本文關鍵詞： Foxp3 TLR4 甲狀腺乳頭狀癌 發(fā)病機制 免疫抑制　出處：《吉林大學》2015年博士論文　論文類型：學位論文

【摘要】：甲狀腺乳頭狀癌是當今世界上發(fā)病率增加最快的惡性腫瘤，已在多個國家上升為發(fā)病率最高的惡性腫瘤，甲狀腺癌的主要病理組織學類型可分為四類：甲狀腺乳頭狀癌(Papillary thyroid carcinoma，PTC)、甲狀腺濾泡狀癌（Follicular thyroidcarcinoma，F(xiàn)TC）、甲狀腺髓樣癌（Medullary thyroid carcinoma，MTC）和甲狀腺未分化癌(Anaplastic thyroid carcinoma，ATC)。其中大多數(shù)為PTC，PTC的分化程度較高、惡性度較低，但復發(fā)和轉移的比例較大，是影響患者預后的主要因素。目前關于PTC的發(fā)病機制已成為醫(yī)學研究的熱點。 Foxp3（Forkhead box protein3）是人體免疫系統(tǒng)發(fā)展和行使功能過程中發(fā)揮重要作用的叉頭/翼狀螺旋轉錄因子家族中的一個成員，尤其是在具有免疫抑制功能的CD4+CD25+調(diào)節(jié)性T細胞(Regulatory T Cells，Tregs)的發(fā)育和發(fā)揮功能過程中發(fā)揮重要作用。Foxp3功能的降低會導致Tregs細胞的減少，并最終導致自我攻擊的免疫細胞的過度繁殖，免疫系統(tǒng)產(chǎn)生自身免疫反應；Foxp3功能的增強會導致Tregs細胞的增加和功能的增強，人體正常的免疫功能受到抑制。故Foxp3在維持機體自身正常發(fā)揮免疫功能的過程中發(fā)揮重要作用。 TLR4屬于Toll樣受體家族(Toll like receptors，TLRs)，最初在免疫細胞的研究中被發(fā)現(xiàn)，參與先天性免疫和后天獲得性免疫。其外源性配體是革蘭氏陰性桿菌胞壁成分脂多糖(Lipopolysaccharide，LPS）。TLR4被證實在多種惡性腫瘤表達，與其在免疫細胞的作用相反，TLR4信號通路介導具有抑制性的細胞因子的釋放，使人體正常的免疫功能被抑制，與惡性腫瘤的臨床進展和不良預后緊密聯(lián)系。并可作為預測部分腫瘤不良預后的一個獨立危險因素。并有研究證實TLR4信號通路與Tregs關系密切，可促進Tregs中Foxp3的表達，增強Tregs的增殖能力和免疫抑制功能。 有研究證實，由于Foxp3免疫抑制功能的特殊性，F(xiàn)oxp3+Tregs還參與了許多腫瘤的免疫逃逸，在包括肺癌、消化系統(tǒng)腫瘤、婦科腫瘤在內(nèi)的多種惡性腫瘤患者的外周血或/和腫瘤局部發(fā)現(xiàn)Foxp3+Tregs功能增強和數(shù)量的增加，且認為與這些惡性腫瘤的臨床進展程度和患者的不良預后顯著正相關。最近有研究通過免疫組織化學方法對PTC患者的甲狀腺組織進行研究發(fā)現(xiàn)：隨著PTC腫瘤細胞的浸潤,Treg細胞介導的免疫抑制有增強趨勢,并與甲狀腺癌的臨床進展密切相關。由此可見，F(xiàn)oxp3+Tregs通過其免疫抑制功能在PTC的發(fā)生發(fā)展過程中充當重要角色。而近年來有研究者證實，不僅Foxp3+Tregs在惡性腫瘤中表達增加，而且人的肺癌、胰腺癌、前列腺癌、乳腺癌等惡性腫瘤細胞自身表達Foxp3，但相關研究較少，認為這可能與部分腫瘤的發(fā)生發(fā)展及預后密切相關，也有部分腫瘤Foxp3表達對于腫瘤發(fā)生發(fā)展的作用存在互相矛盾的結論。可見目前關于Foxp3在腫瘤發(fā)病機制的中作用尚處于起步階段。而截至目前，尚未見關于Foxp3在PTC發(fā)病機制及相關調(diào)控機理方面的研究，故需進一步進行探討。 基于上述已有的研究成果，我們提出如下設想：甲狀腺癌細胞本身是否存在Foxp3表達？如果存在，其可能的臨床意義是？Foxp3在甲狀腺癌發(fā)生發(fā)展的相關機制和其表達的上游調(diào)控通路又是什么？為進一步驗證上述設想，我們選取PTC為研究對象，從臨床病理水平、細胞分子水平和基因水平進行了以下幾個方面實驗研究： 1.從臨床病理水平對Foxp3與TLR4在人PTC組織腫瘤局部的表達進行研究，并就兩者與臨床病理指標相關性進行統(tǒng)計學分析，，以此來對Foxp3與TLR4在人PTC發(fā)病機理中的作用及二者的相互關系進行初步研究。實驗結果顯示：Foxp3和TLR4在人PTC組織腫瘤局部均呈高表達，其中Foxp3蛋白的表達與臨床病理指標中的淋巴結轉移和TNM臨床分期呈正相關，與患者的其他臨床指標無明顯相關性；而TLR4蛋白的表達雖然在人PTC組織腫瘤細胞中呈表達增高，但只與淋巴結轉移明顯相關，與患者的其他臨床指標均無明顯相關性；PTC組織中Foxp3與TLR4的表達呈顯著正相關。研究結果表明：Foxp3和TLR4在人PTC細胞中的表達與PTC的生物學行為顯著正相關，F(xiàn)oxp3和TLR4相互聯(lián)系、共同作用，在PTC的發(fā)生發(fā)展中發(fā)揮重要的作用。 2.進一步研究Foxp3在人PTC的K1細胞系表達的意義。首先在基因和蛋白水平檢測到K1細胞表達Foxp3，然后構建pEGFP-C1-Foxp3重組質(zhì)粒，通過將重組質(zhì)粒瞬時轉染K1細胞研究Foxp3過表達對細胞本身增殖能力、侵襲力和免疫功能的影響。結果顯示轉染Foxp3的K1細胞本身及其培養(yǎng)上清均可顯著抑制T淋巴細胞的增殖，這種抑制作用的途徑之一是通過上調(diào)免疫抑制因子TGF-β1和IL-10的表達來實現(xiàn)的。同時轉染Foxp3對細胞本身增殖能力、侵襲力未見明顯影響。結果表明K1細胞Foxp3表達可能通過抑制機體的免疫功能，間接促進PTC的進展。 3.K1細胞TLR4調(diào)控Foxp3表達的研究。通過LPS活化和多粘菌素B（Polymyxin，PMB）阻斷TLR4信號通路研究TLR4對Foxp3表達的調(diào)控作用。結果顯示LPS刺激K1細胞后可明顯上調(diào)Foxp3和TLR4的表達，阻斷TLR4信號后抑制了Foxp3的表達上調(diào)。因此我們認為：在K1細胞中，TLR4可能作為Foxp3表達的上游調(diào)控分子，激活TLR4信號傳導通路可以上調(diào)Foxp3的表達。 綜上，本研究的意義在于首次就PTC細胞自身Foxp3表達從臨床病理、細胞分子水平、分子基因水平三個層面、從發(fā)病機理以及調(diào)控機制兩個角度進行了初步的研究，為深入全面地認識Foxp3參與人PTC的發(fā)生、發(fā)展、侵襲、轉移的機制提供新的見解，也為臨床PTC的免疫治療提供了新的靶點。
