【作者】 赵莎；

【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:最近几年,生物治疗已成为一种新型的肿瘤治疗方法,并越来越受到人们的重视。其中细胞因子基因治疗是肿瘤生物治疗的研究热点,通过将不同的细胞因子基因导入肿瘤或免疫效应细胞,使其在机体表达来促进肿瘤细胞凋亡或者通过增强免疫系统功能以加速肿瘤的消退。IL-17是目前新发现的主要由CD4+记忆性T淋巴细胞,单核细胞等分泌的一种细胞因子,其在天然免疫和宿主防御中有特殊功能,在炎症反应中对白细胞的迁移和活化、细胞增殖分化、生物因子转录与表达、免疫调节及肿瘤生长方面发挥重要作用。但有关IL-17的作用机理等许多问题还有待于进一步研究。本实验将构建表达IL-17基因的小鼠乳腺癌细胞株,建立荷瘤小鼠模型,研究IL-17基因的生物活性及其抗肿瘤效应。方法:1 IL-17基因转染细胞株的建立采用脂质体转染的方法将插入IL-17基因的pcDNA3.1载体导入小鼠乳腺癌细胞4T1,同时,将pcDNA3.1空载体转染4T1细胞作为对照组细胞,经G418筛选后获得表达IL-17的阳性细胞克隆(4T1/IL-17)。2用光学显微镜观察4T1/IL-17、4T1/pcDNA3.1和4T1细胞的形态变化,RT-PCR检测IL-17基因的表达,激光共聚焦和Western-blot法检测IL-17蛋白的表达。3细胞增殖实验MTS法分析4T1/IL-17细胞体外增殖反应,绘制生长曲线。流式细胞技术检测细胞周期和凋亡率的变化以及细胞表面MHCI、MHCII、LFA-1等分子表达的情况。4 IL-17诱导小鼠巨噬细胞RAW264.7产生IL-6 ELISA法检测4T1/IL-17、4T1/pcDNA3.1和4T1细胞培养上清诱导RAW264.7细胞分泌IL-6水平。5建立荷瘤动物模型,将4T1/IL-17、4T1/pcDNA3.1和4T1细胞分别接种于小鼠右侧背部皮下,观察三种细胞移植瘤在小鼠体内成瘤性、生长情况及小鼠生存期的变化。接种肿瘤细胞6周后分别取三组小鼠脾脏和肿瘤组织,用乳酸脱氢酶法、MTS法分别检测三组小鼠脾细胞CTL杀伤活性、脾细胞增殖反应情况。用ELISA法检测小鼠脾细胞经刺激后产生IFN-γ、IL-12的情况;采用流式细胞技术检测4T1/IL-17细胞接种于小鼠体内的细胞凋亡情况及肿瘤组织细胞表面MHCI、MHCII、LFA-1等分子表达的变化。结果:1以质粒pcDNA3.1为载体成功将IL-17基因转染至小鼠乳腺癌细胞4T1,建立表达IL-17的细胞株4T1/IL-17,该细胞可产生和分泌IL-17分子。光镜下观察三种细胞形态学无明显变化;RT-PCR检测结果显示4T1/IL-17细胞有IL-17基因的表达,激光共聚焦显微镜和Western-blot分析结果证明,4T1/IL-17细胞中有IL-17蛋白表达,而4T1/pcDNA3.1和4T1细胞未见IL-17蛋白表达,从而在基因和蛋白水平证明,成功建立了转染IL-17基因的小鼠乳腺癌细胞株。2 4T1/IL-17细胞培养上清可刺激小鼠巨噬细胞RAW264.7产生IL-6,IL-6水平明显高于4T1/pcDNA3.1和4T1细胞培养上清的作用(P<0.01)。3 4T1/IL-17细胞的体外增殖反应与4T1/pcDNA3.1和4T1细胞无明显差别(P>0.05)。转染IL-17基因不影响细胞周期和细胞凋亡,应用流式细胞技术检测4T1/IL-17和4T1/pcDNA3.1细胞表面MHCI、MHCII、LFA-1的表达水平与4T1细胞相比没有显著性差异(P>0.05)。4将4T1/IL-17细胞接种小鼠体内后,荷瘤小鼠的肿瘤生长速度、体积和重量均明显小于4T1/pcDNA3.1细胞组及4T1细胞组,生存时间也明显延长(P<0.05)。5与4T1/pcDNA3.1及4T1细胞组荷瘤小鼠相比,接种4T1/IL-17细胞组小鼠肿瘤细胞凋亡率明显增高(P<0.05)。接种4T1/IL-17细胞组小鼠肿瘤组织细胞表面MHCI、MHCII、LFA-1分子表达水平显著性增高(P<0.01)。6接种各组肿瘤细胞6周后,4T1/IL-17细胞组小鼠脾细胞的CTL杀伤活性及对ConA刺激的细胞增殖反应性均明显高于接种4T1/pcDNA3.1及4T1细胞组(P<0.01);此外,接种4T1/IL-17细胞组小鼠脾细胞可产生高水平的IFN-γ、IL-12,明显高于接种4T1/pcDNA3.1及4T1细胞组,有显著性差异(P<0.01)。结论:成功构建转染IL-17基因的小鼠乳腺癌细胞株(4T1/IL-17),该细胞可分泌具有生物学活性的IL-17。转染IL-17基因对4T1/IL-17细胞的体外增殖和细胞凋亡无明显影响,外源性IL-17基因的插入对4T1细胞免疫相关分子的表达无影响。转染IL-17基因在小鼠体内具有明显的抗肿瘤作用,其抗肿瘤作用与促进肿瘤细胞细胞凋亡、增强机体免疫应答有关。更多还原

【Abstract】 Objective: In the past few years, the biotherapy is becoming a new method for tumor and a lot of people have paid attention to it. The cytokine gene therapy is one of the biotherapeutic strategies for tumors, in which cytokine genes are transduced into tumor cells or other immune effector cells, which can secret cytokines, induce tumor cells apoptosis and strengthen the immune functions to accelerate tumor regression. In this study, we transfected interleukin-17 gene into mouse breast cancer cells(4T1), set up the animal model to investigate its antitumor mechanisms.Methods: 1 By plasmid vector, IL-17 gene was transfected into mouse breast cancer cells (4T1) and stable clones expressing IL-17(4T1/IL-17) were obtained by selecting with G418. Meanwhile transfected empty plasmid vector(pcDNA3.1) and 4T1 cells