GDF15分子对树突状细胞功能的影响
发布时间：2019-05-29 23:07
【摘要】：肿瘤严重危害人类健康。近年来除手术、放疗、化疗、生物治疗等治疗方法外,肿瘤的免疫治疗越来越受到重视。肿瘤免疫治疗的目的是激发或调动机体的免疫系统,增强肿瘤微环境的抗肿瘤免疫力,从而控制和杀伤肿瘤细胞。但是进一步的研究发现肿瘤细胞可以通过修饰自身表面抗原及改变肿瘤组织周围的微环境以影响免疫细胞的功能,从而逃避机体的免疫识别与攻击。 目前主要从与肿瘤细胞和肿瘤抗原自身相关的机制及与宿主免疫系统相关的机制这两方面来研究肿瘤免疫逃逸。其中,荷瘤机体中的免疫功能障碍是导致机体免疫应答低下和肿瘤逃逸免疫调控的主要机制,是限制肿瘤免疫治疗的重要因素,而抗原递呈细胞(APC)特别是树突状细胞(DC)功能的改变发挥了重要作用。树突状细胞从骨髓造血干细胞到功能完善的成熟DC经过了一系列的过程,肿瘤不但可以直接诱导DC凋亡,还可以通过干扰DC分化的多个环节使肿瘤患者体内DC数量下降,成熟受到抑制,功能发生障碍,从而下调或抑制DC的抗原递呈及免疫起始功能。目前已知部分肿瘤细胞膜分子或分泌的源性因子参与了这一过程,但是在肿瘤复杂的微环境中,肿瘤细胞分泌大量的分子,究竟是肿瘤细胞的哪些表面分子或源性因子与DC相互作用而导致了肿瘤免疫逃逸调控,仍有待进一步研究。 为了系统全面的研究可能参与影响树突状细胞功能的肿瘤细胞相关分子,本研究进行了以下三个方面的工作:首先,通过购买已构建好的人肝癌T7噬菌体cDNA文库,对从体外培养诱导分化的未成熟树突状细胞进行4轮生物淘选,将经ELISA鉴定为与未成熟树突状细胞特异性结合的噬菌体单克隆进行PCR扩增、测序和同源性分析,将获得的候选分子进行生物信息学分析,初步了解其研究进展及功能。然后,拟通过以下实验来鉴定并分析GDF15在肝癌细胞和组织中的表达:(1)采用RT-PCR技术检测肝癌细胞中GDF15的mRNA转录;(2)通过免疫印迹实验检测肝癌细胞中GDF15蛋白的表达;(3)经激光共聚焦检测GDF15在肝癌细胞中的表达和定位;(4)应用免疫组化方法检测GDF15在肝癌组织切片中的表达和定位。最后,拟通过将外源性GDF15与DC共培养,从不同方面来鉴定GDF15影响DC的成熟:(1)流式细胞术检测GDF15对DC表型的影响;(2)扫描电镜观察GDF15对DC形态的影响;(3)流式细胞术检测GDF15对DC吞噬能力的影响;(4)利用ELISA检测GDF15对DC分泌IL-12的影响。本研究获得了以下结果:①获得一系列与树突状细胞特异性结合的肿瘤细胞表面分子和源性因子(人铁蛋白轻链、甲胎蛋白、α1微球蛋白前体、生长分化因子15等),初步评估了筛选蛋白的可能意义、相互结合受体和作用途径以及研究进展后,我们以GDF15为靶分子,以研究其对DC功能的影响;②GDF15主要定位于胞浆中,在肝癌中的表达水平明显高于正常肝脏细胞;③在DC的培养上清中加入GDF15能显著抑制未成熟DC的表型向成熟DC转化,初步证实GDF15参与抑制DC的分化成熟。 综上所述,本研究通过肝癌噬菌体cDNA展示文库技术筛选获得了一些未曾报道的能与DC相互作用的分子,并以GDF15为切入点,初步发现GDF15能抑制DC向成熟表型转化,为进一步探讨GDF15对DC相关功能影响的分子机制奠定基础。
[Abstract]:The tumor is seriously harmful to human health. In recent years, in addition to the treatment methods such as surgery, radiotherapy, chemotherapy and biological treatment, the immunotherapy of the tumor is more and more important. The purpose of the tumor immunotherapy is to stimulate or mobilize the immune system of the body, to enhance the anti-tumor immunity of the tumor microenvironment, thereby to control and kill the tumor cells. However, further studies have found that tumor cells can escape the immune recognition and attack of the immune cells by modifying the surface surface antigen and altering the microenvironment around the tumor tissue to affect the function of the immune cells. At present, the tumor immune escape is studied mainly from the mechanism related to the tumor cell and the tumor antigen and the mechanism related to the host immune system The immune dysfunction in the tumor-bearing body is the main mechanism to control the immune response of the body and the immune regulation of tumor escape, which is an important factor to limit the immunotherapy of the tumor, and the change of the antigen-presenting cell (APC), especially the dendritic cell (DC) function, plays an important role. The dendritic cells from the bone marrow hematopoietic stem cells to the fully functional mature DC have undergone a series of processes, so that the tumor can not only directly induce the DC apoptosis, but also can reduce the DC quantity in the tumor patients by interfering with the multiple links of the DC differentiation, and the maturation is inhibited, and the function generation barrier The antigen-presenting and immune-initiating work of DC is down-regulated or inhibited. It is currently known that some of the tumor cell membrane molecules or secreted sources of origin are involved in this process, but in the complex microenvironment of the tumor, the tumor cells secrete a large amount of It is still to be further research that which surface molecules or the source factors of the tumor cells interact with the DC, leading to the regulation of tumor immune escape. In order to fully study the possible involvement of the system in tumor cell-related molecules that affect the function of dendritic cells, the study carried out three aspects of the study: first, by purchasing the constructed human liver cancer T7 phage cDN A library is selected from the immature dendritic cells that are cultured in vitro to induce differentiation, and