S100B调控巨噬细胞极化而加剧肝纤维化的实验研究

发布时间：2018-03-25 17:22

  本文选题：S100B　切入点：肝纤维化　出处：《吉林大学》2017年博士论文

【摘要】：肝脏纤维化是危害人类健康的最严重的终末期肝病,病毒、酒精、寄生虫等多种病因都可以导致肝脏纤维化。虽然去除或控制病因可以在一定程度上控制疾病的进展,但部分肝纤维化依然持续存在。了解肝纤维化的机制是控制肝纤维化进展及逆转肝纤维化的关键。肝纤维化是肝脏损伤修复失衡的结果。在这个过程中,主要涉及两种重要的细胞:肝星状细胞和枯否细胞。肝星状细胞是产生细胞外基质的主要细胞,它的激活和凋亡是肝纤维化进展和逆转的核心环节。枯否细胞是肝脏固有免疫的重要组成部分,在肝脏损伤和修复过程中发挥重要作用。同时,二者都位于肝脏的窦间隙内。二者从功能上、空间位置上对肝纤维化的作用使二者成为研究的焦点。S100蛋白家族是一个具有EF螺旋结构、钙结合蛋白家族,有20多个家族成员。S100蛋白广泛分布于人体细胞的胞浆和胞核内,参与细胞周期、细胞分化等众多生理活动。S100B蛋白可以调节单核细胞的功能,但是关于S100B蛋白对巨噬细胞的极化以及与肝纤维化的关系没有研究报道。本研究收集自2013年3月至2015年3月于吉林大学第一附属医院肝胆外科住院并因肝硬化需要肝移植而行切除手术的纤维化肝组织和经胆结石手术切除的健康肝组织各35例。我们应用免疫组化、Real-Time PCR和Elisa等方法对比分析纤维化肝组织与健康肝组织内胶原形成及胶原酶活性的差异、肝内巨噬细胞的浸润及极化程度的区别、以及肝组织内S100B蛋白产生的差异。我们建立了体外巨噬细胞诱导的模型,研究了M1和M2两种巨噬细胞不同的诱导条件和细胞表型及功能差异。最后,应用RNA干扰和抗体中和的实验证明了LX2细胞通过分泌S100B蛋白对巨噬细胞极化的调节机制。研究结果表明,与正常肝组织相比,在纤维化肝脏中,促炎性基因(IL-1β、IL-6、IL-12、TNF-α)表达增高,抗炎性基因(IL-10、IL-22)表达降低,纤维化相关基因表达(α-SMA、Col1A1、Col1A2)也增高,而促进胶原降解酶(TIMP-1、TIMP-2)的分泌明显受到抑制。同时检测到纤维化肝组织中巨噬细胞的浸润明显增多,而且表现为M2(IL-10、IL-22、IL-33等高表达)极化形式。S100B蛋白在纤维化肝组织中的表达显著提高。相关性分析结果表明,促炎性基因的表达与纤维化相关基因的表达呈正相关,抗炎性基因的表达与纤维化相关基因的表达呈负相关。S100B蛋白的分泌与纤维化相关基因和M2巨噬巨噬细胞相关基因表达均呈正相关。体外通过对单核细胞的诱导可以获得巨噬细胞,M1型巨噬细胞和M2型巨噬细胞具有不同的细胞形态。M1型巨噬细胞高表达和分泌促炎性细胞因子IL-6、IL-12、TNF-α,抗炎性细胞因子IL-10、IL-22的分泌量较低,同时i NOS的基因表达量很高。M2型巨噬细胞高表达和分泌抗炎性细胞因子IL-10、IL-22,促炎性细胞因子IL-6、IL-12、TNF-α的分泌量较低,同时Arg1的基因表达量很高。LX2细胞抑制M1巨噬细胞的表型、细胞因子分泌和相关转录因子的表达;相反,LX2细胞可以促进M2型巨噬细胞的极化。M1型巨噬细胞对LX2细胞的杀伤作用明显高于M2型巨噬细胞。S100B蛋白体外可以明显地促进M2型巨噬细胞的极化,而抑制M1型巨噬细胞的极化,而且呈现剂量依赖关系。在LX2细胞培养体系中加入S100B的中和性抗体,或用基因沉默的方法将LX2中的S100B基因沉默后,LX2细胞对巨噬细胞极化的影响将消失。S100B通过抑制STAT1的磷酸化水平而抑制M1型巨噬细胞的极化;而通过促进STAT3的磷酸化水平而调节M2型巨噬细胞的极化。综上所述,我们通过实验发现了纤维化肝组织与健康肝组织内巨噬细胞的极化水平的差异,同时在体外实验证明了S100B蛋白可以抑制单核细胞向M1型巨噬细胞极化,而促进单核细胞向M2型巨噬细胞极化及其机制,并指出了可以靶向S100B治疗或缓解肝纤维化的新方向。
[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.缁间笂鎵，

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