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Gu-4靶向整合素CD11b治疗脓毒症及对晚期介质HMGB1调控机制的研究

发布时间:2018-03-20 09:37

  本文选题:Gu-4 切入点:脓毒症 出处:《南京师范大学》2014年博士论文 论文类型:学位论文


【摘要】:脓毒症(spesis)是由感染引起的一种全身炎症反应综合征,目前仍缺乏有效的治疗方法,是临床危重患者的重要死亡原因之一。高迁移率族蛋白B1 (high mobility group protein B1,HMGB1)作为新的“晚期”炎症介质参与了脓毒症的发病过程,在脓毒症发生发展中起十分重要的作用。一方面,炎症刺激可以诱导机体产生大量的晚期炎症介质HMGB1,另一方面释放出来的HGMB1可作为重要炎症因子引发炎症效应。本课题主要围绕可靶向抑制整合素CD11b的乳糖衍生物Gu-4展开研究,探讨了 Gu-4对脓毒症的治疗效应、调控脓毒症时HMGB1释放机制以及抑制HMGB1促炎作用的机制等内容。首先,在盲肠结扎穿刺(CLP)诱导的大鼠脓毒症模型,考察了 Gu-4对脓毒症的治疗效应,发现Gu-4可显著提高脓毒症动物的存活率、改善肺组织损伤。动态观察血清炎症因子TNF-α和HMGB1的变化水平结果提示:Gu-4明显削弱脓毒症时释放到血液循环中HMGB1的水平、抑制由HMGB1触发的TNF-α二次分泌。Gu-4对脓毒症动物的保护作用与动物血清中HMGB1的水平紧密相关。其次,在离体细胞模型,进一步探讨了 Gu-4削弱脓毒症时释放到血液循环中HMGB1水平的作用机制。结果表明:Gu-4或CD11b阻断抗体靶向抑制CD11b能够抑制LPS诱导的HMGB1主动释放;使用shRNA干扰CD11b表达的同时,LPS诱导的HMGB1核质转位以及主动释放亦被影响,并在CD11b敲除小鼠原代细胞中得到了进一步的验证。免疫共沉淀及免疫印迹实验结果提示CD11b通过阻碍cPKC对HMGB1的磷酸化修饰以及CRM1与HMGB1的结合从而调控HMGB1的转位与释放,并且与Src/Syk/PKC信号通路相关。由此可见,LPS诱导巨噬细胞主动释放HMGB1需要白细胞整合素CD11b参与。第三,使用重组人HMGB1蛋白(rhHMGB1)刺激THP-1单核细胞,探讨Gu-4对HMGB1诱导的促炎反应的影响。结果表明,1) Gu-4剂量依赖性地抑制rhHMGB1诱导的TNF-α、IL-1β、IL-6等细胞因子的分泌。2) Gu-4能够抑制rhHMGB1诱导的THP1细胞与血管内皮细胞(HUVECs)的粘附。3) Gu-4可以削弱rhHMGB1引起的粘附分子CD11b分子活化位点暴露的增多。4) Gu-4靶向CD1 1b抑制rhHMGB1介导的ERK与NF-κB的活化。综上所述,本研究查明:乳糖衍生物Gu-4对CLP脓毒症动物模型具有良好治疗效应,其作用机制是通过抑制CD11b从而降低脓毒症时HMGB1的释放水平、削弱HMGB1的促炎活性。本研究首次揭示了脓毒症时巨噬细胞主动释放HMGB1的过程需要白细胞整合素CD11b参与,这为靶向CD11b及HMGB1治疗脓毒症提供了重要的理论依据,具有重要的临床应用价值。
[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- 伪 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- 伪. 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- 伪, 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.
【学位授予单位】:南京师范大学
【学位级别】:博士
【学位授予年份】:2014
【分类号】:R459.7

【参考文献】

相关期刊论文 前2条

1 Geum-Youn Gwak;Tae Gun Moon;Dong Ho Lee;Byung Chul Yoo;;Glycyrrhizin attenuates HMGB1-induced hepatocyte apoptosis by inhibiting the p38-dependent mitochondrial pathway[J];World Journal of Gastroenterology;2012年07期

2 姚咏明;盛志勇;黄立峰;;The Effect of a Novel Cytokine,High Mobility Group Box 1 Protein,on the Development of Traumatic Sepsis[J];Chinese Journal of Integrative Medicine;2009年01期



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