【作者】 谭广；

【导师】 王忠裕；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2005， 博士

【摘要】 胰腺癌是一种高度恶性肿瘤,近几年发病率逐年上升。在欧洲和北美人群中,胰腺癌在癌性死亡原因中列第五位,在中国上海,20 年内发病率增加了4 倍。由于胰腺癌在临床上缺乏特异表现,恶性程度高,极易发生转移,就诊时3/4 病人已属晚期,手术切除为生存期延长提供了最佳机会,可预后并没有明显改善。5 年生存率〈5%,仍有相当多的患者死于复发及转移,传统的化疗及放疗也显得相当无效。因此,基因及免疫治疗在胰腺癌治疗中异军突起,成为广大学者瞩目的焦点。肿瘤复发及难以治愈的根源在于肿瘤的免疫逃逸机制,而免疫逃逸的核心是机体缺乏对肿瘤抗原的有效递呈并诱导特异性抗肿瘤免疫反应。树突状细胞(DC)是目前发现的功能最强大的肿瘤抗原专职递呈免疫细胞(APC),它是唯一既能启动免疫应答又能有效刺激再次应答的APC ,DC 在诱导T 细胞免疫应答和T 细胞依赖性抗体生成中起重要作用,通过DC 递呈抗原诱导的主动特异性抗肿瘤治疗效果显著,DC 疫苗是目前最具应用潜能的治疗性疫苗。另一影响特异性抗肿瘤免疫治疗效果的因素是肿瘤环境中存在的免疫抑制现象,因此DC 及T 淋巴细胞在肿瘤抑制环境中的功能与生存力下降是特异性抗肿瘤治疗效果不佳的主要原因。那么提高DC活性与其在肿瘤抑制环境中的生存力又是当今DC疫苗抗肿瘤治疗的热点。研究发现, IFN-γ,TNF-α及IL-12 等细胞因子在DC 介导的特异免疫应答中起着不可忽视的作用,利用细胞因子基因修饰APC 是当今肿瘤基因治疗中最活跃的领域。IL-23 是近年发现的一种最新细胞因子,IL-23 可显著增强DC 的抗原递呈作用,增强DC 在肿瘤环境中的生存力,同时作为抗血管生成因子影响肿瘤微循环,DC 在IL-23 的作用下不仅诱导宿主产生防御性免疫反应而且增强其自动免疫能力。恶性肿瘤都存在细胞免疫和/或体液免疫的缺陷,并且随病程进展和病变范围的扩大而加重。恶性肿瘤患者的免疫力特别是细胞免疫的降低有以下原因:1)肿瘤患者体内出现免疫因子,包括肿瘤抗原抗体复合物、封闭因子和免疫调节球蛋白等,它们能阻碍免疫活性细胞的抗肿瘤作用,抑制T 细胞的免疫功能;2)恶性肿瘤转移而浸润免疫组织,造成免疫系统功能的损害;3)严重的营养代谢障碍引起免疫系统功能的缺陷;4)抗癌化学药物的免疫抑制反应。那么,是否存在一种既对肿瘤细胞具有显著杀伤作用同时又对机体的免疫系统有保护效应的药物呢?β-榄香烯是从中药温莪术根茎提取的有效单体,β-榄香烯直接作用于肿瘤细胞,抑制其DNA、RNA 及蛋白质的合成,并作用于细胞膜,使细胞破裂而凋亡。实验研究证明β-榄香烯是一种非细胞毒性的抗癌制剂,在对多种恶性肿瘤有确切疗效,同时对机体的免疫系统功能有保护和增强作用。陈剑群等在β-榄香烯对恶性肿瘤患者T 淋巴细胞亚群的影响研究中发现,β-榄香烯能显著升高T 淋巴细胞亚群的功能,对辅助性T 淋巴细胞和杀伤/抑制性T 细胞均有促进,为机体抵抗肿瘤,抑制恶性肿瘤的发展产生积极作用。吴伟忠等发现,β-榄香烯还具有抑制恶性肿瘤细胞浸润与转移的作用,并使肿瘤的诱导分化发生逆转。虽然人体胰腺癌细胞移植裸鼠可以用于胰腺癌的研究,但不能用于探索胰腺癌发生的各个时期[5,6],同时由于裸鼠无免疫系统功能因而缺乏免疫应答能力,因此对裸鼠难以进行免疫抗肿瘤研究。如果能够用相对简单的方法建立一种胰腺癌的模型,而该模型的基因突变类型又符合于人类胰腺癌,它将为人体胰腺癌发病机理和早期诊断及治疗的研究奠定基础[7]。基于上述理论与构想,我们提出以下问题:1)是否能够建立一种稳定且与人类胰腺癌相似具有免疫系统功能的胰腺癌动物模型,并观察β-榄香烯对胰腺癌细胞的体外抑制;2)如何在体外大量扩增DC 并初步探讨DC 诱导的特异性CTLs对胰腺癌细胞的抑制;3)IL-23 作为一种最新发现的功能性细胞因子能否显著增强DC 的活性及其诱导的宿主抗肿瘤免疫反应;4)β-榄香烯和DC 疫苗对实体胰腺癌肿瘤的协同治疗作用如何? 本课题从以上目的出发,探讨了β-榄香烯联合IL-23 基因修饰的DC 疫苗对小鼠胰腺癌体内外的治疗作用,为树突状疫苗治疗胰腺癌的临床应用提供理论基础,为中西医结合临床治疗胰腺癌提供必要的理论依据。分述如下: 目的建立二甲基苯蒽(7,12-dimethylbenz[alpha]anthracene,DMBA)诱发小鼠胰腺癌模型并观察P53 基因的表达。方法于C57BL/6 小鼠胰体尾部以1ml 注射器注射(深2mm)置入二甲基苯蒽(DMBA)混悬液(10mg/100g 体重),在普通环境中饲养小鼠。采用SP 免疫组织化学方法,检测p53 基因的表达情况。更多还原