the ELISA is used for PCR amplification, sequencing and homology analysis of the phage monoclonal antibody specifically combined with the immature dendritic cells, and the obtained candidate molecules are subjected to biological information A Preliminary Study of the Research Progress in the Study of the Study And then, the expression of the GDF15 in the liver cancer cells and tissues is identified and analyzed by the following experiments: (1) the mRNA transcription of the GDF15 in the liver cancer cell is detected by the RT-PCR technology; and (2) the GDF15 protein in the liver cancer cell is detected by the immunoblotting experiment. (3) Expression and localization of GDF15 in liver cancer cells by laser confocal detection; and (4) the expression of GDF15 in the tissue sections of liver cancer was detected by immunohistochemistry. and by co-culturing the exogenous GDF15 with the DC, the maturation of the GDF15 on the DC is identified from different aspects: (1) the effect of the GDF15 on the DC phenotype is detected by flow cytometry; (2) the scanning electron microscope is used to observe the DC morphology of the GDF15 (3) Flow cytometry was used to detect the effect of GDF15 on the phagocytosis of DC, and (4) the secretion of IL-12 by GDF15 was detected by ELISA. The results of this study were as follows: to obtain a series of tumor cell surface molecules and derived factors which specifically bind to the dendritic cells (human ferritin light chain, alpha-fetoprotein,1-1 microglobulin precursor, growth differentiation factor 15, etc.), and the feasibility of screening the protein is preliminarily evaluated. In order to study the effect of GDF15 on the DC function, the expression level of GDF15 was significantly higher than that of normal liver in the cytoplasm. The addition of GDF15 in the culture of DC could significantly inhibit the phenotype of immature DC to the mature DC, and it was confirmed that the GDF15 was involved in the inhibition of DC. To sum up, this study has obtained some non-reported molecules which can interact with the DC through the screening of the phage cDNA library of the liver cancer, and the GDF15 as the starting point, and it is preliminarily found that the GDF15 can inhibit the DC conversion. Molecular machine for further exploring the effect of GDF15 on DC-related function in order to further explore the effect of GDF15 on DC-related function
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
本文编号：2488300
[Abstract]:The tumor is seriously harmful to human health. In recent years, in addition to the treatment methods such as surgery, radiotherapy, chemotherapy and biological treatment, the immunotherapy of the tumor is more and more important. The purpose of the tumor immunotherapy is to stimulate or mobilize the immune system of the body, to enhance the anti-tumor immunity of the tumor microenvironment, thereby to control and kill the tumor cells. However, further studies have found that tumor cells can escape the immune recognition and attack of the immune cells by modifying the surface surface antigen and altering the microenvironment around the tumor tissue to affect the function of the immune cells. At present, the tumor immune escape is studied mainly from the mechanism related to the tumor cell and the tumor antigen and the mechanism related to the host immune system The immune dysfunction in the tumor-bearing body is the main mechanism to control the immune response of the body and the immune regulation of tumor escape, which is an important factor to limit the immunotherapy of the tumor, and the change of the antigen-presenting cell (APC), especially the dendritic cell (DC) function, plays an important role. The dendritic cells from the bone marrow hematopoietic stem cells to the fully functional mature DC have undergone a series of processes, so that the tumor can not only directly