本文關(guān)鍵詞：原發(fā)性膽汁性膽管炎模型鼠腹腔B細(xì)胞調(diào)節(jié)性功能機(jī)制探究　出處：《中國(guó)科學(xué)技術(shù)大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： Bla細(xì)胞 自身免疫性膽管炎 調(diào)節(jié)紊亂 調(diào)節(jié)性B細(xì)胞 腹腔

【摘要】：原發(fā)性膽汁性膽管炎(PBC)是一種自身免疫性肝臟疾病,它的典型特征是肝臟有大量淋巴細(xì)胞浸潤(rùn)在膽管周?chē)?并且血清中有高滴度的由自身反應(yīng)性B細(xì)胞分泌的抗線粒體抗體AMAs。有很多因素參與到PBC的發(fā)病過(guò)程中,包括免疫失調(diào)、遺傳的易感性以及環(huán)境因素等。已有的研究指出B細(xì)胞在PBC的發(fā)病過(guò)程中的作用十分復(fù)雜,根據(jù)情況不同,可能發(fā)揮致病性作用或者免疫調(diào)節(jié)性功能。但是其具體機(jī)制尚不清楚,仍然需要進(jìn)一步研究和探索。B細(xì)胞是一類(lèi)重要的免疫細(xì)胞,主要分為2個(gè)亞群,即B1細(xì)胞(包含B1a細(xì)胞和Blb細(xì)胞)和B2細(xì)胞。這兩個(gè)B細(xì)胞亞群在前體細(xì)胞來(lái)源、發(fā)育途徑、組織分布、表型及功能等方面存在差異。在自身免疫性疾病發(fā)生過(guò)程中,一般認(rèn)為B細(xì)胞通過(guò)分泌抗體發(fā)揮致病性作用。但最近的研究發(fā)現(xiàn)B細(xì)胞除了包含發(fā)揮致病性功能的亞群,同時(shí)還具有免疫調(diào)節(jié)性功能的亞群。調(diào)節(jié)性B細(xì)胞(Breg細(xì)胞)發(fā)揮功能的機(jī)制主要包括產(chǎn)生IL-10和TGF-β、參與次級(jí)抗原遞呈、與致病性T細(xì)胞相互作用以及誘導(dǎo)調(diào)節(jié)性T細(xì)胞(Treg細(xì)胞)的產(chǎn)生等。B1細(xì)胞是一個(gè)特殊的B細(xì)胞亞群,大量存在于腹腔及胸腔中,主要通過(guò)分泌天然抗體、作為抗原遞呈細(xì)胞以及分泌細(xì)胞因子來(lái)發(fā)揮免疫保護(hù)作用。B1細(xì)胞來(lái)源的天然抗體在細(xì)菌和病毒感染過(guò)程中發(fā)揮重要的保護(hù)作用。Bla細(xì)胞是腹腔B1細(xì)胞中的主要亞群,Bla細(xì)胞經(jīng)典的功能包括產(chǎn)生血清中靜息狀態(tài)的IgM、分泌腸道IgA以及發(fā)揮免疫調(diào)節(jié)功能等。B1a細(xì)胞是抗炎性細(xì)胞因子IL-10的主要來(lái)源,并且在接觸性超敏反應(yīng)模型以及病理?xiàng)l件下能夠發(fā)揮免疫調(diào)節(jié)性功能。為了更好的闡明B細(xì)胞亞群在PBC發(fā)病進(jìn)程中的作用,我們以p40-/-IL-2Rα-/-小鼠以及被廣泛研究的dnTGF-βRII小鼠作為PBC的模型鼠展開(kāi)研究。本論文主要探究了p40-/-IL-2Rα-/-小鼠中腹腔B1a細(xì)胞的紊亂與自身免疫性膽管炎的關(guān)系,同時(shí)利用dnTGF-βRⅡ小鼠腹腔B細(xì)胞的共轉(zhuǎn)輸實(shí)驗(yàn)對(duì)模型鼠中腹腔B細(xì)胞的免疫調(diào)節(jié)性功能進(jìn)行驗(yàn)證。目前取得的結(jié)果如下：1.p40-/-IL-2R α-/-模型鼠腹腔徽環(huán)境改變影響腹腔B細(xì)胞數(shù)目并與肝臟炎癥具有相關(guān)性為了探究p40-/-IL-2Rα-/-模型鼠肝臟損傷時(shí)腹腔微環(huán)境的變化,通過(guò)檢測(cè)腹腔中各細(xì)胞亞群數(shù)量,我們發(fā)現(xiàn)模型鼠中腹腔總細(xì)胞數(shù)目顯著增多,且增多的主要是T細(xì)胞,尤其是致病性的CD8+T細(xì)胞,而B(niǎo)細(xì)胞數(shù)目顯著減少。進(jìn)一步研究表明模型鼠腹腔CD4+和CD8+T細(xì)胞分泌Thl型炎性細(xì)胞因子IFN-γ的能力顯著增強(qiáng)。通過(guò)檢測(cè)腹腔灌洗液中炎性細(xì)胞因子的水平,我們發(fā)現(xiàn)促炎性細(xì)胞因子TNF和MCP-1水平顯著升高,這可能是導(dǎo)致腹腔各細(xì)胞亞群改變的原因。通過(guò)相關(guān)性分析,發(fā)現(xiàn)模型鼠中腹腔總細(xì)胞數(shù)與肝臟單個(gè)核細(xì)胞數(shù)呈正相關(guān),腹腔中B1a細(xì)胞占B細(xì)胞的比例與腹腔及肝臟單個(gè)核細(xì)胞數(shù)呈負(fù)相關(guān),且腹腔中Bla細(xì)胞占B細(xì)胞的比例與腹腔及肝臟中致病性的CD8T細(xì)胞的比例呈負(fù)相關(guān)。2. p40-/-IL-2Rα-/-模型鼠腹腔Bla細(xì)胞數(shù)目減少且功能性分子改變通過(guò)對(duì)PBC模型鼠發(fā)病過(guò)程中腹腔B細(xì)胞的詳細(xì)檢測(cè),我們發(fā)現(xiàn)相比于對(duì)照鼠,模型鼠腹腔B1a及B1b細(xì)胞的比例和數(shù)目顯著降低,而且模型鼠腹腔Bla細(xì)胞的比例隨著年齡增長(zhǎng)逐漸降低。進(jìn)一步的機(jī)制研究表明模型鼠腹腔Bla細(xì)胞增殖能力顯著降低且凋亡水平顯著升高。模型鼠腹腔B1a細(xì)胞數(shù)目顯著減少的同時(shí),與Bla細(xì)胞免疫調(diào)節(jié)性功能相關(guān)分子,包括IL-10, CTLA-4和GITR的表達(dá)水平都顯著下降；與B1a細(xì)胞活化及免疫共刺激相關(guān)分子CD44和CD80的表達(dá)水平明顯降低。通過(guò)對(duì)另一種PBC模型鼠,即dnTGF-βRII小鼠的表型檢測(cè),我們發(fā)現(xiàn)與p40-/-IL-2Ra-/-模型鼠類(lèi)似, dnTGF-βRII小鼠腹腔B1a細(xì)胞比例下降,且表達(dá)IL-10、CTLA-4、CD44和CD80的水平同樣顯著低于同窩對(duì)照鼠。