靶向MyD88介导的固有免疫反应抑制胆固醇性胆石形成的实验研究

Because of dramatically increasing in the incidence of metabolic syndrome, the incidence of cholecystolithiasis has been reached as high as 10-15%, consequently resulted directly in a heavy medical burden. Since current treatment of hyperlipidemia is accompanied by excessive cholesterol excretion from the biliary tract, it is difficult to simultaneously achieve the goal of treating hyperlipidemia and preventing cholecystolithiasis by inhibiting cholesterol supersaturation only. Therefore, it is a reasonable approach to prevent cholecystolithiasis by inhibiting the nucleation and promoting the evacuation of cholesterol in the bile. The results in our previous research indicated that similar to atherosclerosis process, the supersaturated cholesterol may directly activate the innate immune response of gallbladder mucosa, increase inflammatory mediators, chemokines and nuclear protein secretion, and promote macrophage infiltration and foam cell formation, which eventually result in gallstone formation. We observed abundant foam cells in mucosa of gallbladder with gallstone, and over-expression of innate immune molecule MyD88 and proinflammatory cytokine IFN1 beta. Moreover, we observed that the formation of gallstone was delayed in MyD88 knockout mice with lithogenic diet. Given that the biliary mucosa and vascular endothelia have a similar innate immunity, we hypothesized that the innate immune response in the gallbladder mucosa to the cholesterol supersaturated bile is the key to the formation of stone. The goal of this study is to elucidate the role of MyD88 mediated innate immunity in gallstone formation. The aims of this study include: 1) to verify the innate immune cell infiltration and molecular expression of innate immunity in gallbladder mucosa in clinical samples with gallstones; 2) to determine the role of innate immune response in cholesterol stone formation in vivo in MyD88 knockout mice of MyD88 inhibitor treated mice; 3) to determine the mechanism of the activation of the innate immune response and cholesterol nucleation by cholesterol supersaturated bile juice in vitro. The research results will provide an important theory for the prevention of gallstone disease, and will have a very important clinical and social-economical significance by reducing the incidence of gallstone disease through effective prevention.

高脂血症一般伴有过多胆固醇从胆道排泄而增加了胆固醇性胆石病的发生，因此必须抑制胆汁中胆固醇成核有效预防胆石病。研究证实动脉粥样硬化板块形成与血管内皮细胞固有免疫反应有关。本课题组前期观察到与动脉粥样硬化板块类似，胆固醇性结石的胆囊粘膜可见大量吞噬脂质的泡沫细胞，固有免疫分子MyD88高表达和炎症介质IFN1β增多。高脂饮食下MyD88基因剔除小鼠能延迟胆石形成。据此假设胆固醇过饱和的胆汁启动胆囊粘膜MyD88介导的固有免疫反应是成石的关键。本研究拟：1）临床病理验证胆囊粘膜中固有免疫细胞浸润和分子的表达激活状态；2）利用MyD88抑制剂和基因敲除小鼠体内证明固有免疫反应在胆固醇成核中的作用；3）体外探讨过饱和胆固醇激活固有免疫反应引起胆石的机制。本研究有望阐明胆固醇成核成石机制，为联合应用MyD88抑制剂预防胆石病提供重要的理论依据。通过有效预防降低胆石病的发病率将有着非常重要的社会意义。

胆固醇结石病是目前临床常见疾病，其发病率逐年上升。胆石病发生机制首要因素是胆汁胆固醇过饱和，此外，胆囊也在结石形成中起到一定的促进作用。本研究：（1）首先利用Myd88基因敲除小鼠观察了对胆囊结石形成的影响，结果显示，Myd88敲除，对于结石形成的进展具有延缓作用，但并不能抑制结石形成。同时对胆囊结石形成过程中胆囊粘膜的病理动态改变进行了揭示。（2）其次，我们观察了不同饮食模型对结石形成的影响，发现：作为夜间活动习性的小鼠，夜间LD饲料摄入对于成石具有重要作用，且LD饲料改变生物钟调节基因。采用限制性饲料（9am-5pm给予lithogenic diet，LD,其余时间禁食,RLD）可改变肠道菌群，调节肝脏胆汁酸-胆固醇代谢进而可影响胆囊胆汁成分。（3）最后，我们利用SRA基因敲除小鼠研究发现：无论野生型小鼠还是KO小鼠，成石饲料均促进胆囊粘膜和肌层增厚，炎症细胞浸润。成石饲料对KO小鼠和C57BL/6小鼠均导致肝脏脂肪变及炎性细胞浸润情况，且这种变化与是否存在SRA无关。本研究结果显示，胆囊病理学改变参与结石形成，Myd88基因只影响结石形成的进展。饮食模式通过改变肠道菌群并调节生物节律进而可影响结石形成。