无统计学意义（P〉0．05）。激光扫描共聚焦显微镜显示，Ad—IL-24组细胞加速凋亡。免疫荧光、Western印迹法和QPCR法结果显示，IL-24过表达后Bax在细胞中的表达明显升高，而Bcl-2在细胞中的表达却明显下降。Western印迹法和qPCR法显示，Ad—IL-24组的Bax、Bcl-2的蛋白及mRNA水平分别与对照组和空载组比较，出现了明显的上升和下降，在蛋白水平上产生与抗cleavedcaspase3抗体结合的特异性条带。结论Ad—IL_24可诱导鳞癌细胞COLO16细胞凋亡，其机制可能与上调Bax基因、下调Bcl-2基因表达，并活化caspase3有关。Objective Toinvestigatethe effect of adenovirus-mediated IL-24 （Ad-IL-24） gene expression on the apoptosis in a human squamous cell carcinoma cell line COLO 16, and to explore the underlying molecular mechanism. Methods Cultured COLO 16 cells were divided into two groups to be transfected with an adenovirus vector carrying the IL-24 gene （Ad-IL-24 group） or green fluorescent protein （Ad-GFP group）, while those receiving no treatment served as the control group. After culture for different durations, qPCR was performed to quantify IL-24 gene expression, methyl thiazolyl tetrazolium （MTY） assay to evaluate the proliferative activity of COLO 16 cells, flow cytometry to detect the apoptosis of COLO 16 cells, laser scanning confocal microscopy （LSCM） to observe the morphological changes of COLO 16 cells, Western blot to determine the levels of Bax and Bcl-2 proteins and to evaluate the activation of caspase-3, qPCR to determine the levels of Bax and Bcl-2 mRNAs, an immunofluorescence assay to observe the expression of Bax and Bcl-2 proteins. Statistical analysis was carried out by a two-sample t-test with the SPSS 19.0 software. Results MTI＇ assay showed that the proliferation of COLO 16 cells in the Ad-IL-24 group was significantly inhibited as early as 4 days after the transfection; thereafter, the inhibitory effect increased in a time- dependent manner, and peaked on day 6 （P 〈 0.05 ）. However, there was no significant difference in cellular proliferative activity between the Ad-GFP group and control group （P 〉 0.05）. Flow cytometry revealed that the apoptosis rate was significantly higher in the Ad-IL-24 group （ 13.10% ±0.92%） than in the control group （3.69% ± 0.36%, P 〈 0.05 ） and Ad-GFP group （3.39% ± 1.06%, P 〈 0.05 ）, but no significant difference was observed between the control group and Ad- GFP group （P〉 0.05）. LSCM demonstrated that the apoptosis of COLO 16 cells was accelerated in the Ad-IL-24 group. The immunofluorescence assay, Western blot and

关 键 词：肿瘤 鳞状细胞 白细胞介素24 细胞凋亡 半胱氨酸天冬氨酸蛋白酶3 COLO-16 

分 类 号：R739.5[医药卫生—肿瘤]