本文選題：Gu-4　切入點(diǎn)：膿毒癥　出處：《南京師范大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：膿毒癥(spesis)是由感染引起的一種全身炎癥反應(yīng)綜合征,目前仍缺乏有效的治療方法,是臨床危重患者的重要死亡原因之一。高遷移率族蛋白B1 (high mobility group protein B1,HMGB1)作為新的“晚期”炎癥介質(zhì)參與了膿毒癥的發(fā)病過程,在膿毒癥發(fā)生發(fā)展中起十分重要的作用。一方面,炎癥刺激可以誘導(dǎo)機(jī)體產(chǎn)生大量的晚期炎癥介質(zhì)HMGB1,另一方面釋放出來的HGMB1可作為重要炎癥因子引發(fā)炎癥效應(yīng)。本課題主要圍繞可靶向抑制整合素CD11b的乳糖衍生物Gu-4展開研究,探討了 Gu-4對(duì)膿毒癥的治療效應(yīng)、調(diào)控膿毒癥時(shí)HMGB1釋放機(jī)制以及抑制HMGB1促炎作用的機(jī)制等內(nèi)容。首先,在盲腸結(jié)扎穿刺(CLP)誘導(dǎo)的大鼠膿毒癥模型,考察了 Gu-4對(duì)膿毒癥的治療效應(yīng),發(fā)現(xiàn)Gu-4可顯著提高膿毒癥動(dòng)物的存活率、改善肺組織損傷。動(dòng)態(tài)觀察血清炎癥因子TNF-α和HMGB1的變化水平結(jié)果提示:Gu-4明顯削弱膿毒癥時(shí)釋放到血液循環(huán)中HMGB1的水平、抑制由HMGB1觸發(fā)的TNF-α二次分泌。Gu-4對(duì)膿毒癥動(dòng)物的保護(hù)作用與動(dòng)物血清中HMGB1的水平緊密相關(guān)。其次,在離體細(xì)胞模型,進(jìn)一步探討了 Gu-4削弱膿毒癥時(shí)釋放到血液循環(huán)中HMGB1水平的作用機(jī)制。結(jié)果表明:Gu-4或CD11b阻斷抗體靶向抑制CD11b能夠抑制LPS誘導(dǎo)的HMGB1主動(dòng)釋放;使用shRNA干擾CD11b表達(dá)的同時(shí),LPS誘導(dǎo)的HMGB1核質(zhì)轉(zhuǎn)位以及主動(dòng)釋放亦被影響,并在CD11b敲除小鼠原代細(xì)胞中得到了進(jìn)一步的驗(yàn)證。免疫共沉淀及免疫印跡實(shí)驗(yàn)結(jié)果提示CD11b通過阻礙cPKC對(duì)HMGB1的磷酸化修飾以及CRM1與HMGB1的結(jié)合從而調(diào)控HMGB1的轉(zhuǎn)位與釋放,并且與Src/Syk/PKC信號(hào)通路相關(guān)。由此可見,LPS誘導(dǎo)巨噬細(xì)胞主動(dòng)釋放HMGB1需要白細(xì)胞整合素CD11b參與。第三,使用重組人HMGB1蛋白(rhHMGB1)刺激THP-1單核細(xì)胞,探討Gu-4對(duì)HMGB1誘導(dǎo)的促炎反應(yīng)的影響。結(jié)果表明,1) Gu-4劑量依賴性地抑制rhHMGB1誘導(dǎo)的TNF-α、IL-1β、IL-6等細(xì)胞因子的分泌。2) Gu-4能夠抑制rhHMGB1誘導(dǎo)的THP1細(xì)胞與血管內(nèi)皮細(xì)胞(HUVECs)的粘附。3) Gu-4可以削弱rhHMGB1引起的粘附分子CD11b分子活化位點(diǎn)暴露的增多。4) Gu-4靶向CD1 1b抑制rhHMGB1介導(dǎo)的ERK與NF-κB的活化。綜上所述,本研究查明:乳糖衍生物Gu-4對(duì)CLP膿毒癥動(dòng)物模型具有良好治療效應(yīng),其作用機(jī)制是通過抑制CD11b從而降低膿毒癥時(shí)HMGB1的釋放水平、削弱HMGB1的促炎活性。本研究首次揭示了膿毒癥時(shí)巨噬細(xì)胞主動(dòng)釋放HMGB1的過程需要白細(xì)胞整合素CD11b參與,這為靶向CD11b及HMGB1治療膿毒癥提供了重要的理論依據(jù),具有重要的臨床應(yīng)用價(jià)值。
[Abstract]:Sepsis is a systemic inflammatory response syndrome caused by infection. High mobility group protein B1HMGB1 is one of the most important causes of death in critically ill patients. As a new "late" inflammatory medium, HMGB1 is involved in the pathogenesis of sepsis and plays a very important role in the development of sepsis. Inflammatory stimulation can induce a large number of advanced inflammatory mediators HMGB1, on the other hand, the released HGMB1 can be used as an important inflammatory factor to trigger inflammation. This study focuses on the lactose derivative Gu-4, which can inhibit integrin CD11b. The therapeutic effect of Gu-4 on sepsis, the mechanism of regulating the release of HMGB1 during sepsis and the mechanism of inhibiting HMGB1 inflammation were discussed. Firstly, the sepsis model of rats was induced by cecal