GDF15分子對樹突狀細胞功能的影響
發(fā)布時間：2019-05-29 23:07
【摘要】：腫瘤嚴重危害人類健康。近年來除手術(shù)、放療、化療、生物治療等治療方法外,腫瘤的免疫治療越來越受到重視。腫瘤免疫治療的目的是激發(fā)或調(diào)動機體的免疫系統(tǒng),增強腫瘤微環(huán)境的抗腫瘤免疫力,從而控制和殺傷腫瘤細胞。但是進一步的研究發(fā)現(xiàn)腫瘤細胞可以通過修飾自身表面抗原及改變腫瘤組織周圍的微環(huán)境以影響免疫細胞的功能,從而逃避機體的免疫識別與攻擊。 目前主要從與腫瘤細胞和腫瘤抗原自身相關(guān)的機制及與宿主免疫系統(tǒng)相關(guān)的機制這兩方面來研究腫瘤免疫逃逸。其中,荷瘤機體中的免疫功能障礙是導致機體免疫應(yīng)答低下和腫瘤逃逸免疫調(diào)控的主要機制,是限制腫瘤免疫治療的重要因素,而抗原遞呈細胞(APC)特別是樹突狀細胞(DC)功能的改變發(fā)揮了重要作用。樹突狀細胞從骨髓造血干細胞到功能完善的成熟DC經(jīng)過了一系列的過程,腫瘤不但可以直接誘導DC凋亡,還可以通過干擾DC分化的多個環(huán)節(jié)使腫瘤患者體內(nèi)DC數(shù)量下降,成熟受到抑制,功能發(fā)生障礙,從而下調(diào)或抑制DC的抗原遞呈及免疫起始功能。目前已知部分腫瘤細胞膜分子或分泌的源性因子參與了這一過程,但是在腫瘤復(fù)雜的微環(huán)境中,腫瘤細胞分泌大量的分子,究竟是腫瘤細胞的哪些表面分子或源性因子與DC相互作用而導致了腫瘤免疫逃逸調(diào)控,仍有待進一步研究。 為了系統(tǒng)全面的研究可能參與影響樹突狀細胞功能的腫瘤細胞相關(guān)分子,本研究進行了以下三個方面的工作:首先,通過購買已構(gòu)建好的人肝癌T7噬菌體cDNA文庫,對從體外培養(yǎng)誘導分化的未成熟樹突狀細胞進行4輪生物淘選,將經(jīng)ELISA鑒定為與未成熟樹突狀細胞特異性結(jié)合的噬菌體單克隆進行PCR擴增、測序和同源性分析,將獲得的候選分子進行生物信息學分析,初步了解其研究進展及功能。然后,擬通過以下實驗來鑒定并分析GDF15在肝癌細胞和組織中的表達:(1)采用RT-PCR技術(shù)檢測肝癌細胞中GDF15的mRNA轉(zhuǎn)錄;(2)通過免疫印跡實驗檢測肝癌細胞中GDF15蛋白的表達;(3)經(jīng)激光共聚焦檢測GDF15在肝癌細胞中的表達和定位;(4)應(yīng)用免疫組化方法檢測GDF15在肝癌組織切片中的表達和定位。最后,擬通過將外源性GDF15與DC共培養(yǎng),從不同方面來鑒定GDF15影響DC的成熟:(1)流式細胞術(shù)檢測GDF15對DC表型的影響;(2)掃描電鏡觀察GDF15對DC形態(tài)的影響;(3)流式細胞術(shù)檢測GDF15對DC吞噬能力的影響;(4)利用ELISA檢測GDF15對DC分泌IL-12的影響。本研究獲得了以下結(jié)果:①獲得一系列與樹突狀細胞特異性結(jié)合的腫瘤細胞表面分子和源性因子(人鐵蛋白輕鏈、甲胎蛋白、α1微球蛋白前體、生長分化因子15等),初步評估了篩選蛋白的可能意義、相互結(jié)合受體和作用途徑以及研究進展后,我們以GDF15為靶分子,以研究其對DC功能的影響;②GDF15主要定位于胞漿中,在肝癌中的表達水平明顯高于正常肝臟細胞;③在DC的培養(yǎng)上清中加入GDF15能顯著抑制未成熟DC的表型向成熟DC轉(zhuǎn)化,初步證實GDF15參與抑制DC的分化成熟。 綜上所述,本研究通過肝癌噬菌體cDNA展示文庫技術(shù)篩選獲得了一些未曾報道的能與DC相互作用的分子,并以GDF15為切入點,初步發(fā)現(xiàn)GDF15能抑制DC向成熟表型轉(zhuǎn)化,為進一步探討GDF15對DC相關(guān)功能影響的分子機制奠定基礎(chǔ)。
[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
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392
本文編號：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
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392
【參考文獻】
相關(guān)期刊論文 前2條
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 范小紅,韓寶惠,董強剛,沙慧芳,包國良,廖美琳;非小細胞肺癌中血管內(nèi)皮生長因子對樹突狀細胞的抑制作用[J];中華結(jié)核和呼吸雜志;2003年09期
，本文編號：2488300
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