【作者】 张征峥；

【导师】 王润田；

【作者基本信息】 河北医科大学 ， 免疫学， 2004， 硕士

【摘要】 目的：肿瘤是当前严重威胁人类健康的疾病之一。目前，手术治疗、放疗、化疗是治疗肿瘤的主要治疗手段。在手术治疗中，由于肿瘤分期、分级和生长部位等因素限制，许多肿瘤不适于手术切除。在肿瘤的放疗、化疗中，并非所有肿瘤细胞均对之敏感或均适宜应用，而且长期的放疗、化疗还可损伤机体的免疫系统，甚至影响其他一些重要组织器官的功能，诱发新的疾病。因此，十分需要发展肿瘤治疗的新方法。免疫治疗不仅能够提高机体免疫力、而且还能特异性杀伤肿瘤细胞而不伤及或极少能伤害正常组织器官，因而，日益受到人们的关注。目前，细胞因子治疗是肿瘤免疫治疗中研究的热点之一。以往有些研究将细胞因子蛋白直接注入体内治疗肿瘤，但由于细胞因子半衰期短、用量较大、并有很大的毒副作用，因而其临床应用受到一定限制。因此，近几年相关研究主要集中于将细胞因子基因导入无或低免疫原性的肿瘤细胞，不仅能使部分肿瘤细胞丧失致瘤性，而且还可使其变为强免疫原性的细胞。这种经过修饰后的肿瘤细胞不仅能够在体内刺激宿主T淋巴细胞产生特异性的免疫反应，杀伤这些经过基因修饰后的肿瘤细胞，而且对未经修饰的野生型肿瘤细胞亦能产生特异性的杀伤作用，在一定程度上展示了细胞<WP=4>因子基因治疗肿瘤的诱人前景。IL-15是1994年Grabstein等发现的一种新的细胞因子。众所周知，NK细胞能广谱的杀伤肿瘤细胞，CD8+T细胞能特异地杀伤肿瘤细胞，而IL-15恰能促进这两种细胞的增殖，提高它们杀伤肿瘤细胞的细胞毒活性。因此，激起了人们探索用IL-15对肿瘤进行免疫治疗的浓厚兴趣。IL-15mRNA组织分布极为广泛，可见于心、肺、肾、胎盘及骨骼肌。另外，单核/巨噬细胞、树突状细胞、成纤维细胞、上皮细胞及骨髓基质细胞中也都有不同程度的IL-15mRNA的表达。但是，研究发现，IL-15的分泌受到多种因素的调控。其中，IL-15的长信号肽是影响IL-15蛋白分泌的主要因素，而以IL-2信号肽替代IL-15信号肽以后，有可能会促进IL-15蛋白的分泌。因此，本研究拟用基因拼接的方法将IL-2信号肽（IL-2sp）基因替代IL-15信号肽（IL-15sp）基因，构建成改型的IL-15基因，将其插入真核表达载体，转染小细胞肺癌细胞，为研究IL-15细胞因子的肿瘤基因治疗奠定基础。方法：以pCI-IL-15为模板进行PCR，以不同的上下游引物分别扩增得到包括其信号肽在内的人IL-15cDNA全长基因片段（FIL-15cDNA）和无信号肽的IL-15成熟肽基因片段（FIL-15mp）；以pcDNA3-IL-2为模板进行PCR，扩增得到人IL-2信号肽基因片段（FIL-2sp）。将FIL-2sp与FIL-15mp用基因拼接（SOEing, Gene splicing by overlap extension）的方法进行PCR扩增，得到改型的IL-15基因片段<WP=5>（FIL-2sp-IL-15mp）。用EcoRⅠ和BamHⅠ限制性内切酶，分别酶切FIL-2sp-IL-15mp扩增产物和真核表达载体pEGFP-N1,将两者的酶切产物用T4 DNA连接酶连接，构建成改型IL-15的重组真核表达载体pEGFP-N1/IL-2sp/IL-15mp (pEGFP-N1/215)。同样方法分别酶切FIL-15mp和FIL-15cDNA，分别与经相同内切酶酶切的载体pEGFP-N1连接，构建成含有FIL-15mp的重组真核表达载体pEGFP-N1/IL-15mp和含有FIL-15cDNA的重组真核表达载体pEGFP-N1/IL-15作为对照。将这三种重组真核表达载体经PCR、双酶切和DNA序列分析鉴定后，以Lipofectamine 2000介导将鉴定基因重组正确的真核表达载体转染人小细胞肺癌细胞株NCI-H446，经G418 (0.8mg/ml)条件培养筛选和有限稀释法克隆化培养，得到被稳定转染的细胞株，用反转录PCR（RT-PCR）方法检测转染细胞株细胞中IL-15mRNA的表达；用完全培养基培养这些转染细胞，待细胞长满后，取细胞的培养上清，冻干法浓缩50倍后，ELISA方法检测分泌至其中的IL-15蛋白。结果1、PCR扩增获得了FIL-15mp、FIL-15cDNA、FIL-2sp、FIL-2sp-IL-15mp；构建了分别含有上述3种基因片段的真核表达载体pEGFP-N1/IL-15mp、 pEGFP-N1/IL-15和pEGFP-N1/215。2、将三种真核重组表达载体分别转染小细胞肺癌细胞株NCI-H446，得到了被稳定转染的细胞株，可检测到IL-15mRNA的表达；将转染细胞株培养上清浓缩50倍后，<WP=6>只在pEGFP-N1/215稳定转染的细胞株培养上清中，检测到IL-15蛋白分泌，分泌量为22pg/ml，而pEGFP-N1/ IL-15mp和pEGFP-N1/IL-15转染的细胞株上清中未检测到分泌的IL-15蛋白。结论：以IL-2信号肽基因代替IL-15原有信号肽基因构建的改型IL-15基因的真核表达载体pEGFP-N1/215，转染人小细胞肺癌细胞株后，确能促进IL-15蛋白的分泌。构建的改型IL-15基因片段和改型IL-15真核表达载体，可以用于进一步研究IL-15的肿瘤基因治疗。更多还原

