【作者】 杨丽娟；

【导师】 单保恩；

【作者基本信息】 河北医科大学 ， 免疫学， 2010， 博士

【摘要】 目的:机体特异性免疫功能包括体液免疫和细胞免疫,其中由各种T细胞介导的细胞免疫是机体抗肿瘤免疫的主体。肿瘤发生与转移的主要原因被认为是机体免疫系统功能减弱,不能对肿瘤细胞产生有效的免疫应答从而使肿瘤细胞逃逸机体的免疫监视。T细胞是一种重要的免疫活性细胞,根据表面CD分子的不同,可以分为CD4+T细胞和CD8+T细胞,其中的CD4+T细胞在整个细胞免疫应答中发挥重要的免疫调节作用。根据分泌细胞因子的不同,传统上将CD4+T细胞分为Th1、Th2、Treg三种类型。2005年新发现了一类CD4+T细胞亚群,因为这类细胞特征性产生IL-17,被称为Th17细胞。研究发现,Th17细胞在抗感染免疫、自身免疫和肿瘤发生发展中发挥重要作用,目前,国外研究机构对Th17细胞在肿瘤中作用的研究方兴未艾,但国内尚未见相关报道。乳腺癌是严重危害女性健康的恶性肿瘤,在发达国家及我国的一些大中型城市,乳腺癌的发病率已居女性恶性肿瘤的首位。目前,手术、化疗和放疗仍是乳腺癌的主要治疗手段,但是都存在一定的局限性。通常采用上述三种治疗方法的综合治疗,但乳腺癌患者的五年生存率仍不十分理想,因此,研究有效的乳腺癌治疗方法和手段是亟待解决的问题。近年来,肿瘤生物治疗发展迅速,因其治疗效果明显、副作用小而被医患双方所重视,已成为肿瘤治疗的新亮点。其中免疫治疗不仅能提高宿主免疫功能,特异性杀伤肿瘤细胞,而且对正常组织细胞无毒副作用或毒副作用甚微,因此,近些年发展很快。为了研究Th17细胞作为乳腺癌免疫治疗新靶标的可行性,本研究选择人乳腺癌组织和癌旁正常组织,对其中的Th17细胞数量及其临床意义进行了研究,进一步采用荷瘤动物模型研究了Th17细胞在乳腺癌发生发展中的作用及其机制,为乳腺癌的免疫治疗提供实验和理论依据。方法:1流式细胞术(FCM)检测30例乳腺癌组织及癌旁正常组织中Th17细胞和Treg细胞百分率,采用多元线性回归分析法统计肿瘤组织中Th17细胞和Treg细胞表达与患者临床病理学特征的相互关系;免疫组化法检测肿瘤组织及癌旁正常组织中IL-17、IL-1β、IL-6、TGF-β和IL-10的表达情况,采用直线相关分析法统计上述细胞因子表达与Th17细胞和Treg细胞数量的相关性。2免疫磁珠法分离小鼠脾初始CD4+T细胞,在多种细胞因子参与下体外诱导和扩增小鼠Th17细胞,并分别于小鼠乳腺癌细胞4T1接种BALB/c小鼠后不同时间(0天、7天和12天)过继Th17细胞进行免疫治疗,观察肿瘤生长情况和小鼠生存期。3将携带mIL-17基因的4T1(4T1/IL-17)细胞接种BALB/c小鼠体内,观察肿瘤生长情况和小鼠生存期;用流式细胞术、实时定量PCR以及Western blot等方法,研究IL-17在肿瘤组织的表达、肿瘤组织Th17细胞数量及其抗肿瘤作用,并对其可能的抗肿瘤机制进行研究。结果:1与癌旁正常组织相比,人乳腺癌组织中Th17细胞数量明显增加,而且TNM分期越低、转移性越低的人乳腺癌组织中Th17细胞表达率越高,提示Th17细胞具有抗肿瘤作用。2人乳腺癌组织高表达IL-1β和IL-6,形成利于Th17细胞分化的细胞因子微环境,导致人乳腺癌组织中Th17细胞数量增加。3与癌旁正常组织相比,人乳腺癌组织中Treg细胞数量明显增加,而且人乳腺癌组织中的Treg细胞通过分泌IL-10发挥促肿瘤作用。4人乳腺癌组织中Th17细胞数量与Treg细胞数量无相关性。5确定了体外诱导小鼠初始CD4+T细胞分化为Th17细胞的最佳细胞因子组合。6过继体外培养的Th17细胞进行免疫治疗可抑制4T1细胞荷瘤小鼠肿瘤的生长、延长荷瘤小鼠生存期。7转染IL-17基因的小鼠乳腺癌细胞4T1可在肿瘤局部形成利于Th17细胞分化的微环境(同时表达TGF-β和IL-6),使肿瘤组织中Th17细胞的数量明显增加。8肿瘤组织Th17细胞数量的增加可通过增加肿瘤组织CTL细胞的数量和功能发挥抗肿瘤作用。结论:与癌旁正常组织相比人乳腺癌组织中Th17细胞数量增加,并具有抗肿瘤活性,通过过继免疫治疗或改变肿瘤组织细胞因子微环境能够增加乳腺癌组织中Th17细胞的数量发挥抗肿瘤作用,这可能是乳腺癌免疫治疗的新策略。更多还原