[Abstract]:Papillary thyroid carcinoma ( PTC ) , thyroid follicular carcinoma ( FTC ) , medullary thyroid carcinoma ( MTC ) and thyroid undifferentiated carcinoma ( ATC ) have been classified into four types : Papillary thyroid carcinoma ( PTC ) , thyroid follicular carcinoma ( FTC ) , thyroid medullary thyroid carcinoma ( MTC ) and thyroid undifferentiated carcinoma ( ATC ) . Most of them are PTC , the differentiation of PTC is higher , the malignancy is low , but the proportion of relapse and metastasis is larger , which is the main factor influencing the prognosis of patients . Foxp3 ( Foxp3 ) plays an important role in the development and function of human immune system , especially in the development and functioning of CD4 + CD25 + regulatory T cells ( Regulatory T Cells ) with immunosuppressive function . Toll like receptors ( TLRs ) , a Toll like receptor family ( TLRs ) , were initially found in the study of immune cells . They are involved in innate immunity and acquired immunity after acquired immunity . The exogenous ligand is lipopolysaccharide ( LPS ) of gram - negative bacillus cell wall component . Toll - like receptor 4 was confirmed to be expressed in a variety of malignant tumors . In contrast to its role in immune cells , the signaling pathway mediated the release of inhibitory cytokines , which was closely related to the clinical progression and poor prognosis of malignant tumors . In recent years , it has been shown that Foxp3 + TVB plays an important role in the development of PTC . In recent years , it has been shown that Foxp3 + TVB plays an important role in the development of PTC . Based on the above - mentioned research results , we suggest that there is Foxp3 expression in the thyroid cancer cell itself ? If it exists , what is the clinical significance of Foxp3 in the development of thyroid cancer and what is the upstream regulatory path of its expression ? In order to further validate the above - mentioned idea , we select PTC as the research object , from the clinical pathology level , cell molecule level and gene level , the following aspects are studied : 1 . The expression of Foxp3 and TL4 in the pathogenesis of human PTC was positively correlated with the clinical pathological indexes , and the expression of Foxp3 protein was positively correlated with the lymph node metastasis and TNM clinical stage in the human PTC tissue , but the expression of Foxp3 was positively correlated with other clinical indexes of the patients . 2 . The expression of Foxp3 in human PTC K1 cell line was further studied . First , the expression of Foxp3 was detected at the level of gene and protein . The effect of Foxp3 overexpression on the proliferation ability , invasion ability and immune function of the cells were investigated . The results showed that the K1 cells transfected with Foxp3 could significantly inhibit the proliferation of T lymphocytes . 3 . The expression of Foxp3 was regulated by LPS activation and polymyxin B ( Polymyxin , PMB ) , and the expression of Foxp3 and Foxp3 was inhibited by LPS activation and polymyxin B ( Polymyxin , PMB ) . In conclusion , the significance of this study is to study the expression of Foxp3 in PTC cells from two aspects : clinical pathology , cell molecular level , molecular gene level , pathogenesis and regulation mechanism . To provide a new insight into the mechanism of human PTC ' s development , development , invasion and metastasis , and provide a new target for the immunotherapy of PTC .

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R736.1

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本文編號：1499508