本文選題：S100B　切入點(diǎn)：肝纖維化　出處：《吉林大學(xué)》2017年博士論文

【摘要】：肝臟纖維化是危害人類健康的最嚴(yán)重的終末期肝病,病毒、酒精、寄生蟲等多種病因都可以導(dǎo)致肝臟纖維化。雖然去除或控制病因可以在一定程度上控制疾病的進(jìn)展,但部分肝纖維化依然持續(xù)存在。了解肝纖維化的機(jī)制是控制肝纖維化進(jìn)展及逆轉(zhuǎn)肝纖維化的關(guān)鍵。肝纖維化是肝臟損傷修復(fù)失衡的結(jié)果。在這個(gè)過(guò)程中,主要涉及兩種重要的細(xì)胞:肝星狀細(xì)胞和枯否細(xì)胞。肝星狀細(xì)胞是產(chǎn)生細(xì)胞外基質(zhì)的主要細(xì)胞,它的激活和凋亡是肝纖維化進(jìn)展和逆轉(zhuǎn)的核心環(huán)節(jié)。枯否細(xì)胞是肝臟固有免疫的重要組成部分,在肝臟損傷和修復(fù)過(guò)程中發(fā)揮重要作用。同時(shí),二者都位于肝臟的竇間隙內(nèi)。二者從功能上、空間位置上對(duì)肝纖維化的作用使二者成為研究的焦點(diǎn)。S100蛋白家族是一個(gè)具有EF螺旋結(jié)構(gòu)、鈣結(jié)合蛋白家族,有20多個(gè)家族成員。S100蛋白廣泛分布于人體細(xì)胞的胞漿和胞核內(nèi),參與細(xì)胞周期、細(xì)胞分化等眾多生理活動(dòng)。S100B蛋白可以調(diào)節(jié)單核細(xì)胞的功能,但是關(guān)于S100B蛋白對(duì)巨噬細(xì)胞的極化以及與肝纖維化的關(guān)系沒(méi)有研究報(bào)道。本研究收集自2013年3月至2015年3月于吉林大學(xué)第一附屬醫(yī)院肝膽外科住院并因肝硬化需要肝移植而行切除手術(shù)的纖維化肝組織和經(jīng)膽結(jié)石手術(shù)切除的健康肝組織各35例。我們應(yīng)用免疫組化、Real-Time PCR和Elisa等方法對(duì)比分析纖維化肝組織與健康肝組織內(nèi)膠原形成及膠原酶活性的差異、肝內(nèi)巨噬細(xì)胞的浸潤(rùn)及極化程度的區(qū)別、以及肝組織內(nèi)S100B蛋白產(chǎn)生的差異。我們建立了體外巨噬細(xì)胞誘導(dǎo)的模型,研究了M1和M2兩種巨噬細(xì)胞不同的誘導(dǎo)條件和細(xì)胞表型及功能差異。最后,應(yīng)用RNA干擾和抗體中和的實(shí)驗(yàn)證明了LX2細(xì)胞通過(guò)分泌S100B蛋白對(duì)巨噬細(xì)胞極化的調(diào)節(jié)機(jī)制。研究結(jié)果表明,與正常肝組織相比,在纖維化肝臟中,促炎性基因(IL-1β、IL-6、IL-12、TNF-α)表達(dá)增高,抗炎性基因(IL-10、IL-22)表達(dá)降低,纖維化相關(guān)基因表達(dá)(α-SMA、Col1A1、Col1A2)也增高,而促進(jìn)膠原降解酶(TIMP-1、TIMP-2)的分泌明顯受到抑制。同時(shí)檢測(cè)到纖維化肝組織中巨噬細(xì)胞的浸潤(rùn)明顯增多,而且表現(xiàn)為M2(IL-10、IL-22、IL-33等高表達(dá))極化形式。S100B蛋白在纖維化肝組織中的表達(dá)顯著提高。相關(guān)性分析結(jié)果表明,促炎性基因的表達(dá)與纖維化相關(guān)基因的表達(dá)呈正相關(guān),抗炎性基因的表達(dá)與纖維化相關(guān)基因的表達(dá)呈負(fù)相關(guān)。S100B蛋白的分泌與纖維化相關(guān)基因和M2巨噬巨噬細(xì)胞相關(guān)基因表達(dá)均呈正相關(guān)。體外通過(guò)對(duì)單核細(xì)胞的誘導(dǎo)可以獲得巨噬細(xì)胞,M1型巨噬細(xì)胞和M2型巨噬細(xì)胞具有不同的細(xì)胞形態(tài)。M1型巨噬細(xì)胞高表達(dá)和分泌促炎性細(xì)胞因子IL-6、IL-12、TNF-α,抗炎性細(xì)胞因子IL-10、IL-22的分泌量較低,同時(shí)i NOS的基因表達(dá)量很高。M2型巨噬細(xì)胞高表達(dá)和分泌抗炎性細(xì)胞因子IL-10、IL-22,促炎性細(xì)胞因子IL-6、IL-12、TNF-α的分泌量較低,同時(shí)Arg1的基因表達(dá)量很高。LX2細(xì)胞抑制M1巨噬細(xì)胞的表型、細(xì)胞因子分泌和相關(guān)轉(zhuǎn)錄因子的表達(dá);相反,LX2細(xì)胞可以促進(jìn)M2型巨噬細(xì)胞的極化。M1型巨噬細(xì)胞對(duì)LX2細(xì)胞的殺傷作用明顯高于M2型巨噬細(xì)胞。S100B蛋白體外可以明顯地促進(jìn)M2型巨噬細(xì)胞的極化,而抑制M1型巨噬細(xì)胞的極化,而且呈現(xiàn)劑量依賴關(guān)系。在LX2細(xì)胞培養(yǎng)體系中加入S100B的中和性抗體,或用基因沉默的方法將LX2中的S100B基因沉默后,LX2細(xì)胞對(duì)巨噬細(xì)胞極化的影響將消失。S100B通過(guò)抑制STAT1的磷酸化水平而抑制M1型巨噬細(xì)胞的極化;而通過(guò)促進(jìn)STAT3的磷酸化水平而調(diào)節(jié)M2型巨噬細(xì)胞的極化。綜上所述,我們通過(guò)實(shí)驗(yàn)發(fā)現(xiàn)了纖維化肝組織與健康肝組織內(nèi)巨噬細(xì)胞的極化水平的差異,同時(shí)在體外實(shí)驗(yàn)證明了S100B蛋白可以抑制單核細(xì)胞向M1型巨噬細(xì)胞極化,而促進(jìn)單核細(xì)胞向M2型巨噬細(xì)胞極化及其機(jī)制,并指出了可以靶向S100B治療或緩解肝纖維化的新方向。
[Abstract]:Liver fibrosis is the most serious human health hazards of end-stage liver disease, virus, alcohol, and other causes of parasites can lead to liver fibrosis. Although the progress of removing or controlling the cause can control the disease to a certain extent, but still persistent in liver fibrosis. Understanding the mechanism of liver fibrosis is the key to control hepatic fibrosis of liver fibrosis progress and reversal of liver fibrosis. Liver damage repair is the result of the imbalance. In this process, mainly related to two kinds of cells: hepatic stellate cells and Kupffer cells. Hepatic stellate cells are the main cells produce extracellular matrix, its activation and apoptosis is a key link of progression and reversal of hepatic fibrosis. Kupffer cells is an important part of liver innate immunity and play an important role in liver injury and repair process. At the same time, the gap between the two are located in the hepatic sinus in two from. The function, space position of hepatic fibrosis to make two become the focus of research of.S100 protein family with EF is a spiral structure, calcium binding protein family, with more than 20 members of the