3. p40-/-IL-2Rα-/-模型鼠腹腔Bla細(xì)胞轉(zhuǎn)錄組學(xué)分析為了全面探究p40-/-IL-2Ra-/-模型鼠腹腔B1a細(xì)胞轉(zhuǎn)錄組變化及其內(nèi)在機(jī)制,利用基因芯片技術(shù)并配合熒光定量PCR驗(yàn)證,我們發(fā)現(xiàn)與對(duì)照鼠相比,模型鼠腹腔Bla細(xì)胞有381個(gè)基因發(fā)生了顯著變化(2倍以上差異),其中包含大量細(xì)胞因子及其受體相互作用相關(guān)、轉(zhuǎn)錄因子活化相關(guān)及細(xì)胞周期相關(guān)基因的變化。且多個(gè)與B細(xì)胞功能相關(guān)的基因都發(fā)生了顯著改變,其中具有免疫抑制性基序(ITIM)的分子CD72 (Cd72)、CD22 (Cd22)等表達(dá)顯著上升,與流式結(jié)果一致的Cd44、Ctla4等表達(dá)顯著下降,以及IL-10的上游抑制分子AHR (Ahr)等表達(dá)顯著上升。這些發(fā)現(xiàn)詳細(xì)地展示了模型鼠與對(duì)照鼠腹腔Bla細(xì)胞存在的差異,揭示了B1a細(xì)胞異常在PBC發(fā)病過(guò)程中的重要作用機(jī)制,并提示了一些在人類(lèi)PBC研究中值得參考的基因和信號(hào)通路。4.PBC模型鼠腹腔B細(xì)胞對(duì)自身免疫性膽管炎治療潛力的評(píng)估為了探討PBC模型鼠腹腔B細(xì)胞對(duì)膽管炎是否具有治療潛能,我們進(jìn)一步利用過(guò)繼轉(zhuǎn)輸實(shí)驗(yàn)發(fā)現(xiàn)單轉(zhuǎn)輸dnTGF-βRⅡ模型鼠脾臟CD8+T細(xì)胞能引起RAGI-/-受體鼠肝臟產(chǎn)生嚴(yán)重的淋巴細(xì)胞浸潤(rùn),而將dnTGF-βRⅡ模型鼠腹腔B細(xì)胞或WT鼠腹腔B細(xì)胞與dnTGF-βⅠ RⅡ模型鼠脾臟CD8+T細(xì)胞共轉(zhuǎn)輸,發(fā)現(xiàn)RAG1-/-受體鼠肝臟淋巴細(xì)胞浸潤(rùn)都有所減輕。該實(shí)驗(yàn)為證明PBC模型鼠腹腔B細(xì)胞在炎性條件下具有強(qiáng)大的免疫調(diào)節(jié)功能提供了有利的證據(jù)。綜上所述,我們發(fā)現(xiàn)在炎性環(huán)境下,p40-/-IL-2Rα-/-模型鼠中腹腔B1a細(xì)胞顯著減少,且B1a細(xì)胞免疫調(diào)節(jié)性功能紊亂,分泌免疫調(diào)節(jié)性分子IL-10和表達(dá)CTLA-4的水平顯著降低,導(dǎo)致腹腔B1a細(xì)胞對(duì)致病性T細(xì)胞的抑制能力下降。dnTGF-βRⅡ模型鼠腹腔Bla細(xì)胞的表型與p40-/-IL-2Rα-/-模型鼠類(lèi)似。因此,我們認(rèn)為PBC模型鼠腹腔B1a細(xì)胞免疫調(diào)節(jié)性功能損傷導(dǎo)致模型鼠病情加重,這可能為PBC的治療提供了一個(gè)新的方向。
[Abstract]:Primary biliary cholangitis (PBC) is an autoimmune liver disease. Its typical feature is a large number of lymphocytes infiltrating around the bile duct in the liver. There is a high titer of anti mitochondrial antibody AMAs secreted by autoreactive B cells in the serum. There are many factors involved in the pathogenesis of PBC, including immune disorders, genetic susceptibility, and environmental factors. Previous studies have pointed out that the role of B cells in the pathogenesis of PBC is very complicated. Depending on the situation, it may play a pathogenic role or an immunomodulatory function. However, its specific mechanism is still unclear, and further research and exploration are still needed. B cells are an important class of immune cells, which are divided into 2 subgroups, that is, B1 cells (including B1a cells and Blb cells) and B2 cells. The two B cell subsets differ in the origin, development pathway, tissue distribution, phenotype and function of the precursor cells. In the process of autoimmune diseases, it is generally believed that B cells play a pathogenicity role by secreting antibodies. However, recent studies have found that B cells contain subsets of pathogenic functions and a subgroup of immunoregulatory functions. Regulatory B cells (Breg cells) play the