Ser-313低磷酸化IκBβ对脓毒症肺损伤保护作用的研究

发布时间：2018-06-05 03:31

本文选题：转基因 + IκBβ　；参考：《第二军医大学》2016年博士论文

【摘要】：研究背景脓毒症是由各种直接或间接感染因素引起的全身性免疫及炎症反应,层级放大的反应程度常可引起多脏器功能障碍甚至衰竭。作为机体气体交换中心的肺脏对炎症反应敏感,脓毒症导致的肺损伤常可带来致命后果。脓毒症所致的肺损伤通常为急性炎症性肺损伤,以肺部炎症细胞浸润为主要表现,脓毒症时因为机体调控出现紊乱,过度放大的炎症反应可加重肺损伤,而且我们认为此时中性粒细胞趋化的适度减弱反而对脏器保护有利。因此探寻减轻脓毒症肺损伤肺部炎症反应的途径是一个可行的保护脓毒症后肺脏的方法。NF-κB是重要的核基因转录因子。正常细胞中NF-κB二聚体与NF-κB抑制蛋白(IκB)相结合,以无活性形式分布于胞浆中。受到LPS刺激后,IκB被降解并活化NF-ΚB,使其移位到细胞核发挥调控基因表达的作用。IκBβ蛋白的磷酸化和去磷酸化是其功能调节的基础。有研究发现IκBβ敲除后的小鼠对过度炎症反应所致脏器功能损伤的保护能力显著下降。这间接说明IκBβ可能对过度炎症反应造成的脏器损伤有保护作用。正常的IκBβ的C端?有2个酪蛋白激酶(casein kinase)活化的丝氨酸磷酸化位点Ser-313和Ser-315,细胞受到LPS刺激时,这种两个位点均磷酸化的IκBβ蛋白降解,随后启动再合成过程,但再合成的IκBβ处于低磷酸化状态,它与细胞核中游离的NF-κB结合,与之持续作用。由此看来,Ser-313或Ser-315位点低磷酸化的IκBβ可能对NF-κB相关通路引起的针对内源性和外源性毒素的免疫应答存在作用。Ser-313低磷酸化的IκBβ对趋化因子是否有调控作用未见明确报道。通过Ser-313位点突变,使Ser-313位无法磷酸化生成低磷酸化IκBβ,可以针对这些设想进行研究。我们构建了过表达Ser-313低磷酸化IκBβ的转基因小鼠,建立其脓毒症模型后对Ser-313低磷酸化IκBβ的存在、分布和对炎症的调控、脓毒症后小鼠死亡率、脓毒症肺损伤的评估及肺部中性粒细胞趋化进行研究,模拟临床患者脓毒症的过程,以期揭示Ser-313低磷酸化的IκBβ在机体脓毒症后对肺损伤的保护作用和其机制。第一部分Ser-313低磷酸化IκBβ转基因小鼠构建、低磷酸化IκBβ相关研究及其对脓毒症小鼠的保护作用研究目的:对Ser-313低磷酸化IκBβ的表达、分布进行探究;初步研究其对脓毒症后全身炎症反应的作用;通过死亡率探究其对脓毒症后小鼠的保护作用。研究方法:构建Ser-313点突变而使该位不能磷酸化的低磷酸化IκBβ转基因小鼠。建立小鼠的由盲肠结扎穿刺(CLP)导致的脓毒症模型。对转基因和野生型小鼠脓毒症模型后组织行IκBβ基因转录及蛋白的检测。针对IκBβ进行免疫共沉淀检测。研究转基因小鼠和野生型小鼠的死亡率。对脓毒症模型后小鼠循环内的两种主要炎症因子TNF-α和IL-6进行检测。研究结果:成功构建Ser-313低磷酸化IκBβ转基因小鼠,自行繁殖、鉴定,并验证其可持续表达Ser-313低磷酸化的IκBβ。建立小鼠CLP脓毒症模型,发现转基因小鼠的生存率显著优于野生型小鼠,术后24小时,野生型和转基因小鼠的生存率分别是60%和100%,术后64小时,野生型小鼠完全死亡,转基因小鼠的生存率达到50%。发现在野生型小鼠CLP后,组织内Ser-313位磷酸化的IκBβ占总IκBβ的比值分别为3h—0,6h—17.5%,12h—54.1%,24h—73.9%,说明脓毒症早期正常的IκBβ均被降解,新合成的IκBβ大多是Ser-313低磷酸化的IκBβ。脓毒症后野生型小鼠组织中IκBβm RNA的水平稳定地增加,在术后12小时达到极大值,与脓毒症后总IκBβ蛋白量的变化趋势相符。发现Ser-313低磷酸化IκBβ在细胞核内与p105/p50,p65(Rel A)和Rel B 4种NF-κB蛋白存在相互作用,与c-Rel之间可能不存在相互作用。发现Ser-313低磷酸化IκBβ的转基因小鼠脓毒症后血清中TNFα和IL-6浓度均较野生型小鼠低,说明Ser-313低磷酸化的IκBβ能够降低促炎细胞因子的表达。研究结论:Ser-313低磷酸化IκBβ在细胞核内与p105/p50,p65(Rel A)和Rel B存在相互作用;能降低小鼠脓毒症后死亡率,调控炎症反应。第二部分Ser-313低磷酸化IκBβ通过调控小鼠脓毒症后促炎趋化作用而对脓毒症肺损伤起保护作用研究目的:探索Ser-313低磷酸化IκBβ对脓毒症后小鼠肺组织的保护效果;探索其与脓毒症后肺组织炎症细胞趋化的关系。研究方法:对两种小鼠脓毒症后的肺部组织行HE染色观察肺部损伤程度,进行肺损伤评分。测定肺组织干湿比。对肺组织切片行CD11b免疫组化染色。制备两种小鼠肺组织基因芯片,对基因芯片提示有表达量变化的主要趋化因子进行realtimePCR检测。选取最主要的中性粒细胞趋化因子CXCL1和CXCL2进行肺组织蛋白的ELISA检测和Western Blotting检测。研究结果:发现Ser-313低磷酸化IκBβ转基因小鼠CLP后肺损伤较野生型小鼠明显减轻,中性粒细胞浸润减少。CLP术后24h时,野生型小鼠的肺损伤评分明显高于转基因小鼠(10分vs 6分,p0.01),肺干湿比野生型小鼠vs转基因小鼠为3.79±0.12vs 2.56±0.11(n=3,p0.01)。CD11b免疫组化染色表明转基因小鼠在脓毒症后肺部中性粒细胞浸润较野生型小鼠少。两种小鼠CLP术后12小时肺组织基因芯片KEGG富集分析显示,转基因小鼠脓毒症后在趋化因子信号通路的基因转录明显低于于野生型小鼠,最主要的中性粒细胞趋化因子CXCL1和CXCL2均相对于野生型小鼠转录下调。小鼠肺组织Realtime PCR检测显示转基因小鼠脓毒症后肺组织9种趋化因子m RNA的转录下调,结果与基因芯片相似。ELISA和Western Blotting的结果基本一致,证实两种小鼠CLP后中性粒细胞的主要趋化因子CXCL1和CXCL2表达差异明显,在CLP术后6小时和12小时,转基因小鼠表达均明显低于野生型小鼠。研究结论:Ser-313低磷酸化的IκBβ对小鼠脓毒症肺损伤有明确的保护作用。其保护机制可能是通过下调趋化因子表达,特别是下调与中性粒细胞相关的趋化因子CXCL1、CXCL2的表达,从而抑制肺炎性反应时中性粒细胞趋化而实现的。
[Abstract]:Background sepsis is a systemic immune and inflammatory response caused by a variety of direct or indirect infection factors. The degree of cascade amplification often causes multiple organ dysfunction or failure. The lungs are sensitive to inflammation as the gas exchange center of the body. The lung injury caused by sepsis can often lead to fatal consequences. Sepsis Lung injury is usually acute inflammatory lung injury, with infiltration of inflammatory cells in the lungs as the main manifestation. Sepsis can aggravate lung injury because of the disorder in the regulation of organism and excessive enlargement of inflammation, and we think that the moderate weakening of neutrophil chemotaxis is beneficial to the protection of organs at this time. The way to damage the inflammatory response of the lungs is a feasible way to protect the lungs after sepsis..NF- kappa B is an important nuclear gene transcription factor. The NF- kappa B two polymer in normal