TLR4-补体相互作用促发脂肪细胞代谢性炎症的机制研究

The new conception metaflammation is proposed recently and defined as low-grade, chronic inflammation orchestrated by metabolic cells(adipocytes,liver) in response to excess nutrients and energy.It is well known that adipose tissue releases many inflammatory mediators and macrophage infiltrate into adipose tissue under obese state.. The first feature of obese inflammation is that the initiation is metabolic-it originates from metabolic signals and within metabolic cells such as the adipocyte. In the study in vitro 3T3-L1 adiocytes are treated by oleate, LPS, C5a，LPS + C5a separately, Than to detect the following content:.(1)the secretion of adipokines such as adiponectin, IL-6,IL-8,KC, MCP-1,ASP will be measured by Elisa in the media and adipokines mRNA expression will be assayed by RT-PCR; (2)The expression of Receptor TLR4, C5L2, C5aR will be test by RT-PCR and western blot, at the same time to evaluate C5L2-C5aR heterodimerization by confocal microscopy;(3) ASP- C5L2 pathway function is detected by measure glucose transport and TG synthesis stimulated by ASP(4) The change of insulin signaling pathway, including expression of IR,IRS-1,IRS-2,PI3K and their activity. (5) The change of IKK/NF-kappaB inflammtory signaling pathway, including Total unclear NF-κB, IKK，IκB mRNA and protein expression, phosphorylation of IKKand IκB, NF-κB-p65 translocation and NF-κB activity by electrophoretic mobility shift assay, immunofluorescent staining, NF-κB responsive luciferase assay and etc.. Through these studys we hope we can test and verify our following hypothesis：1. C5a affects the metaflammation in adipocytes;2. TLR -C5a interaction initiates the inflammation reaction in adipocytes and TLR activation enhance the inflammatory affection of C5a; 3.The change of C5L2 expression or/and heterodimerization of C5L2-c5aR induced by metabolic or inflammatory factor have regulating function on inflammatory reaction initiated by TLR-C5a interaction.. The discovery that obesity and related disease results in an inflammatory satae ushered in a research field that examines the inflammatory mechanisms.This study explore that early mechenism of inflammation in adipocytes. It is the First time to reach the TLR-complements interaction in adipocytes. The completion of the subject will show new field of the cross-talking between metabolism and immune system. Further knowledg on these function will help to understand the mechanism of metaflammation and supply the new pharmacy target for pretecting and treatment of obesity and related disease.

近年来脂肪组织代谢与免疫的交叉对话成为研究的热点问题之一，脂肪组织的低水平慢性炎症与胰岛素抵抗的发生密切相关。本研究以体外培养的3T3-L1 脂肪细胞为研究对象，采用生物化学，分子生物学及共聚焦显微镜等的实验方法，从细胞因子、受体及受体间相互作用、代谢和炎症信号等多个层次，首先评价TLR4活化与C5a共同作用对炎症反应的影响;然后检测异常代谢信号（高油酸）对IKK/NF-κB炎症信号的影响及炎症刺激因子（LPS，C5a，LPS+C5a）对代谢功能的影响；分析不同干预下C5L2表达及与C5aR二聚体化与代谢及炎症反应间的关系。最后采用C5L2基因敲除小鼠模型，通过检测系统的代谢稳态、脂肪组织炎症及原代培养细胞对炎症刺激因子的反应等，进一步验证C5L2在代谢性炎症中发挥的调节作用。该课题的完成，将深化对代谢-免疫交叉对话的认识，并为探求肥胖及其相关疾病的防治提供新思路。

随着大量脂肪细胞因子的发现及其功能研究，脂肪组织作为内分泌器官进一步深化了对肥胖，代谢综合征及其相关疾病发生发展的认识。脂肪因子的复杂多样性，使得脂肪组织成为多学科研究的交叉点，越来越多的证据表明，代谢与免疫之间存在密切联系，脂肪组织作为免疫器官再次成为关注的热点。.3T3-L1 脂肪细胞分别给予油酸, LPS, C5a，LPS+C5a刺激，研究发现 1.C5a诱导脂肪细胞释放炎症因子及上调炎症信号通路关键因子的表达。2. LPS+C5a刺激加强了C5a诱导脂肪细胞释放炎症因子及上调炎症信号通路的表达，TLR -C5a 具有协同作用。3.oleate,LPS或\和C5a诱导脂肪细胞C5L2-c5aR的表达及其二聚体的形成参与脂肪细胞代谢性验证的发生发展，是促发脂肪细胞验证的关键调节点。本研究分别从代谢及炎症两个途径证实脂肪组织代谢性炎症中代谢与免疫之间存的密切联系及其可能的相关机制，为探求肥胖及其相关疾病的发病机制提供新的理论基础，并为相应的疾病防治提供新思路。