HMGB1的重组及其在血管生成中的作用

发布时间：2018-04-04 05:22

本文选题：高迁移率族蛋白B1(HMGB1)　切入点：Toll样受体(TLR)4　出处：《汕头大学》2011年博士论文

【摘要】：血管生成与多种疾病的发生、发展密切相关。炎症是导致血管异常增生的关键因素,而Toll样受体(Toll-like receptors,TLRs)在炎症应答中起重要作用。因此,本研究拟通过角膜血管生成(corneal neovascularization,CNV)模型,探讨TLR4及其内源性配体——高迁移率族蛋白B1(high-mobility group box-1,HMGB1)在炎症导致的血管生成中的作用。实验首先通过分子克隆方法构建HMGB1全长、HMGB1 A box和B box的表达载体,提纯出具有生物活性的重组蛋白,并免疫新西兰兔制备抗HMGB1多克隆抗体,体内、外实验鉴定抗体的纯度、特异性、活性和效价。之后建立小鼠碱烧伤CNV模型,通过基因敲除和拮抗剂阻断等手段,应用实时定量聚合酶链式反应(polymerase chain reaction,PCR)、组织病理学等检测方法,从分子、细胞和整体水平上探讨内源性HMGB1-TLR4信号通路在调节生物活性介质的表达、巨噬细胞的局部活化浸润等方面的作用。研究结果显示,TLR4基因的缺失可明显抑制碱烧伤CNV的形成,减少烧伤角膜局部巨噬细胞浸润和血管内皮生长因子(vascular endothelial growth factor,VEGF)等生长因子的生成;而TLR2基因的缺失对CNV形成无明显影响。研究还发现野生型(wild type,WT)CNV模型小鼠角膜局部HMGB1和TLR4表达上调。烧伤角膜局部外用HMGB1活性蛋白可加重WT小鼠CNV的形成及角膜局部巨噬细胞浸润,但却对TLR4-/-小鼠无明显效果;HMGB1刺激体外收集的WT小鼠腹腔巨噬细胞后,其VEGF等生长因子表达上调的幅度显著高于TLR4-/-腹腔巨噬细胞,印证了体内CNV模型HMGB1给药的观察结果。HMGB1和TLR4的特异性拮抗剂眼表给药抑制WT小鼠CNV的形成及角膜局部巨噬细胞浸润,从另一角度说明了内源性HMGB1-TLR4信号通路在CNV形成中的重要作用。总之,研究结果表明碱烧伤角膜局部内源性HMGB1-TLR4信号通路可增强巨噬细胞的活化浸润以及炎症介质、生长因子的分泌,从而介导CNV的形成。此发现为进一步阐明CNV的发生机理提供新的理论依据,同时也期望可为病理性新生血管相关疾病的防治提供新的思路和方法。
[Abstract]:Angiogenesis is closely related to the occurrence and development of many diseases.Inflammation is a key factor in vascular dysplasia, and Toll-like receptors (TLRs) play an important role in the inflammatory response.Therefore, the purpose of this study was to investigate the role of TLR4 and its endogenous ligand, B1(high-mobility group box-1 HMGB1, in angiogenesis induced by inflammation through the corneal angiogenesis model.Firstly, the expression vectors of HMGB1 full-length HMGB1A box and B box were constructed by molecular cloning method. The recombinant proteins with biological activity were purified, and the polyclonal antibodies against HMGB1 were prepared by immunizing New Zealand rabbits. The purity of the antibodies was identified in vitro and in vivo.Specificity, activity and titer.Then the CNV model of mice with alkali burn was established. By means of gene knockout and antagonist blocking, real-time quantitative polymerase chain reaction and histopathology were used.To explore the role of endogenous HMGB1-TLR4 signaling pathway in regulating the expression of bioactive mediators and the local activation and infiltration of macrophages.The results showed that the deletion of TLR4 gene could significantly inhibit the formation of CNV, decrease the formation of macrophages and vascular endothelial growth factor (VEGF) in cornea.However, the deletion of TLR2 gene had no significant effect on the formation of CNV.It was also found that the expression of HMGB1 and TLR4 were up-regulated in the cornea of wild type type WT-CNV mice.Topical application of HMGB1 protein in burn cornea could aggravate the formation of CNV and the infiltration of macrophages in the cornea of WT mice, but had no obvious effect on TLR4-r-mice after stimulation with HMGB1 to stimulate the peritoneal macrophages collected from WT mice in vitro.The up-regulated expression of VEGF and other growth factors was significantly higher than that of TLR4-r-peritoneal macrophages, which confirmed the observation results of HMGB1 administration in vivo. The specific antagonists of HMGB1 and TLR4 inhibited the formation of CNV and the infiltration of local macrophages in the cornea of WT mice.The important role of endogenous HMGB1-TLR4 signaling pathway in the formation of CNV is explained from another point of view.In conclusion, the results suggest that local endogenous HMGB1-TLR4 signaling pathway in alkali burn cornea can enhance the activation and infiltration of macrophages and the secretion of inflammatory mediators and growth factors, thus mediating the formation of CNV.The findings provide a new theoretical basis for further elucidation of the pathogenesis of CNV and hope to provide new ideas and methods for the prevention and treatment of pathological angiogenic diseases.
【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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