induce the DC apoptosis, but also can reduce the DC quantity in the tumor patients by interfering with the multiple links of the DC differentiation, and the maturation is inhibited, and the function generation barrier The antigen-presenting and immune-initiating work of DC is down-regulated or inhibited. It is currently known that some of the tumor cell membrane molecules or secreted sources of origin are involved in this process, but in the complex microenvironment of the tumor, the tumor cells secrete a large amount of It is still to be further research that which surface molecules or the source factors of the tumor cells interact with the DC, leading to the regulation of tumor immune escape. In order to fully study the possible involvement of the system in tumor cell-related molecules that affect the function of dendritic cells, the study carried out three aspects of the study: first, by purchasing the constructed human liver cancer T7 phage cDN A library is selected from the immature dendritic cells that are cultured in vitro to induce differentiation, and the ELISA is used for PCR amplification, sequencing and homology analysis of the phage monoclonal antibody specifically combined with the immature dendritic cells, and the obtained candidate molecules are subjected to biological information A Preliminary Study of the Research Progress in the Study of the Study And then, the expression of the GDF15 in the liver cancer cells and tissues is identified and analyzed by the following experiments: (1) the mRNA transcription of the GDF15 in the liver cancer cell is detected by the RT-PCR technology; and (2) the GDF15 protein in the liver cancer cell is detected by the immunoblotting experiment. (3) Expression and localization of GDF15 in liver cancer cells by laser confocal detection; and (4) the expression of GDF15 in the tissue sections of liver cancer was detected by immunohistochemistry. and by co-culturing the exogenous GDF15 with the DC, the maturation of the GDF15 on the DC is identified from different aspects: (1) the effect of the GDF15 on the DC phenotype is detected by flow cytometry; (2) the scanning electron microscope is used to observe the DC morphology of the GDF15 (3) Flow cytometry was used to detect the effect of GDF15 on the phagocytosis of DC, and (4) the secretion of IL-12 by GDF15 was detected by ELISA. The results of this study were as follows: to obtain a series of tumor cell surface molecules and derived factors which specifically bind to the dendritic cells (human ferritin light chain, alpha-fetoprotein,1-1 microglobulin precursor, growth differentiation factor 15, etc.), and the feasibility of screening the protein is preliminarily evaluated. In order to study the effect of GDF15 on the DC function, the expression level of GDF15 was significantly higher than that of normal liver in the cytoplasm. The addition of GDF15 in the culture of DC could significantly inhibit the phenotype of immature DC to the mature DC, and it was confirmed that the GDF15 was involved in the inhibition of DC. To sum up, this study has obtained some non-reported molecules which can interact with the DC through the screening of the phage cDNA library of the liver cancer, and the GDF15 as the starting point, and it is preliminarily found that the GDF15 can inhibit the DC conversion. Molecular machine for further exploring the effect of GDF15 on DC-related function in order to further explore the effect of GDF15 on DC-related function
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 Mabood Qureshi;Jim Xiang;;Dendritic Cell-Derived Exosomes Stimulate Stronger CD8~+ CTL Responses and Antitumor Immunity than TVimor Cell-Derived Exosomes[J];Cellular & Molecular Immunology;2006年03期
2 范小红,韩宝惠,董强刚,沙慧芳,包国良,廖美琳;非小细胞肺癌中血管内皮生长因子对树突状细胞的抑制作用[J];中华结核和呼吸杂志;2003年09期
，本文编号：2488300
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