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

【摘要】：甲狀腺乳頭狀癌是當(dāng)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(xué)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(fù)發(fā)和轉(zhuǎn)移的比例較大，是影響患者預(yù)后的主要因素。目前關(guān)于PTC的發(fā)病機(jī)制已成為醫(yī)學(xué)研究的熱點(diǎn)。 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(zhuǎn)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(xì)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(dǎo)致Tregs細(xì)胞的減少，并最終導(dǎo)致自我攻擊的免疫細(xì)胞的過度繁殖，免疫系統(tǒng)產(chǎn)生自身免疫反應(yīng)；Foxp3功能的增強(qiáng)會導(dǎo)致Tregs細(xì)胞的增加和功能的增強(qiáng)，人體正常的免疫功能受到抑制。故Foxp3在維持機(jī)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(xì)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(shí)在多種惡性腫瘤表達(dá)，與其在免疫細(xì)胞的作用相反，TLR4信號通路介導(dǎo)具有抑制性的細(xì)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(jìn)展和不良預(yù)后緊密聯(lián)系。并可作為預(yù)測部分腫瘤不良預(yù)后的一個獨(dú)立危險因素。并有研究證實(shí)TLR4信號通路與Tregs關(guān)系密切，可促進(jìn)Tregs中Foxp3的表達(dá)，增強(qiáng)Tregs的增殖能力和免疫抑制功能。 有研究證實(shí)，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(qiáng)和數(shù)量的增加，且認(rèn)為與這些惡性腫瘤的臨床進(jìn)展程度和患者的不良預(yù)后顯著正相關(guān)。最近有研究通過免疫組織化學(xué)方法對PTC患者的甲狀腺組織進(jìn)行研究發(fā)現(xiàn)：隨著PTC腫瘤細(xì)胞的浸潤,Treg細(xì)胞介導(dǎo)的免疫抑制有增強(qiáng)趨勢,并與甲狀腺癌的臨床進(jìn)展密切相關(guān)。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(dāng)重要角色。而近年來有研究者證實(shí)，不僅Foxp3+Tregs在惡性腫瘤中表達(dá)增加，而且人的肺癌、胰腺癌、前列腺癌、乳腺癌等惡性腫瘤細(xì)胞自身表達(dá)Foxp3，但相關(guān)研究較少，認(rèn)為這可能與部分腫瘤的發(fā)生發(fā)展及預(yù)后密切相關(guān)，也有部分腫瘤Foxp3表達(dá)對于腫瘤發(fā)生發(fā)展的作用存在互相矛盾的結(jié)論�？梢娔壳瓣P(guān)于Foxp3在腫瘤發(fā)病機(jī)制的中作用尚處于起步階段。而截至目前，尚未見關(guān)于Foxp3在PTC發(fā)病機(jī)制及相關(guān)調(diào)控機(jī)理方面的研究，故需進(jìn)一步進(jìn)行探討。 基于上述已有的研究成果，我們提出如下設(shè)想：甲狀腺癌細(xì)胞本身是否存在Foxp3表達(dá)？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(guān)機(jī)制和其表達(dá)的上游調(diào)控通路又是什么？為進(jìn)一步驗(yàn)證上述設(shè)想，我們選取PTC為研究對象，從臨床病理水平、細(xì)胞分子水平和基因水平進(jìn)行了以下幾個方面實(shí)驗(yàn)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(dá)進(jìn)行研究，并就兩者與臨床病理指標(biāo)相關(guān)性進(jìn)行統(tǒng)計(jì)學(xué)分析，，以此來對Foxp3與TLR4在人PTC發(fā)病機(jī)理中的作用及二者的相互關(guān)系進(jìn)行初步研究。實(shí)驗(yàn)結(jié)果顯示：Foxp3和TLR4在人PTC組織腫瘤局部均呈高表達(dá)，其中Foxp3蛋白的表達(dá)與臨床病理指標(biāo)中的淋巴結(jié)轉(zhuǎn)移和TNM臨床分期呈正相關(guān)，與患者的其他臨床指標(biāo)無明顯相關(guān)性；而TLR4蛋白的表達(dá)雖然在人PTC組織腫瘤細(xì)胞中呈表達(dá)增高，但只與淋巴結(jié)轉(zhuǎn)移明顯相關(guān)，與患者的其他臨床指標(biāo)均無明顯相關(guān)性；PTC組織中Foxp3與TLR4的表達(dá)呈顯著正相關(guān)。研究結(jié)果表明：Foxp3和TLR4在人PTC細(xì)胞中的表達(dá)與PTC的生物學(xué)行為顯著正相關(guān)，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(jìn)一步研究Foxp3在人PTC的K1細(xì)胞系表達(dá)的意義。首先在基因和蛋白水平檢測到K1細(xì)胞表達(dá)Foxp3，然后構(gòu)建pEGFP-C1-Foxp3重組質(zhì)粒，通過將重組質(zhì)粒瞬時轉(zhuǎn)染K1細(xì)胞研究Foxp3過表達(dá)對細(xì)胞本身增殖能力、侵襲力和免疫功能的影響。結(jié)果顯示轉(zhuǎn)染Foxp3的K1細(xì)胞本身及其培養(yǎng)上清均可顯著抑制T淋巴細(xì)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(dá)來實(shí)現(xiàn)的。同時轉(zhuǎn)染Foxp3對細(xì)胞本身增殖能力、侵襲力未見明顯影響。結(jié)果表明K1細(xì)胞Foxp3表達(dá)可能通過抑制機(jī)體的免疫功能，間接促進(jìn)PTC的進(jìn)展。 3.K1細(xì)胞TLR4調(diào)控Foxp3表達(dá)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(dá)的調(diào)控作用。結(jié)果顯示LPS刺激K1細(xì)胞后可明顯上調(diào)Foxp3和TLR4的表達(dá)，阻斷TLR4信號后抑制了Foxp3的表達(dá)上調(diào)。因此我們認(rèn)為：在K1細(xì)胞中，TLR4可能作為Foxp3表達(dá)的上游調(diào)控分子，激活TLR4信號傳導(dǎo)通路可以上調(diào)Foxp3的表達(dá)。 綜上，本研究的意義在于首次就PTC細(xì)胞自身Foxp3表達(dá)從臨床病理、細(xì)胞分子水平、分子基因水平三個層面、從發(fā)病機(jī)理以及調(diào)控機(jī)制兩個角度進(jìn)行了初步的研究，為深入全面地認(rèn)識Foxp3參與人PTC的發(fā)生、發(fā)展、侵襲、轉(zhuǎn)移的機(jī)制提供新的見解，也為臨床PTC的免疫治療提供了新的靶點(diǎn)。
[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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R736.1

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本文編號：1499508