【作者】 张哲；

【导师】 单保恩； 艾军；

【作者基本信息】 河北医科大学 ， 免疫学， 2010， 硕士

【摘要】 目的:乳腺癌是女性最常见的恶性肿瘤,严重影响着妇女的身心健康。近年来,生物治疗已成为肿瘤治疗的一种新模式,其中免疫基因治疗是肿瘤生物治疗的研究热点,通过将不同免疫分子基因导入肿瘤或免疫效应细胞,使其在机体表达并分泌有活性的细胞因子,促使肿瘤细胞的凋亡或通过增强免疫系统功能,以加速肿瘤的消退。白细胞介素27(IL-27)是2002年Pflanz报道的一种IL-6/IL-12家族细胞因子[1],作用于固有免疫和适应性免疫系统的多种细胞,发挥广泛的免疫调节作用。IL-27除在Th1反应中起重要作用外,还具有抗感染﹑诱导炎症反应等多种生物学活性[2]。树突状细胞(dendritic cells, DC)是机体功能最强的专职抗原提呈细胞,能高效地摄取、加工处理和提呈抗原,具有较强的迁移能力,并能激活初始T细胞,处于启动、调控和维持免疫应答的中心环节[3-4]。本实验将IL-27基因转染人外周血来源的DC,制备IL-27/DC疫苗,观察其对乳腺癌细胞的杀伤活性,为抗乳腺癌免疫治疗提供实验依据。方法:1建立转染IL-27基因的DC疫苗(IL-27/DC)首先,分离、培养人外周血DC并诱导其成熟,用光学显微镜观察成熟DC细胞的形态,流式细胞技术检测及比较成熟与未成熟DC细胞的表型及成熟状况;将携带IL-27基因的逆转录病毒转染成熟DC细胞,用光学显微镜观察DC﹑IL-27/DC细胞的形态变化,RT-PCR法检测IL-27/DC、DC中IL-27 mRNA表达水平,ELISA法检测IL-27/DC培养上清中IL-27含量。2 IL-27诱导干扰素(IFN-γ)水平分析ELISA法检测细胞培养上清诱导人外周血单个核细胞(human peripheral blood mononuclear cells, hPBMCs)产生IFN-γ和IL-4水平。3淋巴细胞增殖反应从人外周血分离和培养T细胞;用MTT法检测DC﹑IL-27/DC分别对T细胞增殖能力的刺激作用,并绘制生长曲线。4细胞毒性T细胞(cytotoxic T lymphocyte, CTL)杀伤效应检测MTT法检测DC﹑IL-27/DC疫苗活化的CTL在体外杀伤乳腺癌细胞4T1的活性。5动物模型的建立[5]及IL-27/DC体内抗肿瘤活性研究把BALB/c小鼠随机分成三组,分别接种疫苗IL-27/DC、DC于小鼠皮下,未接种细胞小鼠作为对照组。两周之后,于三组小鼠皮下接种乳腺癌细胞4T1,观察、比较其在小鼠体内的成瘤性﹑生长情况和小鼠的生存期。ELISA法检测各组小鼠脾细胞分泌IFN-γ的能力。结果:1从外周血分离、诱导的成熟DC与未成熟DC比较,高表达CD80﹑CD86分子。将携带IL-27基因的逆转录病毒转染人DC,获得表达IL-27的IL-27/DC。光镜下观察,转染IL-27基因后,与成熟DC细胞形态学无明显差异;RT-PCR分析结果显示,IL-27/DC细胞有IL-27p28和IL-27EBI3表达,而野生型成熟DC细胞未见IL-27p28和IL-27EBI3表达;ELISA法检测结果显示,IL-27/DC细胞培养上清中可检测出高水平IL-27,而DC细胞未检出IL-27。该结果提示从基因和蛋白水平证明,成功建立了转染IL-27基因的人DC疫苗。2 IL-27/DC细胞培养上清可刺激hPBMCs产生IFN-γ,IFN-γ水平明显高于DC细胞(P<0.01),然而,两种细胞产生的IL-4水平无显著性差异(P>0.05)。3 IL-27/DC细胞组刺激自体T淋巴细胞增殖,与DC细胞组相比差异显著,有统计学意义(P<0.05)。4在不同效靶比条件下,IL-27/DC细胞均能增强CTL对4T1细胞的杀伤活性,其杀伤能力与效应细胞数量成正比,显著高于DC组(P<0.05)。5在接种IL-27/DC疫苗小鼠组中,4T1细胞的生长速度较接种DC和未接种细胞组明显减慢(P<0.01),且接种IL-27/DC疫苗组荷瘤小鼠的生存期也明显长于接种DC疫苗和未接种细胞组(P<0.01)。接种IL-27/DC疫苗组小鼠脾细胞分泌IFN-γ水平明显高于接种DC疫苗和未接种细胞组(P<0.01)。结论:成功建立了IL-27基因修饰的人DC疫苗(IL-27/DC),该疫苗能显著刺激T细胞增殖,对4T1细胞有明显的杀伤活性;该疫苗在小鼠体内具有明显抗肿瘤活性可能与刺激荷瘤小鼠脾细胞分泌IFN-γ等因素有关。更多还原

