本文選題：LPS + 膿毒癥　； 參考：《吉林大學(xué)》2015年博士論文

【摘要】：膿毒癥，是臨床上死亡率較高的疾病之一，在臨床各科室尤其是呼吸科、ICU科中發(fā)病率很高。臨床上導(dǎo)致敗血癥的細(xì)菌有很多種，以大腸埃希菌更為常見(jiàn)，且死亡率很高。膿毒癥導(dǎo)致患者死亡的最主要原因是導(dǎo)致急性多器官功能損傷，最終導(dǎo)致多器官功能衰竭死亡。對(duì)于膿毒癥的發(fā)病機(jī)制及治療的探討目前仍然是國(guó)內(nèi)外研究的熱門問(wèn)題，希望通過(guò)對(duì)機(jī)制的研究，為臨床治療膿毒癥提供有效的輔助治療藥物。 參與急性炎癥的主要細(xì)胞成分有中性粒細(xì)胞、單核細(xì)胞、巨噬細(xì)胞等。常見(jiàn)的炎癥因子有白介素（IL）、腫瘤壞死因子（TNF）、血小板活化因子（PAF）、前列腺素等。血管反應(yīng)被認(rèn)為是炎癥過(guò)程的中心環(huán)節(jié)。在膿毒癥中，中性粒細(xì)胞等炎癥細(xì)胞激活，可導(dǎo)致多種促炎癥因子及粘附分子增多，血管通透性增高，白細(xì)胞滲出并導(dǎo)致組織水腫和炎癥。 內(nèi)毒素導(dǎo)致的炎癥反應(yīng)的主要信號(hào)通路有NF-kB pathway，MAPK/ERKpathway等等。NF-kB可與多種基因啟動(dòng)子或增強(qiáng)子部位的kB位點(diǎn)特異性結(jié)合，促進(jìn)基因轉(zhuǎn)錄，是炎癥反應(yīng)的主要介質(zhì)和轉(zhuǎn)錄因子。MAPK的持續(xù)激活可以導(dǎo)致炎癥因子不斷產(chǎn)生，誘導(dǎo)“瀑布”效應(yīng)，引起炎癥持續(xù)及損傷不斷加重。其中較為重要的是p38MAPK信號(hào)，它與NF-kB pathway關(guān)系密切，是激活NF-kB pathway的主要因素�；罨腘F-kB pathway和p38MAPK pathway可以導(dǎo)致炎癥因子瀑布式級(jí)聯(lián)反應(yīng)、氧自由基增多，導(dǎo)致細(xì)胞損傷甚至死亡。 西洛他唑是常見(jiàn)的一種抗血小板凝集的藥物，在近期國(guó)內(nèi)外的很多實(shí)驗(yàn)中均證明西洛他唑?qū)τ跍p輕炎癥損傷具有重要作用。為了進(jìn)一步研究LPS誘導(dǎo)的膿毒癥進(jìn)展過(guò)程中的炎癥改變情況，進(jìn)一步研究西洛他唑減輕LPS誘導(dǎo)的肺腎損傷的機(jī)制，我們以C57小鼠為研究對(duì)象，，通過(guò)腹腔內(nèi)注射LPS的方法制作膿毒癥模型，并通過(guò)西洛他唑腹腔內(nèi)注射的方法進(jìn)行研究，應(yīng)用病理及免疫熒光染色、ELISA、Western Blot等方法進(jìn)行檢測(cè)。 實(shí)驗(yàn)第一部分：對(duì)于LPS腹腔內(nèi)注射導(dǎo)致肺損傷和腎損傷的研究。我們通過(guò)腹腔內(nèi)注射內(nèi)毒素（LPS）的方法，成功制作了膿毒癥的模型�；诓煌难芯磕康模覀冎谱髁藘煞N模型：為觀察組織病理改變，我們制作了第一模型組（低劑量組）：腹腔內(nèi)注射LPS5mg/kg造模，24小時(shí)后處死小鼠并留取組織標(biāo)本，通過(guò)腎組織和肺組織病理染色和冰凍切片的免疫熒光染色，觀察內(nèi)毒素?fù)p傷時(shí)對(duì)于肺組織和腎組織的結(jié)構(gòu)改變的影響；為了研究促炎癥因子的變化情況，我們制作了第二模型組（高劑量組），腹腔內(nèi)注射LPS7.5mg/kg造模，分別于注射LPS6小時(shí)和24小時(shí)后處死小鼠，收集血液及肺組織及腎組織樣本，測(cè)定肺組織MPO和血清BUN水平，評(píng)價(jià)臟器損傷程度變化，通過(guò)ELISA方法，測(cè)定促炎癥因子IL-6和TNF-α的水平變化，通過(guò)Western Blot測(cè)定炎癥相關(guān)因子ICAM-1和VCAM-1、炎癥通路ERK激活情況以及Mst-1的變化。 通過(guò)實(shí)驗(yàn)我們發(fā)現(xiàn)，在LPS誘導(dǎo)的膿毒癥模型早期，病理上主要以炎癥細(xì)胞浸潤(rùn)為主，炎癥細(xì)胞透過(guò)血管壁向組織內(nèi)浸潤(rùn)，局部血管上皮及組織細(xì)胞損傷，但對(duì)于組織整體結(jié)構(gòu)的破壞并不明顯。肺組織及腎臟功能損傷，肺組織MPO6小時(shí)和24小時(shí)均明顯增高，但兩者無(wú)統(tǒng)計(jì)學(xué)差異，腎功能BUN水平隨時(shí)間推移，明顯增高。