【Abstract】 Objective To study the pathogenesis of pancreatic carcinoma and investigate the p53 genes expression in carcinogenesis and development of pancreatic carcinoma induced by DMBA. Methods High dose (10mg/100gweight) of dimethylbenzanthracene (DMBA) was put into the parenchyma of mouse pancreas to develop a model of pancreatic carcinoma in mice. Immunohistochemical SP method and was used to study the expression of P53 in pancreatic carcinoma induced by DMBA. Results The prevalence of pancreatic carcinoma among DMBA-treated mice within 3-5 months was 30% ( 12/40 ) . 3 mice developed pancreatic ductal adenocarcinomas with glandular duct-like distribution of cancer cells, various sizes of glandular cavities, obvious nucleiheterotpic and typical cocus of cancer cells under the microscope. There was no expression of P53 in normal pancreatic tissues. The overexpression of P53 was detected in pancreatic carcinoma (10/12). Conclusion High dose DMBA was put into the parenchyma of rat pancreas could obtained a pancreatic cancer model with high incidence in a short time. There are P53 protein overexpressions in the pancreatic carcinoma induced by DMBA in mice, and P53 protein overexpression may relate to the degree of malignancy of the tumors , the result obtained in this study was quiet similar with the human pancreatic cancer gene mutation types. Objective To study the method of obtaining a large number of dendritic cells. To study the specific CTLs effect against tumor cells initiated by dendritic cells pulsed with peptide of cancer cell . To offer the basic theory for the treatment of pancreatic cancer with DC vaccine combined with β-elemene. Methods Development of cells with cytologic features of dendritic cell in bone marrow cultures supplemented with GM-CSF and IL-4. determining the DC phenotype and the specific structure of DC by electronic microscopy. To observe the CTLs effect against pancreatic carcinoma leading by the Dendritic cell pulsed with tumor cell lysate in vitro. Results A large number of typical dendritic cells proliferated by supplementing with GM-CSF and IL-4 cytokines. Dendritic cell has specific cell appearance and structure , and highly express various cell surface molecules. rmTNF-αhas the ability of stimulating DC mature, the mature DC has the enhancing abilities of antigen presenting and IL-12 self-secreting ,as well as, express higher levels of CD54、MHC-II 及CD86 molecules than control group, P<0.05. T lymphoid cell stimulated by dendritic cells without tumor antigen could not recognize and kill the target cells , only if dendritic cells pulsed with peptide of cancer cell can lead a strong immune response to special tumor cells. The inhibiting ratio of CTLs is significant higher than that in other groups. Conclusion Bone marrow DC has strong ability of inducing special CTLs against determined cancer cells after they were pulsed with tumor cell lysate. DC vaccine is probably a new immunotherapeutic method against tumor in the near future.Objective To clone two subunits P19 and P40 of the Interleukin 23 gene, to construct the dual-expression vector pcDNA3-IL-23 in order to study the anti-tumor therapy inducing by the dendritic cells genetically transfect by Interleukin 23. Methods total RNA of P19 and P40 were extracted from thymus and spleen of mice respectively by one step way, and cDNA was amplified by RT-PCR, the PCR product was linked with pMD18-T vector and E.Coli JM109 was transformed by pMD18-P19 and pMD18-P40, positive plasmid was selected by PCR method. P19 and P40 gene were digested from pMD18-P19 and pMD18-P40, the product was inserted into pcDNA3.0 expression vector, selected positive plasmid was tested by XhoI/KpnI restrict enzyme. Result Total RNA of P19 and P40 from thymus and spleen respectively was complete, The sequence of cloned gene is consistent with the reported sequence of the Genebank. The constructed pcDNA3-IL-23 eukaryotic dual-expression vector was proved to have P19 and P40 subunits of 1.62kbp by restriction endonuclease analysis. Conclusion We successful更多还原