family of.S100 protein is widely distributed in the cytoplasm and nucleus, is involved in cell cycle, cell differentiation and other physiological.S100B protein can regulate the function of monocyte, but on the polarization of S100B protein on macrophages and hepatic fibrosis have not been reported. This study collected from March 2013 to March 2015 in the First Affiliated Hospital of Jilin University and Department of hepatobiliary surgery hospital due to liver cirrhosis requiring liver transplantation and resection of liver fibrosis and after surgical resection of liver health gallstones the tissues of 35 cases. We used immunohistochemistry, compared with Real-Time PCR and Elisa analysis of liver fibrosis and healthy liver tissue The formation of collagen and collagenase activity difference, difference between infiltration and degree of polarization of macrophages in the liver, and the difference of S100B protein in liver tissue produced. We established in vitro macrophage induced model of M1 and M2 two macrophages induced by different conditions and cell phenotypic and functional differences. Finally, the application of RNA interference and antibody neutralization experiment proves that LX2 cells secrete S100B protein regulating mechanism of macrophage polarization. The results show that, compared with the normal liver tissue in liver fibrosis, proinflammatory gene (IL-1 beta, IL-6, IL-12, TNF- alpha) and increased expression of inflammatory genes (IL-10, IL-22) decreased expression, expression fibrosis related genes (alpha -SMA, Col1A1, Col1A2) also increased, and promote collagen degradation enzymes (TIMP-1, TIMP-2) secretion was significantly inhibited. To detect macrophage infiltration of fibrotic liver tissues increased obviously at the same time Many, but also for the M2 (IL-10, IL-22, IL-33 and other high expression of.S100B protein expression) polarization in fibrotic liver tissues increased significantly. The results of correlation analysis showed that the expression of fibrosis related gene expression and positively promote inflammatory gene expression related to inflammatory gene expression and fibrosis related genes were negative.S100B protein secretion and fibrosis related genes and M2 macrophage macrophage related gene expression were positively correlated. In vitro by inducing monocytes can obtain macrophages and M1 macrophages and M2 macrophages with high expression of.M1 macrophages in different cell morphology and secretion of proinflammatory cytokines IL-6, IL-12, TNF- alpha and anti inflammatory cytokines IL-10, IL-22 secretion was low, and I high expression of NOS gene had a high expression of.M2 macrophages and the secretion of inflammatory cytokines IL-10, IL-22, pro IL-12 IL-6, inflammatory cytokines, secretion of TNF- alpha is low, while the expression of Arg1 gene was very high phenotype.LX2 cells inhibit macrophage M1 expression and secretion of cytokines and related transcription factors; on the contrary, LX2 can promote the cell killing effect of polarized.M1 type macrophage M2 macrophages on LX2 cells M2 type was higher than that of.S100B protein in macrophage in vitro can obviously promote the polarization of M2 macrophages, polarization and inhibition of M1 macrophages, but also in a dose-dependent manner. The neutralizing antibody added S100B system in LX2 cells, or by gene silencing S100B gene silencing in LX2, the effect of LX2 cell on macrophage polarization will disappear polarization suppression of M1 macrophages.S100B through inhibition of STAT1 phosphorylation and; can promote the level of STAT3 phosphorylation and regulation of macrophage M2 鏋佸寲.緇間笂鎵，

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