促炎癥細(xì)胞因子IL-6和TNF-α均明顯增高，IL-66小時(shí)和24小時(shí)增加量無(wú)明顯統(tǒng)計(jì)差異，而TNF-α6小時(shí)含量高于24小時(shí)；Mst-1隨時(shí)間推移，明顯減少。 實(shí)驗(yàn)第二部分：西洛他唑減輕肺及腎臟損傷的研究。為了明確西洛他唑減輕炎癥損傷的機(jī)制，我們應(yīng)用西洛他唑治療膿毒癥。腹腔內(nèi)注射LPS（5mg/Kg）造模，對(duì)照組腹腔內(nèi)注射PBS，在注射LPS1小時(shí)后注射西洛他唑0.3mg/只,為避免西洛他唑溶劑DMSO的實(shí)驗(yàn)干擾，PBS組和LPS組1小時(shí)后注射同等體積的DMSO。造模24小時(shí)后收集血液及組織，通過(guò)ELISA方法，測(cè)定促炎癥因子TNF-α的水平變化。對(duì)于腎組織，我們通過(guò)Western Blot測(cè)定西洛他唑?qū)ρ装Y相關(guān)因子VCAM-1、p-ERK、p-p38的影響，了解西洛他唑?qū)ρ装Y通路ERK、p-p38MAPK途徑激活情況的影響；為了明確西洛他唑?qū)ρ趸瘧?yīng)激的作用，測(cè)定了HO-1、MnSOD及TBARS。同時(shí)，我們還應(yīng)用Western Blot及ELISA方法對(duì)肺組織進(jìn)行了研究。 通過(guò)實(shí)驗(yàn)我們發(fā)現(xiàn)，治療組小鼠（LPS/CSZ組）與模型組（LPS組）小鼠血清中的TNF-α水平相比明顯下降。LPS/CSZ組與LPS組相比，肺組織中MPO檢測(cè)無(wú)明顯差異，ICAM-1、VCAM-1表達(dá)無(wú)明顯差異；腎組織中，LPS/CSZ組HO-1含量降低，TBARS實(shí)驗(yàn)中與硫代巴比妥酸物質(zhì)反應(yīng)減少，MnSOD的水平較LPS組有所升高，三者證明西洛他唑具有抗氧化應(yīng)激作用；ERK，p38MAPK通路激活減少，VCAM-1減少，腎臟損傷減輕。實(shí)驗(yàn)證明，西洛他唑可以減輕LPS導(dǎo)致的腎臟損傷，但對(duì)于肺損傷的減輕作用不明顯。
[Abstract]:Sepsis is one of the diseases with high mortality in clinic. The incidence of ICU is very high in all clinical departments, especially in the Department of respiration. There are many bacteria in the clinic which cause sepsis. Escherichia coli is more common and the death rate is high. The most important reason for the death of patients with sepsis is the cause of acute multiple organ damage. Finally, the cause of acute multiple organ damage is caused by sepsis. The pathogenesis and treatment of sepsis are still a hot issue at home and abroad. We hope to provide effective adjuvant therapy for clinical treatment of sepsis through the study of the mechanism.
The main components involved in acute inflammation are neutrophils, monocytes, macrophages, etc.. Common inflammatory factors include IL, tumor necrosis factor (TNF), platelet activating factor (PAF), prostaglandin and so on. Vascular response is considered to be the central link in the inflammatory process. In sepsis, inflammatory cells like neutrophils are activated in sepsis. It can lead to a variety of proinflammatory and adhesion molecules, vascular permeability, leukocyte exudation and tissue edema and inflammation.