cells is combined with the NF- kappa B inhibitor protein (I kappa B) and distributes in the cytoplasm in an inactive form. After being stimulated by LPS, I kappa B is degraded and activated to the cell. The phosphorylation and dephosphorylation of.I kappa B beta protein in the regulatory gene expression is the basis for its functional regulation. Some studies have found that the protective ability of mice after I kappa B beta knockout is significantly lower in the protection of organ dysfunction caused by excessive inflammatory response. This indirectly indicates that the I kappa B beta may have protective effects on the organ damage caused by excessive inflammatory reaction. Use. The C terminal of normal I kappa B beta? There are 2 casein kinase (casein kinase) activated serine phosphorylation sites Ser-313 and Ser-315. When LPS is stimulated by LPS, the I kappa B beta protein, which is phosphorylated at the two loci, is degraded and then initiates the resynthesis process, but the resynthesized I append B beta is in the low phosphorylation state. It seems that the low phosphorylation of I kappa B beta of the Ser-313 or Ser-315 loci may have no clear coverage of the regulatory role of I kappa B beta of the low phosphorylation of.Ser-313 in the immune response to endogenous and exogenous toxins induced by NF- kappa B related pathways. No phosphorylation of I kappa B beta could be used to study these assumptions. We constructed transgenic mice expressing Ser-313 low phosphorylation of I kappa B beta, and established the presence, distribution and regulation of Ser-313 low phosphorylation of I kappa B beta, and the mortality of sepsis, and the evaluation of sepsis lung injury. Pulmonary neutrophil chemotaxis is studied to simulate the process of clinical sepsis to reveal the protective effect and mechanism of Ser-313 low phosphorylation of I kappa B beta on lung injury after sepsis. The first part of the Ser-313 low phosphorylation I kappa B beta transgenic mice, the low phosphorylation of I kappa B beta related research and the protection of sepsis mice. The purpose of the study was to explore the expression and distribution of I kappa B beta of Ser-313, and to investigate its effect on the systemic inflammatory response after sepsis; to explore the protective effect of its effect on sepsis after the death of sepsis. The research method: to construct Ser-313 point mutation to make the I kappa B beta transgenic mice that can not be phosphorylated. To establish a mouse model of sepsis caused by cecal ligation puncture (CLP). Detection of I kappa B beta gene transcription and protein in transgenic and wild type mice after sepsis. Immunoprecipitation was carried out against I kappa B beta. The mortality of transgenic mice and wild type mice was studied. Two species in the cycle of sepsis model mice were used. The main inflammatory factors TNF- alpha and IL-6 were detected. The result of the study: successfully constructed Ser-313 low phosphorated I kappa B beta transgenic mice, self propagated and identified, and verified the I kappa B beta of Ser-313 low phosphorylation, established the mouse CLP sepsis model, and found that the survival rate of the transgenic mice was significantly better than that of the wild type mice, 24 hours after the operation, wild. The survival rate of the type and transgenic mice was 60% and 100% respectively. 