ligation and puncture. The therapeutic effects of Gu-4 on sepsis were investigated. It was found that Gu-4 could significantly improve the survival rate of septic animals. The dynamic observation of the levels of serum inflammatory factors TNF- 偽 and HMGB1 suggested that the level of HMGB1 released into the blood circulation was significantly reduced when the sepsis occurred. The protective effect of inhibiting the secondary secretion of TNF- 偽. Gu-4 triggered by HMGB1 on septic animals is closely related to the level of HMGB1 in animal serum. The mechanism of Gu-4 decreasing the level of HMGB1 released into blood circulation during sepsis was further investigated. The results showed that the inhibition of LPS induced active release of HMGB1 by LPS could be inhibited by the targeting inhibition of CD11b by the blocking antibody of 1: Gu-4 or CD11b. LPS-induced nuclear and cytoplasmic translocation and active release of HMGB1 were also affected by shRNA interference with CD11b expression. The results of immunoprecipitation and immunoblotting showed that CD11b regulated the translocation and release of HMGB1 by blocking the phosphorylation modification of HMGB1 by cPKC and the binding of CRM1 to HMGB1. It can be seen that LPS-induced active release of HMGB1 by macrophages requires the involvement of leukocyte integrin CD11b. Third, recombinant human HMGB1 protein rhHMGB1 is used to stimulate THP-1 monocytes. To investigate the effect of Gu-4 on the pro-inflammatory response induced by HMGB1, the results showed that Gu-4 dose-dependently inhibited the secretion of cytokines such as TNF- 偽, IL-1 尾, IL-6 and other cytokines induced by rhHMGB1. 2) Gu-4 could inhibit the adhesion of rhHMGB1 induced THP1 cells to vascular endothelial cells (HUVECs). 3) Gu-4. Gu-4 targeted CD11b to inhibit rhHMGB1 mediated activation of ERK and NF- 魏 B by weakening the increased exposure of CD11b activation sites induced by rhHMGB1. It was found that lactose derivative Gu-4 has a good therapeutic effect on CLP sepsis animal model, and its mechanism is to reduce the level of HMGB1 release by inhibiting CD11b. This study first revealed that leukocyte integrin CD11b is involved in the process of active release of HMGB1 by macrophages during sepsis, which provides an important theoretical basis for targeted CD11b and HMGB1 in the treatment of sepsis. It has important clinical application value.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R459.7

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相關(guān)期刊論文 前2條

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本文編號(hào)：1638510