functional mechanisms, mainly including the production of IL-10 and TGF- beta, participation in secondary antigen presentation, interaction with pathogenic T cells, and induction of regulatory T cells (Treg cells). B1 cell is a special B cell subgroup, which exists in the abdominal cavity and thoracic cavity. It mainly plays the role of immune protection by secreting natural antibodies, antigen presenting cells and secreting cytokines. Natural antibodies from B1 cells play an important role in the process of bacterial and viral infection. Bla cells are the main subsets of B1 cells in peritoneal cavity. The classical functions of Bla cells include the production of IgM in resting state, secretion of intestinal IgA and immune regulation function. B1a cells are the main source of anti-inflammatory cytokine IL-10, and can play an immunoregulatory function under the contact hypersensitivity model and pathological conditions. In order to better clarify the role of B cells in the pathogenesis of PBC in the process of subsets, we used p40-/-IL-2R and alpha - / - mice are widely studied dnTGF- beta RII mice as a model of PBC rats. This paper mainly explores the relationship between peritoneal B1a cells p40-/-IL-2R alpha - / - mice in disorder and autoimmune cholangitis, while the use of dnTGF- beta R II mouse peritoneal B cells were transferred immune experiments on peritoneal B cells in rat model of regulatory function verification. The results are as follows: 1.p40-/-IL-2R alpha - rat model of abdominal Hui environmental changes affecting the number of B cells and peritoneal and liver inflammation associated with peritoneal microenvironment in order to explore the changes of p40-/-IL-2R alpha - rat model of liver injury, by detecting the peritoneal cell subsets number, we found that the total number of cells in a rat model of abdominal cavity increased significantly the increase is mainly, and T cells, especially pathogenic CD8+T cells, and the number of B cells decreased significantly. Further study showed that the ability of CD4+ and CD8+T cells to secrete type Thl inflammatory cytokine IFN- gamma in the rat peritoneal cavity was significantly enhanced. By detecting the levels of inflammatory cytokines in peritoneal lavage fluid, we found that the levels of proinflammatory cytokines TNF and MCP-1 increased significantly, which may be the reason leading to the change of peritoneal cell subsets. Through correlation analysis, found that positive peritoneal mouse models of liver cells and total number of mononuclear cells, B1a cells accounted for the proportion of B cells was negatively correlated with the number of mononuclear cells of liver and abdominal abdominal cavity, abdominal cavity and Bla cells for pathogenic CD8T cells B cells and the proportion of abdominal cavity and in the liver the ratio was negatively correlated. 