腺病毒介导的白介素24联合放疗对CNE-2Z细胞株及其裸鼠移植瘤的抑瘤增效作用

Adenovirus-mediated interleukin-24 enhanced the anfitumor effect of radiotherapy on nasopharyngeal carcinoma in vitro and in vivo

目的 研究以腺病毒为载体的白细胞介素24（IL-24）基因（简称AdVIL-24）过表达对人CNE-2Z鼻咽癌细胞体内外放疗的抑癌增效作用及潜在的作用机制.方法 将AdVIL-24感染CNE-2Z细胞,用反转录聚合酶链反应（RT-PCR）法及Western blot法检测IL-24在CNE-2Z细胞中的转录和表达.体外实验：将PBS组、AdV组、AdVIL-24组、放疗组及AdVIL-24联合放疗组的CNE-2Z细胞,采用四甲基偶氮唑蓝（MTT）法检测CNE-2Z细胞的生长情况；碘化丙啶（PI）染色检测细胞周期,Annexin-V-PE/7-AAD染色检测CNE-2Z细胞凋亡率的变化；RT-PCR检测CNE-2Z细胞中P21、P27、cyclin E、CDK2、Bcl-2、Bax的转录；Western blot检测CNE-2Z细胞中P21、P27、cyclin E、CDK2、Bcl-2、Bax、Caspase-3的表达.体内实验：采用CNE-2Z细胞株建立人鼻咽癌裸鼠模型,然后将各组荷瘤裸鼠瘤体进行相应处理,动态测量肿瘤体积及最后取瘤称量重量；免疫组化检测P21、P27、cyclin E、CDK2、Bcl-2、Bax、Caspase-3、CD34、VEGF等因子的表达以及微脉管密度.采用金正均法（Q值）计算联合用药后的最终效应.结果 体外实验：AdVIL-24联合放疗组能使CNE-2Z细胞生长出现抑制（P＜0.05,Q3d=0.916,Q4d=1.050）；G1期阻滞（50.37％,P＜0.05,Q=1.042）;并诱导其凋亡（48.82％,P＜0.05,Q=1.042）；能明显上调P21、P27、Bax/Bcl-2、Caspase-3等基因转录和表达,下调cyclin E、CDK2基因的转录和表达（P＜0.05；QP21 =0.959,QP27 =0.956,Qcyclin E=1.078,QCDK2=1.046,QBax/Bcl-2=0.995）.体内实验：AdVIL-24联合放疗组能使CNE-2Z移植瘤生长出现抑制（P＜0.05,Qvolume14=1.053,Qweight=1.004）；诱导其凋亡（P＜0.05,Q=0.974）；上调P21、P27、Bax/Bcl-2、cleaved-Caspase-3的表达,下调cyclin E、CDK2的表达（P ＜0.05,QP21=0.920,QP27=0.937,Qcyclin E=1.060,QCDK2=1.019,QBax/Bcl-2=0.982,Qcleaved-Caspase-3=0.927）；CD34、VEGF的表达以及微脉管密度较弱（P＜0.05；QCD34=0.990,QMVD=0.988）.放疗组VEGF的表达出现了明显的表达增强（P＜0.05）,然而AdVIL-24能够明显抑制其表达（P＜0.05）.结论 AdVIL-24联合放疗能有效抑制肿瘤的生长促进其凋亡,是治疗鼻咽癌的一种可能有效的方法.

Objective To investigate the inhibitory effect of adenovirus-mediated interleukin-24 （AdVIL-24） in conjunction with ionizing radiation on the growth of CNE-2Z human NPC cells in vitro and in vivo and underlying mechanisms.Methods The CNE-2Z cells were transfected with AdVIL-24 alone or combined with radiotherapy.The transfection efficacy of AdVIL-24 in CNE-2Z cells was determined by RT PCR and Western blot.Cell growth was assessed by MTT assay,apoptosis was detected by flow cytometry through double staining of cells with propidium iodide （PI） and the expressions of P21,P27,cyclin E,CDK2,Bax,Bcl-2 and caspase-3 were detected with semi-quantitative RT-PCR and western blot,respectively.Anti-tumor effect of AdVIL-24 was observed using CNE-2Z human nasopharyngeal carcinoma transplanted tumor in athymic nude mouse model.The volume and weight of the xenografted tumors were measured and the expressions of P21,P27,cyclin E,CDK2,Bax,Bcl-2,caspase-3,CD34 and VEGF and the microvessel density in xenografted tumors were determined by immunohistochemistry analysis.Results AdVIL-24 plus radiotherapy induced cell growth inhibition （P 〈 0.05,Q3d,4d =0.916,1.050）,cell cycle G1 phase arrest （50.37%,P〈0.05,Q =1.042） and apoptosis（48.82%,P 〈0.05,Q =1.042）,substantial upregulations of P21,P27,the ratio of Bax/Bcl-2 and cleaved caspase-3 and downregulations of cyclin E and CDK2（P 〈0.05,QP21 =0.959,QP27 =0.956,Qcyclin E =1.078,QCDK2 =1.046,QBax/Bcl-2 =0.995）in vitro.In the xenografted tumors,AdVIL-24 plus radiotherapy induced cell growth inhibition（P 〈 0.05,Qvolume14 =1.053,Qweight =1.004）,apoptosis （P 〈 0.05,Q =0.974）,substantial upregulation of P21,P27,the ratio of Bax/Bcl-2 and cleaved caspase-3 and downregulation of cyclin E and CDK2 （P 〈 0.05 ; QP21 =0.920,QP27 =0.937,QcyclinE =1.060,QCDK2 =1.019,QBax/Bcl-2 =0.982,Qcleaved-Caspase-3 =0.927）,decreased the tumor vessel CD34 expression and microvessel density.AdVIL-24 potentially blocked the radiation-induced enhancement of VEGF.Conclusion AdVIL-24 gene therapy combined with radiotherapy may be a novel and effective treatment strategy for human NPC.