【Abstract】 Objective: Breast cancer is one of the most common tumors in women, which is a life-threatening disease that is seriously affecting the physical and mental health of the female patients. In recent years, tumor biotherapy has gained more attention from the people all over the world as a new mode and method for carcinoma treatment, in which cytokine gene therapy has always been the hot spots in this area. The routine theory is that cytokine genes which are transduced into tumor cells or other immune effector cells can secrete cytokines, which could induce tumor cell apoptosis or accelerate tumor regression by enhancing the immune activities in the body. Interleukin-27 (IL-27) is a novel IL-6/IL-12 family member[1], reported by Pflanz in 2002. In natural and adaptive immune systems, IL-27 plays a broad role in the immunological regulation of various kinds of cells. Besides the important function to the Th1 reaction, IL-27 has various biological activities[2], including anti-infection, anti-tumor and inflammation-inducing reaction, et al. Dendritic cells (DC) are the most powerful professional antigen presenting cells (APC) that can intake, process and present antigen effectively, and possess a high potency of migration and obvious activation to naive T cells. Thus, DC is the core in the process of initiating, regulating and maintaining of primary immune responses[3-4]. In this study, we set up the IL-27/DC vaccine by transfecting IL-27 gene into human dendritic cells (DC), and investigated its activities on tumor-killing. We are anticipating that our research could provide a new vision and experimental guide for the clinical immunotherapy on breast cancer in the future.Methods: 1 Establishment of IL-27/DC vaccine Preparation of dendritic cells (DC) from human peripheral blood mononuclear cells (hPBMCs), Observe morphous of mature DC by light microscope, and analyze the expression of CD80, CD86 and MHCⅠ, MHCⅡmolecul on the surface of DC and the mature condition of DC by flow cytometry. Transfect IL-27 gene carried by retrovirus vector into mature human DC and assess; Observe morphologic changes of DC and IL-27/DC cells by light microscope; IL-27 gene expression was confirmed by RT-PCR and the secretion of IL-27 in the supernatant of IL-27/DC cells was detected by ELISA.2 Analysis on IFN-γinduced by IL-27 ELISA was used to detect the secretion of IFN-γand IL-4 in cell supernatant.3 Lymphocyte proliferation analysis Differentiation and cultivation of T lymphocyte, MTT was used to detect the proliferation potency of T lymphocyte stimulated respectively by DC and IL-27/DC in vitro, and draw the growth curve.4 The assessment of the lethal effects of cytotoxic T lymphocyte (CTL) MTT was used to measure the lethal effect to 4T1 cells in vitro of activited cytotoxic lymphocyte (CD8+T) (CTL) stimulated by IL-27/DC and DC cells respectively.5 Establishment of animal model and analysis on antitumor activity of IL-27/DC in vivo Divide mice to three groups: IL-27/DC group, DC group and control group. Mice were immunized respectively with DC and IL-27/DC cells administered subcutaneously in the right flank, and challenged in the left flank after the last immunization with 4T1 cells. The tumor volume and the survival time were observed. ELISA was used to detect the quantity of IFN-γsecreted by splenocytes.Results: 1 There was an obviously higher level of expression of CD80, CD86 molecul in mature DC than immature. The IL-27/DC vaccine constantly expressing IL-27 was obtained by transfection. Positive expression of IL-27p28 and IL-27EBI3 gene in IL-27/DC cell were showed by RT-PCR. There was no significant difference except for some decrease of the quantity of cells in the morphous of DC and IL-27/DC observed by light microscope. ELISA result indicated that the supernatant of IL-27/DC cell could produce higher level of IL-27 compared to DC cells (P<0.01), suggesting that IL-27 gene was transfected successfully into dendritic cell lines in gene and protein level.2 ELISA results indicated that there was a higher level of IFN-γsecretion in the supernatant of IL-27/DC cell compared to DC cells (P<0.01). However , there was no significant difference in the two groups on the results of IL-4 production(P>0.05).3 MTT results showed that IL-27/DC could notably stimulate the proliferation of T lymphocyts in vitro, which had made a statistic significance compared to other groups (P<0.05).4 Under different effect-target ratios (E/T), the group of IL-27/DC always showed a higher level of performance of killing-activity to 4T1 cells compared to other groups (P<0.05), whose potency was demonstrated to be an obvious enhancing trend as the increasing of the effector cells.5 The growth of tumor in mice immunized with IL-27/DC vaccine was markedly retarded compared to the DC and control group (P<0.05), and the survival time of the mice immunized with IL-27/DC vaccine was longer than the other groups (P<0.01). The quantitation of IFN-γproduction in vitro from splenocytes of IL-27/DC vaccine group demonstrated a higher level compared to the other groups (P<0.01).Conclusions: Successfully establish IL-27/DC vaccine by transfecting IL-27 gene into human dendritic cells (DC). IL-27/DC vaccine could notably stimulate the proliferation of T lymphocyts in vitro. IL-27/DC vaccine had a higher killing-activity to 4T1 cells. The obvious antitumor activities of the vaccine in vivo was speculated to be associated with its function to stimulate IFN-γproduction in mice splenocytes.更多还原