中华耳鼻咽喉头颈外科杂志
中華耳鼻嚥喉頭頸外科雜誌
중화이비인후두경외과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
2013年
11期
942-950
,共9页
鼻咽肿瘤%白细胞介素类%基因疗法%放射疗法%细胞系,肿瘤
鼻嚥腫瘤%白細胞介素類%基因療法%放射療法%細胞繫,腫瘤
비인종류%백세포개소류%기인요법%방사요법%세포계,종류
Nasopharyngeal neoplasms%Interleukins%Gene therapy%Radiotherapy%Cell line,tumor
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.
目的 研究以腺病毒為載體的白細胞介素24(IL-24)基因(簡稱AdVIL-24)過錶達對人CNE-2Z鼻嚥癌細胞體內外放療的抑癌增效作用及潛在的作用機製.方法 將AdVIL-24感染CNE-2Z細胞,用反轉錄聚閤酶鏈反應(RT-PCR)法及Western blot法檢測IL-24在CNE-2Z細胞中的轉錄和錶達.體外實驗:將PBS組、AdV組、AdVIL-24組、放療組及AdVIL-24聯閤放療組的CNE-2Z細胞,採用四甲基偶氮唑藍(MTT)法檢測CNE-2Z細胞的生長情況；碘化丙啶(PI)染色檢測細胞週期,Annexin-V-PE/7-AAD染色檢測CNE-2Z細胞凋亡率的變化；RT-PCR檢測CNE-2Z細胞中P21、P27、cyclin E、CDK2、Bcl-2、Bax的轉錄；Western blot檢測CNE-2Z細胞中P21、P27、cyclin E、CDK2、Bcl-2、Bax、Caspase-3的錶達.體內實驗:採用CNE-2Z細胞株建立人鼻嚥癌裸鼠模型,然後將各組荷瘤裸鼠瘤體進行相應處理,動態測量腫瘤體積及最後取瘤稱量重量；免疫組化檢測P21、P27、cyclin E、CDK2、Bcl-2、Bax、Caspase-3、CD34、VEGF等因子的錶達以及微脈管密度.採用金正均法(Q值)計算聯閤用藥後的最終效應.結果 體外實驗:AdVIL-24聯閤放療組能使CNE-2Z細胞生長齣現抑製(P＜0.05,Q3d=0.916,Q4d=1.050)；G1期阻滯(50.37％,P＜0.05,Q=1.042);併誘導其凋亡(48.82％,P＜0.05,Q=1.042)；能明顯上調P21、P27、Bax/Bcl-2、Caspase-3等基因轉錄和錶達,下調cyclin E、CDK2基因的轉錄和錶達(P＜0.05；QP21 =0.959,QP27 =0.956,Qcyclin E=1.078,QCDK2=1.046,QBax/Bcl-2=0.995).體內實驗:AdVIL-24聯閤放療組能使CNE-2Z移植瘤生長齣現抑製(P＜0.05,Qvolume14=1.053,Qweight=1.004)；誘導其凋亡(P＜0.05,Q=0.974)；上調P21、P27、Bax/Bcl-2、cleaved-Caspase-3的錶達,下調cyclin E、CDK2的錶達(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的錶達以及微脈管密度較弱(P＜0.05；QCD34=0.990,QMVD=0.988).放療組VEGF的錶達齣現瞭明顯的錶達增彊(P＜0.05),然而AdVIL-24能夠明顯抑製其錶達(P＜0.05).結論 AdVIL-24聯閤放療能有效抑製腫瘤的生長促進其凋亡,是治療鼻嚥癌的一種可能有效的方法.
목적 연구이선병독위재체적백세포개소24(IL-24)기인(간칭AdVIL-24)과표체대인CNE-2Z비인암세포체내외방료적억암증효작용급잠재적작용궤제.방법 장AdVIL-24감염CNE-2Z세포,용반전록취합매련반응(RT-PCR)법급Western blot법검측IL-24재CNE-2Z세포중적전록화표체.체외실험:장PBS조、AdV조、AdVIL-24조、방료조급AdVIL-24연합방료조적CNE-2Z세포,채용사갑기우담서람(MTT)법검측CNE-2Z세포적생장정황；전화병정(PI)염색검측세포주기,Annexin-V-PE/7-AAD염색검측CNE-2Z세포조망솔적변화；RT-PCR검측CNE-2Z세포중P21、P27、cyclin E、CDK2、Bcl-2、Bax적전록；Western blot검측CNE-2Z세포중P21、P27、cyclin E、CDK2、Bcl-2、Bax、Caspase-3적표체.체내실험:채용CNE-2Z세포주건립인비인암라서모형,연후장각조하류라서류체진행상응처리,동태측량종류체적급최후취류칭량중량；면역조화검측P21、P27、cyclin E、CDK2、Bcl-2、Bax、Caspase-3、CD34、VEGF등인자적표체이급미맥관밀도.채용금정균법(Q치)계산연합용약후적최종효응.결과 체외실험:AdVIL-24연합방료조능사CNE-2Z세포생장출현억제(P＜0.05,Q3d=0.916,Q4d=1.050)；G1기조체(50.37％,P＜0.05,Q=1.042);병유도기조망(48.82％,P＜0.05,Q=1.042)；능명현상조P21、P27、Bax/Bcl-2、Caspase-3등기인전록화표체,하조cyclin E、CDK2기인적전록화표체(P＜0.05；QP21 =0.959,QP27 =0.956,Qcyclin E=1.078,QCDK2=1.046,QBax/Bcl-2=0.995).체내실험:AdVIL-24연합방료조능사CNE-2Z이식류생장출현억제(P＜0.05,Qvolume14=1.053,Qweight=1.004)；유도기조망(P＜0.05,Q=0.974)；상조P21、P27、Bax/Bcl-2、cleaved-Caspase-3적표체,하조cyclin E、CDK2적표체(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、VEGF적표체이급미맥관밀도교약(P＜0.05；QCD34=0.990,QMVD=0.988).방료조VEGF적표체출현료명현적표체증강(P＜0.05),연이AdVIL-24능구명현억제기표체(P＜0.05).결론 AdVIL-24연합방료능유효억제종류적생장촉진기조망,시치료비인암적일충가능유효적방법.
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.
