LPS-TLR4通路在酒精性慢性胰腺炎人胰腺星状细胞介导胰腺纤维化作用机制及TLR4拮抗剂抑制胰腺纤维化的研究

The morbidity of alcoholic chronic pancreatitis (ACP) has been steadily increasing in China, which has been causing great harm to human health. Pancreatic fibrosis is a key pathological feature of ACP. Previously ours and other studies indicated that activated rat pancreatic stellate cells (PSC) synthesized almost all of the extracellular matrix of the pancreas during fibrogenesis. It's therefore that PSC activation might be suitalbe targets for antifibrotic therapies. The pathogenesis of ACP has so far not been clarified clearly. Drinking alcohol usually doesn't cause ACP. Lipopolysaccharide (LPS), the principal endotoxin of gram- negative bacteria, mediated signaling through toll-like receptor 4 (TLR4) has played an important role in the pathogensis of alcohol-related hepatic fibrosis. However, the role of LPS-TLR4 signaling pathway on human PSC (hPSC)-mediated pancreatic fibrosis and antifibrotic therapies has not yet been clarified. In this study we will determine the signaling trasduction, transcriptional regulation and gene expression of TLR4 signaling in hPSC under stimulation with LPS. The key factors and response gene products and fibrotic markers related to TLR4 signaling pathway of hPSC will also be determined in pancreas tissues from ACP patiens. Therafter, the antifibrotic effect of TLR4 antogonist E5564 in hPSC-mediated pancreatic fibrosis will be evaluated through its blockade of LPS-TLR4 signaling in coculture of hPSC and macrophages. This study will provide an experimental basis for anti-pancreatic fibrosis in human.

酒精性慢性胰腺炎（ACP）在我国发病率逐年升高，对人们健康危害极大。胰腺纤维化是ACP的主要病理改变。我们及他人先前的研究显示大鼠胰腺星状细胞（PSC）合成胰腺几乎全部的细胞外基质，故PSC是研究抗胰腺纤维化的靶细胞。ACP发病机制尚不明确，单纯饮酒通常不至发病, G-菌内毒素脂多糖（LPS）-Toll样受体4（TLR4）通路在酒精性肝纤维化的作用已被证实, 其在人PSC(hPSC)介导胰腺纤维化的作用及抗胰腺纤维化的治疗研究亟待进行。本研究将检测LPS作用下hPSC内TLR4信号传导通路，转录调控及基因表达；测定ACP患者胰腺组织hPSC内TLR4信号通路关键因子，应答基因产物及纤维化标志物，明确其与胰腺纤维化的关系。首次采用TLR4拮抗剂E5564通过阻断LPS作用下巨噬细胞和hPSC内TLR4信号通路抑制hPSC介导胰腺纤维化的作用，为其通过抗胰腺纤维化而防治ACP提供实验依据。

近4年来，我们研究了内毒素脂多糖(LPS)在人类慢性胰腺炎(CP)，酒精性慢性胰腺炎(ACP)发生学的作用，在人胰腺星状细胞(hPSC),巨噬细胞水平研究了LPS-TLR4通路的传导特征,具体结果及意义分别如下：. (1) 45例CAP患者中，27例(60%)伴有内毒素血症血清化学趋化因子MCP-1、MIP-1α、Rantus与纤维源性因子TGF-β1 水平明显高于无内毒素血症CAP患者水平。体外分离单核细胞经GM-CSF 6天的诱导转化为巨噬细胞。酒精加LPS处理巨噬细胞其上清MCP-1、MIP-1α、Rantus及TGF-β1水平明显高于单纯酒精组（P＜0.01）。本研究结果表明LPS可诱导巨噬细胞产生化学趋化因子及纤维源性因子，进而在CAP炎症及纤维化发病过程中发挥作用。.(2)采用酶消化法成功分离了hPSC，培养激活的hPSC可表达TLR4和其应答基因产物包括致炎因子(IL-1, IL-6) 和化学趋化因子 (Rantes)，而LPS可明显增加这些因子的合成。结果表明LPS可使hPSC内TLR4通路上调，进而增加炎症因子的产生。. (3)我们对CP患者胰腺组织和血清进行了TLR4信号通路关键因子的检测，结果显示CP患者肝组织TLR4信号通路关键因子上调，伴有内毒素血症CP患者血清TLR4通路应答基因产物TNF-α,IL-6,IL-12水平明显升高，提示LPS-TLR4通路及应答基因产物在CP患者炎症进展过程中发挥作用。.(4)我们首次采用RSV启动子驱动SV40大抗原成功构建了大鼠PSC系(RP-2细胞)，研究结果表明该细胞系与原代激活的PSC具有良好的一致性，可作为一种独特的工具用于胰腺纤维化、炎症及免疫发病学的研究。.(5)我们对48只雄性ＳＤ大鼠随机分为4组，分别喂养等热量的Lieber-Decarli酒精饲料和对照饲料8周或10周，成功建立了酒精性胰腺炎和酒精性肝炎模型，检测大鼠门静脉血LPS水平。这项研究证明肠源性内毒素在长期酒精喂养大鼠胰腺纤维化和肝纤维化发生中发挥重要的作用。.(6)我们的研究了吉林地区IgG4相关疾病，特别是IgG4相关自身免疫性胰腺炎的发病情况;首次报道了3例罕见的IgG4相关疾病，这些罕见病例的发现对临床医生如何合理鉴别胰腺肿块、颌下肿块等, 早期诊断IgG4相关疾病，避免不必要的外科手术具有重要的指导