64 hours after the operation, the wild type mice died completely. The survival rate of the wild type mice was complete. The survival rate of the transgenic mice reached 50%.. After the wild type mice CLP, the ratio of I kappa B beta to total I kappa B beta in the tissues was 3H 0,6h 17.5%, 12h - 54.1%, 24h - 73.9%, indicating the early positive of sepsis. The normal I kappa B beta was degraded, and the newly synthesized I kappa B beta was mostly Ser-313 low phosphorylated I kappa B beta. The level of I kappa B beta m RNA in the tissues of the wild type mice after sepsis was steadily increased, and reached a maximum at 12 hours after the operation, which was in line with the trend of the total I kappa protein content after sepsis. The interaction between 4 NF- kappa B proteins of p65 (Rel A) and Rel B may not interact with c-Rel. It is found that the serum TNF alpha and concentration in the serum of the transgenic mice with Ser-313 low phosphorylation of I kappa B beta are lower than those of the wild type mice. -313 low phosphorylation of I kappa B beta interacts with p105/p50, p65 (Rel A) and Rel B in the nucleus; it can reduce the mortality of sepsis in mice and regulate the inflammatory response. Second part Ser-313 I kappa B beta by regulating the chemotaxis of sepsis in mice by regulating the chemotaxis of sepsis, the purpose of the study is to explore the low phosphorus level. The protective effect of acidified I kappa B beta on the lung tissue of mice after sepsis; explore the relationship between the inflammatory cell chemotaxis and the pulmonary tissue after sepsis. Methods: the lung injury degree of the lung tissues of two mice after sepsis was observed by HE staining, the lung injury score was evaluated and the ratio of lung tissue to dry and wet was measured. The CD11b immunohistochemical staining of the lung tissue sections was performed. Two kinds of mouse lung tissue gene chips were prepared, and the major chemotactic factors indicating the changes in expression were detected by realtimePCR. The most important neutrophil chemotactic factor CXCL1 and CXCL2 were selected for ELISA detection and Western Blotting detection of lung tissue protein. The results showed that the Ser-313 low phosphorylation I kappa B beta transformation was found. The lung injury of mice after CLP was significantly lower than that of the wild type mice. When the neutrophils infiltration reduced the 24h after.CLP, the lung injury score of the wild type mice was significantly higher than that of the transgenic mice (10 vs 6, P0.01), and the lung dry and wet was 3.79 + 0.12vs 2.56 + 0.11 (n=3, P0.01).CD11b immuno histochemical staining for the transgenic mice. The pulmonary neutrophils infiltration in mice was less than that of the wild type mice after sepsis. The gene chip KEGG enrichment analysis in two mice after CLP 12 hours showed that the gene transcription of the chemokine signaling pathway in the transgenic mice was significantly lower than that in the wild type mice after sepsis, the most important neutrophil chemokines CXCL1 and CXCL2 The Realtime PCR detection of lung tissue in mice showed that the 9 chemokine m RNA transcriptional downregulation of lung tissue after sepsis was found in mice. The results were basically consistent with the results of gene chip similar.ELISA and Western Blotting, which confirmed the main chemokines CXCL1 and CXCL2 of the neutrophils in two mice after CLP. The expression of the transgenic mice was obviously lower than that of the wild type mice at 6 hours and 12 hours after CLP. The study concluded that the Ser-313 low phosphorylation of I kappa B beta has a clear protective effect on the lung injury of sepsis in mice. Its protective mechanism may be by down regulation of chemokine expression, especially the chemotaxis associated with neutrophils. The expression of factor CXCL1 and CXCL2 inhibited the chemotaxis of neutrophils in pneumonia.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R459.7

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