本文選題：轉(zhuǎn)基因 + IκBβ�。� 參考：《第二軍醫(yī)大學(xué)》2016年博士論文

【摘要】：研究背景膿毒癥是由各種直接或間接感染因素引起的全身性免疫及炎癥反應(yīng),層級放大的反應(yīng)程度�？梢鸲嗯K器功能障礙甚至衰竭。作為機體氣體交換中心的肺臟對炎癥反應(yīng)敏感,膿毒癥導(dǎo)致的肺損傷�？蓭碇旅蠊Ｄ摱景Y所致的肺損傷通常為急性炎癥性肺損傷,以肺部炎癥細(xì)胞浸潤為主要表現(xiàn),膿毒癥時因為機體調(diào)控出現(xiàn)紊亂,過度放大的炎癥反應(yīng)可加重肺損傷,而且我們認(rèn)為此時中性粒細(xì)胞趨化的適度減弱反而對臟器保護(hù)有利。因此探尋減輕膿毒癥肺損傷肺部炎癥反應(yīng)的途徑是一個可行的保護(hù)膿毒癥后肺臟的方法。NF-κB是重要的核基因轉(zhuǎn)錄因子。正常細(xì)胞中NF-κB二聚體與NF-κB抑制蛋白(IκB)相結(jié)合,以無活性形式分布于胞漿中。受到LPS刺激后,IκB被降解并活化NF-ΚB,使其移位到細(xì)胞核發(fā)揮調(diào)控基因表達(dá)的作用。IκBβ蛋白的磷酸化和去磷酸化是其功能調(diào)節(jié)的基礎(chǔ)。有研究發(fā)現(xiàn)IκBβ敲除后的小鼠對過度炎癥反應(yīng)所致臟器功能損傷的保護(hù)能力顯著下降。這間接說明IκBβ可能對過度炎癥反應(yīng)造成的臟器損傷有保護(hù)作用。正常的IκBβ的C端?有2個酪蛋白激酶(casein kinase)活化的絲氨酸磷酸化位點Ser-313和Ser-315,細(xì)胞受到LPS刺激時,這種兩個位點均磷酸化的IκBβ蛋白降解,隨后啟動再合成過程,但再合成的IκBβ處于低磷酸化狀態(tài),它與細(xì)胞核中游離的NF-κB結(jié)合,與之持續(xù)作用。由此看來,Ser-313或Ser-315位點低磷酸化的IκBβ可能對NF-κB相關(guān)通路引起的針對內(nèi)源性和外源性毒素的免疫應(yīng)答存在作用。Ser-313低磷酸化的IκBβ對趨化因子是否有調(diào)控作用未見明確報道。通過Ser-313位點突變,使Ser-313位無法磷酸化生成低磷酸化IκBβ,可以針對這些設(shè)想進(jìn)行研究。我們構(gòu)建了過表達(dá)Ser-313低磷酸化IκBβ的轉(zhuǎn)基因小鼠,建立其膿毒癥模型后對Ser-313低磷酸化IκBβ的存在、分布和對炎癥的調(diào)控、膿毒癥后小鼠死亡率、膿毒癥肺損傷的評估及肺部中性粒細(xì)胞趨化進(jìn)行研究,模擬臨床患者膿毒癥的過程,以期揭示Ser-313低磷酸化的IκBβ在機體膿毒癥后對肺損傷的保護(hù)作用和其機制。第一部分Ser-313低磷酸化IκBβ轉(zhuǎn)基因小鼠構(gòu)建、低磷酸化IκBβ相關(guān)研究及其對膿毒癥小鼠的保護(hù)作用研究目的:對Ser-313低磷酸化IκBβ的表達(dá)、分布進(jìn)行探究;初步研究其對膿毒癥后全身炎癥反應(yīng)的作用;通過死亡率探究其對膿毒癥后小鼠的保護(hù)作用。研究方法:構(gòu)建Ser-313點突變而使該位不能磷酸化的低磷酸化IκBβ轉(zhuǎn)基因小鼠。建立小鼠的由盲腸結(jié)扎穿刺(CLP)導(dǎo)致的膿毒癥模型。對轉(zhuǎn)基因和野生型小鼠膿毒癥模型后組織行IκBβ基因轉(zhuǎn)錄及蛋白的檢測。針對IκBβ進(jìn)行免疫共沉淀檢測。研究轉(zhuǎn)基因小鼠和野生型小鼠的死亡率。對膿毒癥模型后小鼠循環(huán)內(nèi)的兩種主要炎癥因子TNF-α和IL-6進(jìn)行檢測。研究結(jié)果:成功構(gòu)建Ser-313低磷酸化IκBβ轉(zhuǎn)基因小鼠,自行繁殖、鑒定,并驗證其可持續(xù)表達(dá)Ser-313低磷酸化的IκBβ。建立小鼠CLP膿毒癥模型,發(fā)現(xiàn)轉(zhuǎn)基因小鼠的生存率顯著優(yōu)于野生型小鼠,術(shù)后24小時,野生型和轉(zhuǎn)基因小鼠的生存率分別是60%和100%,術(shù)后64小時,野生型小鼠完全死亡,轉(zhuǎn)基因小鼠的生存率達(dá)到50%。