【Abstract】 Objective: Cancer is one of the serious disease threatening human health currently. At present, operation, radiotherapy and chemotherapy are the main methods to treat tumors. But because of the limit of stage classification and location of the tumor,operation is restricted. In the course of radiotherapy or chemotherapy, not all of the tumors are sensitive or applicable to these methods. At the same time, long-term therapy of radiotherapy or chemotherapy can also damage immune system, even influence the function of other organs, further induce a new disease. Therefore, to develop some new approach to overcome cancers is necessary. Immunotherapy can not only enhance the immune function of body, but also kill tumors in special pathway, and does not influence the function of other normal tissues. At present, more and more people have pay attention to immunotherapy. Cytokine therapy is one of the focus in immunotherapy research. In previous research, researchers used to injected the protein of cytokine into body with tumor to treat cancer. Because of the short half-life, large usages, strong side-effect, it was limited in clinical application. Therefore, in recent <WP=8>years, researchers have transfer cytokine genes into tumors which have no or less immunogenicity. By this way, some tumor cells had lost the tumorigenicity，and became strong immunogenicity. These modified tumor cells can not only stimulate the immune response of T lymphocytes to kill them, but also to kill unmodified wild tumors specially. To some extent, it exhibits an attractive foreground to treat tumors with cytokine gene therapy.Interleukin 15 is a new cytokine that was found by Grabstein in 1994. It is well known that natural killer cells can kill tumor cells in a broad-spectrum way, CD8+T cells can kill tumor cells in a special way. IL-15 has a critical role in development and function of NK and CD8+ T cells. So it evokes the stong interesting of tumor immunological treatment by using IL-15. IL-15 mRNA is not only widely expressed in heart, lung, kidney, placenta and skeleton muscle, but also expressed in monocytes/macrophages, dendritic cells, fibroblasts，epithelia cells and marrow stroma cells. But researchers found that the expression of IL-15 was regulated by many factors. Long signal peptides of IL-15 is the main disadvantage factor that in the process of secreting IL-15. To substitute IL-2 signal peptide (IL-2sp) for IL-15 signal peptide (IL-15sp) can promote secretion of IL-15. In present study, we substituted with IL-2 signal peptide for IL-15 <WP=9>peptide and constructed a modified IL-15 gene (FIL-2sp-IL-15mp), inserted FIL-2sp-IL-15mp into a eukaryotic expression vector and transfered into tumor cells. Which established a foundation for investigating the tumor therapy with IL-15 gene .Methods: The complete cDNA fragment of hIL-15（FIL-15cDNA） and the DNA fragment encoding hIL-15 mature peptide（FIL-15mp） were amplified by PCR from pCI-IL-15. The DNA fragment of hIL-2 sp was amplified by PCR from pcDNA3-IL-2. Using the method of gene splicing by overlap extension, the fragment of modified IL-15 gene（FIL-2sp-IL-15mp） was acquired. The FIL-2sp-IL-15mp and the eukaryotic expression vector, pEGFP-N1, , were digested with EcoR Ⅰand BamH Ⅰ respectively and the FIL-2sp-IL-15mp was inserted into EcoR Ⅰand BamH Ⅰsites of pEGFP-N1 to construct pEGFP-N1/ IL-2sp/IL-15mp (pEGFP-N1/215). By the same means the recombined eukaryotic expression vector pEGFP-N1/IL-15mp ,which contained hIL-15 mature peptide coding sequence, and the recombined eukaryotic expression vector pEGFP-N1/IL-15 , which include FIL-15cDNA were acquired respectively for controls. After identified by PCR, restriction endonuclease and DNA sequencing analysis, the three eukaryotic expression vectors were transfected by the Lipofectamine 2000 into NCI-H446 respectively. These stable transfectsed cells were selected respectively in complete culture medium <WP=10>supplemented with 0.8mg/ml G418 and limited dilute. When the cell lines that tranfected with the e更多还原