【作者】 潘新亭；

【导师】 李德春；

【作者基本信息】 苏州大学 ， 普通外科学， 2008， 博士

【摘要】 目的:1.构建携带IL-24基因片段的重组复制缺陷型腺病毒Ad-IL-24,为探讨治疗胰腺癌的方法提供实验基础。2.观察重组腺病毒介导的IL-24基因对人胰腺癌细胞patu8988生长的抑制作用,探讨IL-24抑制胰腺肿瘤生长的机理。3.研究重组腺病毒介导的IL-24基因对小鼠髓系树突状细胞成熟活化的影响,探讨IL-24基因在肿瘤免疫治疗中的作用。4.观察重组腺病毒介导的IL-24基因对裸鼠体内胰腺肿瘤生长的抑制作用,探讨IL-24对胰腺癌的治疗潜力。方法:从Genbank中查得IL-24基因cDNA全长序列,选择5’端转录起始位点附近长621bp的基因序列为目的基因片段,设计特异性克隆引物。从人外周血单个核细胞(PBMC)中提取总RNA,以总RNA为模板用逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR )扩增IL-24基因片段。将经基因测序证实为正确的基因片段反向克隆到重组腺病毒载体AdEasy系统中的穿梭质粒pAdTrack-CMV的多克隆位点(multiple cloning site,MCS ),构建携带IL-24基因片段的重组穿梭质粒pAdTrack-CMV-IL-24;再将pAdTrack-CMV-IL-24与重组腺病毒载体AdEasy系统中的骨架质粒pAdEasy-1在大肠杆菌DH5a内进行同源重组,经293细胞包装生成携带IL-24基因片段的重组腺病毒Ad-IL-24,同时构建空病毒Ad-GFP。通过在荧光显微镜下观察发绿色荧光数检测绿色荧光蛋白(green fluorescence protein,GFP)的表达,测定Ad-IL-24和Ad-GFP的滴度。用感染复数(multiplicities of infection,MOI)为100的Ad-IL-24感染人胰腺癌细胞株patu8988,用Western blot、ELISA检测细胞培养液中IL-24的蛋白表达;用MTT法、流式细胞术(FCM)分别检测Ad-IL-24对人胰腺癌细胞株patu8988生长情况的影响。采用小鼠髓系树突状细胞(BMDCs)开展研究,从小鼠骨髓中获取和培养未成熟树突状细胞(dendritic cells,DC),通过FCM、ELISA法检测DC经IL-24转染后表型和功能状态的变化,研究IL-24促进DC成熟活化的作用。裸鼠成瘤实验,观察人胰腺癌patu8988细胞的成瘤情况及IL-24对裸鼠体内胰腺肿瘤的治疗作用;通过免疫组化检测肿瘤组织中微血管密度MVD、VEGF、MMP-2的表达。结果:1.成功的从人外周血单个核细胞(PBMC)中提取并扩增出621bp的IL-24基因片段,经DNA测序证实该基因片段序列与Genbank中IL-24基因cDNA序列5’端转录起始位点附近621bp的基因序列完全一致。2.成功地构建出携带IL-24基因片段的重组腺病毒Ad-IL-24,病毒滴度>5×1010pfu/ml。3.Ad-IL-24感染的patu8988细胞增殖能力降低,凋亡率明显升高。4.IL-24刺激BMDCs 48h后,细胞表现出成熟DCs的表型和功能特征,即共刺激分子CD40, CD80, CD86上调表达;并能有效拮抗负性调控因子IL-10下调DCs上CD40、CD80、CD86表达的作用,细胞培养液中IL-12、TNF-α的表达水平升高。5.瘤内注射Ad-IL-24后胰腺肿瘤瘤体生长减慢,肿瘤内微血管密度MVD、VEGF、MMP-2较对照组明显减少,荷瘤裸鼠的生存期明显延长。结论:1.重组腺病毒介导的IL-24基因可明显抑制胰腺癌细胞的生长、促进胰腺癌细胞凋亡。2.重组腺病毒介导的IL-24基因可促进树突状细胞的活化及功能成熟。3.重组腺病毒介导的IL-24基因能抑制胰腺肿瘤血管生成,延长荷瘤裸鼠的生存期。更多还原

【Abstract】 Objective:This study was conducted (1) to construct a recombinant adenoviral vector carrying IL-24 gene(Ad-IL-24);(2) to detect the change of the human pancreatic carcinoma cell patu8988 infected with Ad-IL-24 and elucidate the molecular mechanism of Ad-IL-24 in inhibiting the growth of pancreatic carcinoma(PC);(3)to analyze the variations of the surface phenotype and immuno-status of dendritic cells that they had been IL-24 gene modified;(4) to observe the growth of PC in vivo,and demonstrat the effect of Ad-IL-24 in treatment of PC.Methods:Total RNA was extracted from PBMC, and then a 621bp fragment at the 5’ end of human IL-24 cDNA was synthesized by reverse-transcription polymerase chain reaction (RT-PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV,with the resultant plasmid and the backbone plasmid pAdEasy-l,the homologous recombination took place in the E.coli DH5a and the recombinant adenoviral plasmid carrying the IL-24 gene was constructed was generated. The adenoviruses(Ad-IL-24) were packaged and amplified in the HEK 293 ce1ls. Ad-IL-24 infected the human pancreatic carcinoma cell line patu8988. Then the production of IL-24 in the human pancreatic carcinoma patu8988 cells was detected with Western Blot Analysis,ELISA and FCM. According to Inaba’smethods,bone marrow-derived Dcs were induced. The surface molecules of DCs are tested by FCM. Following the Ad-IL-24-infected cells was subcutaneously inoculated in nude mice, and injected intratumorally into pre-existing tumors.After four weeks,the tumor tissue was detected with IHC and RT-PCR.Results:The recombinant adenovirus vector carrying IL-24 was constructed Successfully,fluorescence the strong green fluorescence was microscopy. The viral titer was observed in HEK 293 cells under a fluorescence microscopy.The viral titer was 5×1010 pfu/ml. Compared with PBS or Ad-GFP-infected patu8988 cells, infection of patu8988 cells with Ad-IL-24 significantly reduced the growth of human pancreatic carcinoma patu8988 cells,and the tumor volume resulted in reduction in nude mice.In addition,direct intratumoral injection of Ad-IL-24 into pre-existing tumors significantly impaired the further expansion of the tumor mass and resulted in a reduction in tumor.Ad-IL-24 therapy resulted in a reduction in tumor vessel density.In vitro,Ad-IL-24 induce the phenotypic and functional maturation of DC. Beside these results, the growth rate of PC significantly reduced and the survivals prolonged in the Ad-IL-24-infected nude rate modelsConclusion:(1)The recombinant adenovirus with IL-24 can effectively inhibit cells growth and promote the apoptosis of human pancreatic carcinoma patu8988 cells. (2)Ad-IL-24 induce the phenotypic and functional maturation of DC.(3)Ad-IL-24 can effectively prolong survivals and attenuate angiogenesis of pancreatic carcinoma in vivo models and demonstrat a therapeutic potential for human pancreatic carcinoma.更多还原