發(fā)現(xiàn)在野生型小鼠CLP后,組織內(nèi)Ser-313位磷酸化的IκBβ占總IκBβ的比值分別為3h—0,6h—17.5%,12h—54.1%,24h—73.9%,說明膿毒癥早期正常的IκBβ均被降解,新合成的IκBβ大多是Ser-313低磷酸化的IκBβ。膿毒癥后野生型小鼠組織中IκBβm RNA的水平穩(wěn)定地增加,在術(shù)后12小時達(dá)到極大值,與膿毒癥后總IκBβ蛋白量的變化趨勢相符。發(fā)現(xiàn)Ser-313低磷酸化IκBβ在細(xì)胞核內(nèi)與p105/p50,p65(Rel A)和Rel B 4種NF-κB蛋白存在相互作用,與c-Rel之間可能不存在相互作用。發(fā)現(xiàn)Ser-313低磷酸化IκBβ的轉(zhuǎn)基因小鼠膿毒癥后血清中TNFα和IL-6濃度均較野生型小鼠低,說明Ser-313低磷酸化的IκBβ能夠降低促炎細(xì)胞因子的表達(dá)。研究結(jié)論:Ser-313低磷酸化IκBβ在細(xì)胞核內(nèi)與p105/p50,p65(Rel A)和Rel B存在相互作用;能降低小鼠膿毒癥后死亡率,調(diào)控炎癥反應(yīng)。第二部分Ser-313低磷酸化IκBβ通過調(diào)控小鼠膿毒癥后促炎趨化作用而對膿毒癥肺損傷起保護(hù)作用研究目的:探索Ser-313低磷酸化IκBβ對膿毒癥后小鼠肺組織的保護(hù)效果;探索其與膿毒癥后肺組織炎癥細(xì)胞趨化的關(guān)系。研究方法:對兩種小鼠膿毒癥后的肺部組織行HE染色觀察肺部損傷程度,進(jìn)行肺損傷評分。測定肺組織干濕比。對肺組織切片行CD11b免疫組化染色。制備兩種小鼠肺組織基因芯片,對基因芯片提示有表達(dá)量變化的主要趨化因子進(jìn)行realtimePCR檢測。選取最主要的中性粒細(xì)胞趨化因子CXCL1和CXCL2進(jìn)行肺組織蛋白的ELISA檢測和Western Blotting檢測。研究結(jié)果:發(fā)現(xiàn)Ser-313低磷酸化IκBβ轉(zhuǎn)基因小鼠CLP后肺損傷較野生型小鼠明顯減輕,中性粒細(xì)胞浸潤減少。CLP術(shù)后24h時,野生型小鼠的肺損傷評分明顯高于轉(zhuǎn)基因小鼠(10分vs 6分,p0.01),肺干濕比野生型小鼠vs轉(zhuǎn)基因小鼠為3.79±0.12vs 2.56±0.11(n=3,p0.01)。CD11b免疫組化染色表明轉(zhuǎn)基因小鼠在膿毒癥后肺部中性粒細(xì)胞浸潤較野生型小鼠少。兩種小鼠CLP術(shù)后12小時肺組織基因芯片KEGG富集分析顯示,轉(zhuǎn)基因小鼠膿毒癥后在趨化因子信號通路的基因轉(zhuǎn)錄明顯低于于野生型小鼠,最主要的中性粒細(xì)胞趨化因子CXCL1和CXCL2均相對于野生型小鼠轉(zhuǎn)錄下調(diào)。小鼠肺組織Realtime PCR檢測顯示轉(zhuǎn)基因小鼠膿毒癥后肺組織9種趨化因子m RNA的轉(zhuǎn)錄下調(diào),結(jié)果與基因芯片相似。ELISA和Western Blotting的結(jié)果基本一致,證實兩種小鼠CLP后中性粒細(xì)胞的主要趨化因子CXCL1和CXCL2表達(dá)差異明顯,在CLP術(shù)后6小時和12小時,轉(zhuǎn)基因小鼠表達(dá)均明顯低于野生型小鼠。研究結(jié)論:Ser-313低磷酸化的IκBβ對小鼠膿毒癥肺損傷有明確的保護(hù)作用。其保護(hù)機制可能是通過下調(diào)趨化因子表達(dá),特別是下調(diào)與中性粒細(xì)胞相關(guān)的趨化因子CXCL1、CXCL2的表達(dá),從而抑制肺炎性反應(yīng)時中性粒細(xì)胞趨化而實現(xiàn)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R459.7

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