The main signal pathways of endotoxin induced inflammatory response are NF-kB pathway, MAPK/ERKpathway and so on, which can specifically bind to the kB loci of various gene promoters or enhancers, promote gene transcription, which is the main medium of the inflammatory response and the sustained activation of the transcription factor.MAPK, which can lead to the continuous production of inflammatory factors. The effect of waterfall causes inflammation and damage continuously. The more important of which is the p38MAPK signal, which is closely related to NF-kB pathway, which is the main factor to activate NF-kB pathway. The activated NF-kB pathway and p38MAPK pathway can lead to the cascade cascade of inflammatory factors, the increase of oxygen free radicals, and cell damage and even death.
Cilostazol is a common antiplatelet aggregation drug. It has been proved to play an important role in reducing inflammatory damage in a number of experiments both at home and abroad. In order to further study the inflammatory changes in the progress of LPS induced sepsis, cilostazol was further studied to reduce LPS induced lung and renal injury. The mechanism, we take C57 mice as the research object, by intraperitoneal injection of LPS method to make sepsis model, and through the method of intraperitoneal injection of cilostazol to study, using pathological and immunofluorescence staining, ELISA, Western Blot and other methods to detect.
Part 1: the study of LPS intraperitoneal injection of lung injury and renal injury. We successfully made a model of sepsis by intraperitoneal injection of endotoxin (LPS). Based on different research purposes, we made two models: To observe histopathological changes, we made the first model group (low dose group). Intraperitoneal injection of LPS5mg/kg model, 24 hours after the death of mice and left tissue specimens, through the renal tissue and lung tissue pathological staining and frozen section immunofluorescence staining, observe the effects of endotoxin injury on the structural changes in the lung tissue and renal tissue, in order to study the changes in the proinflammatory factors, we made second The model group (high dose group), intraperitoneally injected with LPS7.5mg/kg model, killed mice after LPS6 hours and 24 hours respectively, collected blood and lung tissue and renal tissue samples, measured the level of MPO and serum BUN in lung tissue, evaluated the changes in the degree of organ damage, and measured the level of the level of pro-inflammatory factor IL-6 and TNF- alpha by ELISA method, through Weste. RN Blot was used to measure inflammation related factors ICAM-1 and VCAM-1, ERK activation in inflammatory pathway and Mst-1 changes.
In the early stage of LPS induced sepsis, we found that in the early stage of the sepsis model, the main pathological changes were infiltration of inflammatory cells, inflammatory cells infiltrated into the tissue through the vascular wall, local vascular epithelium and tissue cells were damaged, but the damage to the whole structure of the tissues was not obvious. Lung tissue and kidney function damage, lung tissue MPO6 hours and 24 small There was no significant difference between the two groups, but the BUN level of renal function increased significantly as time went on. The proinflammatory cytokines IL-6 and TNF- alpha increased significantly, and there was no significant statistical difference between IL-66 hours and 24 hours, while the content of TNF- alpha 6 hours was higher than 24 hours, and Mst-1 decreased with time.
The second part of the experiment: cilostazol alleviates the injury of lung and kidney. To clarify the mechanism of cilostazol alleviating inflammatory damage, we use cilostazol in the treatment of sepsis. Intraperitoneal injection of LPS (5mg/Kg) model, intraperitoneal injection of PBS in the control group, injection of cilostazol 0.3mg/ only after LPS1 hours, in order to avoid cilostazol solvent DM SO experimental interference, group PBS and LPS group 1 hours after the injection of the same volume of DMSO. model 24 hours after the collection of blood and tissue, the ELISA method, the determination of the level of the level of pro-inflammatory factor TNF- alpha. For renal tissue, we used Western Blot to determine the effect of cilostazol on the inflammatory related factors, VCAM-1, p-ERK, p-p38, to understand cilostazol. The effect of ERK, p-p38MAPK pathway activation in the inflammatory pathway; to determine the effect of cilostazol on oxidative stress, and to determine HO-1, MnSOD and TBARS., we also used Western Blot and ELISA methods to study lung tissue.
Through the experiment, we found that the mice in the treatment group (group LPS/CSZ) and the model group (group LPS) had a significant decrease in the level of TNF- alpha in the serum of the mice. There was no significant difference in the MPO detection in the lung tissue between the group.LPS/CSZ and the LPS group, and there was no significant difference in the expression of ICAM-1 and VCAM-1 in the lung tissue; the HO-1 content in the LPS /CSZ group was lower in the renal tissue and in the TBARS experiment with the thiobarbituric acid. The level of MnSOD was higher than that in group LPS. The three showed that cilostazol had antioxidant stress, ERK, p38MAPK pathway activation decreased, VCAM-1 decreased, and renal injury alleviated. The experiment proved that cilostazol could reduce the renal damage caused by LPS, but the reduction of lung injury was not obvious.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R459.7

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