were as control groups.2 The morphologic changes of 4T1/IL-17, 4T1/ pcDNA3.1 and 4T1 cells were observed by light microscope, IL-17 gene expression was detected by RT-PCR and the secretion of IL-17 protein was measured by LSM and Western-blot .3 The proliferation of cells in vitro was detected by MTS and then draw the growth curve. Flow cytometry was used to analyze the expression of MHCI, MHCII and LFA-1 molecular on the surface of 4T1/IL-17、4T1/pcDNA3.1 and 4T1 cells.4 The production of IL-6 from mouse macrophage RAW264.7 induced by IL-17 was detected by ELISA.5 4T1/IL-17, 4T1/pcDNA3.1 and 4T1 cells were subcutaneously inoculated into mice respectively and the tumor volumn and the survival time were observed. Cell apoptosis in vitro and in vivo were analyzed by flow cytometry. The expression of the MHCI, MHCII and LFA-1 molecules on the surface of tumor cells were also detected by flow cytometry. LDH releaese, MTS were used to determine CTL activity, proliferation of the splenocytes respectively. The production of IFN-γand IL-12 from splenocytes were detected by ELISA.Results: 1 4T1/IL-17 cell stable expressing IL-17 were successfully set up. IL-17 gene expression in 4T1/IL-17 cell was detected by RT-PCR.The protein level was showed by LSM and Western-blot, which suggested that IL-17 gene transfected into breast cancer cell lines and could express in gene and protein level.2 The growth of 4T1/IL-17 cell was similar to 4T1 cell and 4T1/pcDNA3.1 cell in vitro (P>0.05). There were no differences of expression of MHCI, MHCII and LFA-1 molecular in 4T1/IL-17, 4T1/pcDNA3.1, 4T1 cells (P>0.05).3 The supernatant of 4T1/IL-17 cell could induce RAW264.7 cell to produce higher level of IL-6 than 4T1/pcDNA3.1 and 4T1 cells (P<0.01).4 The growth of tumor in mice inoculted with 4T1/IL-17 cells was significantly retarded (P<0.05), and the survival time was longer compared to 4T1/pcDNA3.1 and 4T1 groups (P<0.05).5 There was no difference of cell apoptosis rate in 4T1/IL-17, 4T1/pcDNA3.1, 4T1 cells in vitro (P>0.05). But, in the mice of inoculation, cell apoptosis rate of 4T1/IL-17 group was higher than those of 4T1/pcDNA3.1, 4T1 groups (P<0.05). The expression of MHCI, MHCII and LFA-1 molecules in the tumor tissues from 4T1/IL-17 inoculated mice were also higher than two other groups (P<0.01).6 The CTL activity and proliferation of the splenocytes from 4T1/IL-17 inoculated mice was higher than those of 4T1/pcDNA3.1, 4T1 groups (P<0.01). The splenocytes from the mice inoculated with 4T1/IL-17 cells can secret higher IFN-γand IL-12 than two other groups (P<0.01). Conclusions: Successfully constructed the IL-17 transfected breast cancer cell line (4T1/IL-17) with the pcDNA3.1 vector. The gene transfection of IL-17 had no effect on cell growth and apoptosis in vitro; But in the mice inoculated with 4T1/IL-17, IL-17 could enhance the immune function and induce the antitumor activity in mice.更多还原