2. p40-/-IL-2R alpha - rat model of peritoneal Bla cells decreased in number and function of molecular changes through detailed detection of peritoneal B cells in the pathogenesis of PBC model rats, we found that compared to control mice, rat model of peritoneal B1a and B1b cells and the ratio of the number decreased significantly, but the proportion of peritoneal Bla cells in rat model with increasing age reduce. Further mechanism studies showed that the proliferation of Bla cells in the peritoneal cavity was significantly reduced and the level of apoptosis was significantly increased. The number of B1a cells in the abdominal cavity of model rats was significantly reduced, while the expression levels of Bla, including IL-10, CTLA-4 and GITR, were significantly decreased. The expression levels of CD44 and CD80 correlated with B1a cell activation and costimulatory molecules were significantly decreased. Through the phenotypic detection of another PBC model mouse, dnTGF- beta RII, we found that compared with p40-/-IL-2Ra-/- model mice, the proportion of peritoneal B1a cells in dnTGF- beta RII mice decreased, and the levels of IL-10, CTLA-4, CD44 and CD80 were also significantly lower than those in the same control mice. 3. p40-/-IL-2R alpha - / - SD rats Bla cell transcriptome analysis in order to explore the changes of p40-/-IL-2Ra-/- in rat model of peritoneal B1a cell transcriptome and its intrinsic mechanism using gene chip technology and fluorescence quantitative PCR test, we found that compared with the control rats, rats peritoneal Bla cells have undergone significant changes in 381 genes (more than 2 times the difference), which contains a large number of cell factor and its receptor interactions, changes related to activation of transcription factors and cell cycle related genes. And related to B cell function genes have undergone significant changes, which has immune inhibitory motifs (ITIM) molecular CD72 (Cd72), CD22 (Cd22) expression was significantly increased, and the flow cytometry results consistent with Cd44 and Ctla4 expression decreased significantly, and the IL-10 upstream inhibitor AHR (Ahr) the expression increased significantly. These findings show the difference between the Bla cells in the abdominal cavity of the model mice and the control mice, revealing the important mechanism of B1a cell abnormality in the pathogenesis of PBC, and suggesting that some of them are in humans.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R575.7;R-332

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