本文選題：高遷移率族蛋白B1(HMGB1)　切入點(diǎn)：Toll樣受體(TLR)4　出處：《汕頭大學(xué)》2011年博士論文

【摘要】：血管生成與多種疾病的發(fā)生、發(fā)展密切相關(guān)。炎癥是導(dǎo)致血管異常增生的關(guān)鍵因素,而Toll樣受體(Toll-like receptors,TLRs)在炎癥應(yīng)答中起重要作用。因此,本研究擬通過(guò)角膜血管生成(corneal neovascularization,CNV)模型,探討TLR4及其內(nèi)源性配體——高遷移率族蛋白B1(high-mobility group box-1,HMGB1)在炎癥導(dǎo)致的血管生成中的作用。 實(shí)驗(yàn)首先通過(guò)分子克隆方法構(gòu)建HMGB1全長(zhǎng)、HMGB1 A box和B box的表達(dá)載體,提純出具有生物活性的重組蛋白,并免疫新西蘭兔制備抗HMGB1多克隆抗體,體內(nèi)、外實(shí)驗(yàn)鑒定抗體的純度、特異性、活性和效價(jià)。之后建立小鼠堿燒傷CNV模型,通過(guò)基因敲除和拮抗劑阻斷等手段,應(yīng)用實(shí)時(shí)定量聚合酶鏈?zhǔn)椒磻?yīng)(polymerase chain reaction,PCR)、組織病理學(xué)等檢測(cè)方法,從分子、細(xì)胞和整體水平上探討內(nèi)源性HMGB1-TLR4信號(hào)通路在調(diào)節(jié)生物活性介質(zhì)的表達(dá)、巨噬細(xì)胞的局部活化浸潤(rùn)等方面的作用。 研究結(jié)果顯示,TLR4基因的缺失可明顯抑制堿燒傷CNV的形成,減少燒傷角膜局部巨噬細(xì)胞浸潤(rùn)和血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor,VEGF)等生長(zhǎng)因子的生成;而TLR2基因的缺失對(duì)CNV形成無(wú)明顯影響。研究還發(fā)現(xiàn)野生型(wild type,WT)CNV模型小鼠角膜局部HMGB1和TLR4表達(dá)上調(diào)。燒傷角膜局部外用HMGB1活性蛋白可加重WT小鼠CNV的形成及角膜局部巨噬細(xì)胞浸潤(rùn),但卻對(duì)TLR4-/-小鼠無(wú)明顯效果;HMGB1刺激體外收集的WT小鼠腹腔巨噬細(xì)胞后,其VEGF等生長(zhǎng)因子表達(dá)上調(diào)的幅度顯著高于TLR4-/-腹腔巨噬細(xì)胞,印證了體內(nèi)CNV模型HMGB1給藥的觀察結(jié)果。HMGB1和TLR4的特異性拮抗劑眼表給藥抑制WT小鼠CNV的形成及角膜局部巨噬細(xì)胞浸潤(rùn),從另一角度說(shuō)明了內(nèi)源性HMGB1-TLR4信號(hào)通路在CNV形成中的重要作用。 總之,研究結(jié)果表明堿燒傷角膜局部?jī)?nèi)源性HMGB1-TLR4信號(hào)通路可增強(qiáng)巨噬細(xì)胞的活化浸潤(rùn)以及炎癥介質(zhì)、生長(zhǎng)因子的分泌,從而介導(dǎo)CNV的形成。此發(fā)現(xiàn)為進(jìn)一步闡明CNV的發(fā)生機(jī)理提供新的理論依據(jù),同時(shí)也期望可為病理性新生血管相關(guān)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.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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本文編號(hào)：1708547