【Abstract】 Objective: Iit is presumede that the development of tumors is related to a defect of a host immunosurveillance system and an escape mechanism of tumors from host immune responses. The adaptive immunity in human being include humoral immunity and cell immunity, and the cell immunity mediated by T cells plays a crucial role in tumor immunosurveillance. T cells are significant immune competent cell, and according to the CD molecule on cellular membrane, T cells can be divided into CD4+T and CD8+T cells. CD4+T cells play central roles in regulation of the immune system in mammals, and CD4+T cells include distinct effector lineages, Th1, Th2 and Treg, each characterized by the production of a unique profile of effector cytokines. A new subset of CD4+ T cells, termed Th17 cells, has been characterized by the prodution of IL-17 in 2005. The primary function of Th17 cells is to fight infection by bacterial and fungal pathogens, and Th17 cells are also involved in inflammation and autoimmune disease. Recently, more and more research has shown that Th17 cells are found in both mouse and human tumors. However, the biologic role of Th17 cells is poorly understood in the tumor microenvironment.Breast carcinoma is a lethal malignant tumor, and in the first place of the serious diseases threatening female’s health in developed country and some big cities of our country. At present, operation, chemothreapy and radiotherapy are the main methods to treat tumors, while all have some limitations. In order to enhance the therapeutic efficacy of breast carcinoma, usually employ the combined therapy of the three main methods by turns. But the five year survival rate of breast carcinoma patient still is not satisfactory. It is the day to look for the better methods to treat breast carcinoma.Immunotherapy of tumors which can not only enhance the immune function of host, kill tumor cells in specific way, but also has less side-effects on normal tissues and more and more people have pay attention to immunotherapy. In order to investigate the possibility that if Th17 cells can be used as an new therpeutic target of breast carcinoma, in this study, we examined the express rate of Th17 cells in the cancer tissue and non-cancerous tissue of patients with invasive breast carcinoma, and determined if the express rate of Th17 cells in tumor tissues contribute to influence the clinical pathology factors of the homologous patients. In order to research the antitumor activity, relate mechanisms and provided basis for clinical application of Th17 cells, we study further the effects of Th17 cells in animal model.Methods:1 Expression of Th17 cells and Treg cells in the breast cancer tissues and non-cancerous tissues were detected by flow cytometry (FCM) in 30 breast carcinoma patients, and their correlation with clinical pathology factors was statistical analyzed by multiple linear regression analysis.The expression of interleukin-17 (IL-17), interleukin-1β(IL-1β), interleukin-6 (IL-6), transforming growth factor-β(TGF-β) and interleukin-10 (IL-10) in the breast cancer tissues were measured by immunohistochemical staining, and their correlation with the expression of Th17 cells or Treg cells were statistical analyzed by linear correlation dependablity analysis.2 The CD4+CD62L+ T cells purified by MACS were stimulated under different cytokine conditions to induce and amplify mice Th17 cells in vitro. At the 0, 7 or 12 day after mice were injected with tumor cells, the Th17 cells (experiment group) or PBS (control group) was infused through tumor adjacent into tumor barring mice. The tumor changing and the survival curves of tumor model were detected.3 4T1/IL-17, 4T1/pcDNA3.1 and the parental 4T1 cells were respectively inoculated to mice in the subcutaneous tissue. Three mice of every group were killed on the six weeks after tumor cells inoculation, the tumors were obtained from mice. The other mice of every group were used to observe survival and tumor size. FCM, real time PCR and Western blot were used to determine IL-17 protein expression and Th17 cells ratio in tumor tissues to explain the anti-tumor effect of 4T1/IL-17 cells.Results:1 Compared with the non-cancerous breast tissues, there are significant augment of Th17 cells in the malignant breast tissues, and the Th17 cells display anti-tumor effect in human breast cancer.2 Th17 cells skewing depend on the locally cytokines microenviroment in human breast cancer tissues.3 Compared with the non-cancerous breast tissues, there are significant augment of Treg cells in the malignant breast tissues, and the IL-10 secreted by Treg cells may involve in their promote-tumor effect.4 In human breast cancer tissues, the ratio of Th17 cells has nothing with the ratio of Treg cells.5 Ascertain the best cytokine combination inducing the mice Th17 cells from CD4+CD62L+ T cells in vitro.6 Adoptive transfer of Th17 cells cultured in vitro through tumor adjacent into tumor barring mice, the tumors of mice had accepted Th17 cells adoptive immunotherapy developed obviously slower than the mice had not. And the survival time of tumor barring mice had accepted Th17 cells adoptive immunotherapy also was more longer than the tumor barring mice had not.7 IL-17 secreted from 4T1/IL-17 cells can induce the express of TGF-βand IL-6, this cytokines microenviroment can benefit the skew of Th17 cells.8 The accrescence of Th17 cells in tumor tissues can promote the tumor infiltration and function of CTL, and this may involve in the anti-tumor effect of Th17 cells.Conclusion: There are significant augment of Th17 cells in the malignant breast tissues compared with the non-cancerous breast tissues. And the increased Th17 cells in the tumor tissue of breast cancer patients display anti-tumor effect.To augment the cells population of Th17 cells in the breast tumor tissues by adoptive immunotherapy or altering the cytokine microenvironment in breast tumor tissues may be the novel immunotherapy method of breast carcinoma.更多还原