Foxp3在甲状腺乳头状癌中的表达及TLR4对其调控的研究

发布时间：2018-02-10 03:04

本文关键词： Foxp3 TLR4 甲状腺乳头状癌发病机制免疫抑制　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：甲状腺乳头状癌是当今世界上发病率增加最快的恶性肿瘤，已在多个国家上升为发病率最高的恶性肿瘤，甲状腺癌的主要病理组织学类型可分为四类：甲状腺乳头状癌(Papillary thyroid carcinoma，PTC)、甲状腺滤泡状癌（Follicular thyroidcarcinoma，FTC）、甲状腺髓样癌（Medullary thyroid carcinoma，MTC）和甲状腺未分化癌(Anaplastic thyroid carcinoma，ATC)。其中大多数为PTC，PTC的分化程度较高、恶性度较低，但复发和转移的比例较大，是影响患者预后的主要因素。目前关于PTC的发病机制已成为医学研究的热点。 Foxp3（Forkhead box protein3）是人体免疫系统发展和行使功能过程中发挥重要作用的叉头/翼状螺旋转录因子家族中的一个成员，尤其是在具有免疫抑制功能的CD4+CD25+调节性T细胞(Regulatory T Cells，Tregs)的发育和发挥功能过程中发挥重要作用。Foxp3功能的降低会导致Tregs细胞的减少，并最终导致自我攻击的免疫细胞的过度繁殖，免疫系统产生自身免疫反应；Foxp3功能的增强会导致Tregs细胞的增加和功能的增强，人体正常的免疫功能受到抑制。故Foxp3在维持机体自身正常发挥免疫功能的过程中发挥重要作用。 TLR4属于Toll样受体家族(Toll like receptors，TLRs)，最初在免疫细胞的研究中被发现，参与先天性免疫和后天获得性免疫。其外源性配体是革兰氏阴性杆菌胞壁成分脂多糖(Lipopolysaccharide，LPS）。TLR4被证实在多种恶性肿瘤表达，与其在免疫细胞的作用相反，TLR4信号通路介导具有抑制性的细胞因子的释放，使人体正常的免疫功能被抑制，与恶性肿瘤的临床进展和不良预后紧密联系。并可作为预测部分肿瘤不良预后的一个独立危险因素。并有研究证实TLR4信号通路与Tregs关系密切，可促进Tregs中Foxp3的表达，增强Tregs的增殖能力和免疫抑制功能。有研究证实，由于Foxp3免疫抑制功能的特殊性，Foxp3+Tregs还参与了许多肿瘤的免疫逃逸，在包括肺癌、消化系统肿瘤、妇科肿瘤在内的多种恶性肿瘤患者的外周血或/和肿瘤局部发现Foxp3+Tregs功能增强和数量的增加，且认为与这些恶性肿瘤的临床进展程度和患者的不良预后显著正相关。最近有研究通过免疫组织化学方法对PTC患者的甲状腺组织进行研究发现：随着PTC肿瘤细胞的浸润,Treg细胞介导的免疫抑制有增强趋势,并与甲状腺癌的临床进展密切相关。由此可见，Foxp3+Tregs通过其免疫抑制功能在PTC的发生发展过程中充当重要角色。而近年来有研究者证实，不仅Foxp3+Tregs在恶性肿瘤中表达增加，而且人的肺癌、胰腺癌、前列腺癌、乳腺癌等恶性肿瘤细胞自身表达Foxp3，但相关研究较少，认为这可能与部分肿瘤的发生发展及预后密切相关，也有部分肿瘤Foxp3表达对于肿瘤发生发展的作用存在互相矛盾的结论。可见目前关于Foxp3在肿瘤发病机制的中作用尚处于起步阶段。而截至目前，尚未见关于Foxp3在PTC发病机制及相关调控机理方面的研究，故需进一步进行探讨。基于上述已有的研究成果，我们提出如下设想：甲状腺癌细胞本身是否存在Foxp3表达？如果存在，其可能的临床意义是？Foxp3在甲状腺癌发生发展的相关机制和其表达的上游调控通路又是什么？为进一步验证上述设想，我们选取PTC为研究对象，从临床病理水平、细胞分子水平和基因水平进行了以下几个方面实验研究： 1.从临床病理水平对Foxp3与TLR4在人PTC组织肿瘤局部的表达进行研究，并就两者与临床病理指标相关性进行统计学分析，，以此来对Foxp3与TLR4在人PTC发病机理中的作用及二者的相互关系进行初步研究。实验结果显示：Foxp3和TLR4在人PTC组织肿瘤局部均呈高表达，其中Foxp3蛋白的表达与临床病理指标中的淋巴结转移和TNM临床分期呈正相关，与患者的其他临床指标无明显相关性；而TLR4蛋白的表达虽然在人PTC组织肿瘤细胞中呈表达增高，但只与淋巴结转移明显相关，与患者的其他临床指标均无明显相关性；PTC组织中Foxp3与TLR4的表达呈显著正相关。研究结果表明：Foxp3和TLR4在人PTC细胞中的表达与PTC的生物学行为显著正相关，Foxp3和TLR4相互联系、共同作用，在PTC的发生发展中发挥重要的作用。 2.进一步研究Foxp3在人PTC的K1细胞系表达的意义。首先在基因和蛋白水平检测到K1细胞表达Foxp3，然后构建pEGFP-C1-Foxp3重组质粒，通过将重组质粒瞬时转染K1细胞研究Foxp3过表达对细胞本身增殖能力、侵袭力和免疫功能的影响。结果显示转染Foxp3的K1细胞本身及其培养上清均可显著抑制T淋巴细胞的增殖，这种抑制作用的途径之一是通过上调免疫抑制因子TGF-β1和IL-10的表达来实现的。同时转染Foxp3对细胞本身增殖能力、侵袭力未见明显影响。结果表明K1细胞Foxp3表达可能通过抑制机体的免疫功能，间接促进PTC的进展。 3.K1细胞TLR4调控Foxp3表达的研究。通过LPS活化和多粘菌素B（Polymyxin，PMB）阻断TLR4信号通路研究TLR4对Foxp3表达的调控作用。结果显示LPS刺激K1细胞后可明显上调Foxp3和TLR4的表达，阻断TLR4信号后抑制了Foxp3的表达上调。因此我们认为：在K1细胞中，TLR4可能作为Foxp3表达的上游调控分子，激活TLR4信号传导通路可以上调Foxp3的表达。综上，本研究的意义在于首次就PTC细胞自身Foxp3表达从临床病理、细胞分子水平、分子基因水平三个层面、从发病机理以及调控机制两个角度进行了初步的研究，为深入全面地认识Foxp3参与人PTC的发生、发展、侵袭、转移的机制提供新的见解，也为临床PTC的免疫治疗提供了新的靶点。
[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 .

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R736.1

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