本文選題：脂多糖　切入點(diǎn)：內(nèi)毒素　出處：《天津醫(yī)科大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：目的血小板減少癥(thrombocytopenia, TCP)是膿毒癥的嚴(yán)重并發(fā)癥之一,常會(huì)增加患者的出血傾向。同時(shí)它也是膿毒癥嚴(yán)重程度的評(píng)價(jià)指標(biāo)及判斷膿毒癥患者預(yù)后的預(yù)測(cè)指標(biāo)。但膿毒癥合并TCP的發(fā)生機(jī)制并未完全明確。已有的研究結(jié)果表明,膿毒癥時(shí)TCP的發(fā)生是多因素的,可能涉及到炎癥反應(yīng)、凝血系統(tǒng)及免疫反應(yīng)。用大腸桿菌脂多糖(lipopolysaccharide, LPS)攻擊小鼠可誘導(dǎo)出快速的TCP,但這種TCP的發(fā)生是LPS介導(dǎo)的炎癥反應(yīng)或凝血系統(tǒng)過(guò)度活化所致,還是LPS與血小板Toll樣受體4相互作用后直接引起的血小板破壞所致仍不明確。因此,我們通過(guò)建立單次腹腔注射不同劑量LPS誘導(dǎo)小鼠TCP模型,觀察LPS誘導(dǎo)的快速TCP的變化趨勢(shì)、檢測(cè)血漿炎癥反應(yīng)及凝血系統(tǒng)活化的標(biāo)志物水平以及LPS對(duì)分離血小板凋亡指標(biāo)的影響,進(jìn)而判斷TCP與LPS誘導(dǎo)快速TCP時(shí)機(jī)體的炎癥反應(yīng)和凝血系統(tǒng)狀態(tài)的關(guān)系,及LPS與血小板直接作用引起的血小板生存狀態(tài)的改變?cè)赥CP發(fā)生中的作用。內(nèi)容1、單次腹腔注射LPS誘導(dǎo)小鼠TCP模型的建立及評(píng)價(jià)。2、LPS誘導(dǎo)小鼠TCP時(shí)機(jī)體炎癥反應(yīng)和凝血狀態(tài),及與TCP發(fā)生的關(guān)系的研究。3、體外條件下,LPS誘導(dǎo)血小板凋亡的研究。方法第一部分：建立單次腹腔注射LPS誘導(dǎo)小鼠TCP模型,觀察不同劑量LPS腹腔注射后不同時(shí)間點(diǎn)小鼠PLT、MPV及PDW的變化,并觀察各組動(dòng)物的死亡率第二部分：經(jīng)腹腔注射非致死劑量(0.5mg/kg)和致死劑量(50mg/kg) LPS,于給藥后4h和24h采集標(biāo)本,應(yīng)用ELISA方法檢測(cè)血漿IL-6、TNF-α、TAT、 FDP、D-Dimer和TPO濃度,觀察肺組織病理形態(tài)學(xué)改變,應(yīng)用real-time PCR測(cè)定腎TPO mRNA表達(dá)量。第三部分：制備洗滌血小板,室溫條件下與LPS共同孵育3Omin,應(yīng)用化學(xué)發(fā)光法測(cè)定ATP水平和Caspase-3活性,應(yīng)用流式細(xì)胞技術(shù)測(cè)定PS外翻和AΨm去極化,應(yīng)用Western Blot測(cè)定Caspase-8、-9、 ak、Bax和 Bcl-2的表達(dá)。結(jié)果第一部分：?jiǎn)未胃骨蛔⑸銵PS,可穩(wěn)定地誘導(dǎo)小鼠TCP的發(fā)生,TCP的程度取決于LPS的劑量。根據(jù)對(duì)死亡率的影響,將LPS劑量分為非致死劑量(0.05mg/kg-5mg/kg)和致死劑量(50mg/kg)。非致死劑量各組隨LPS劑量不同,PLT組間差異有統(tǒng)計(jì)學(xué)意義,而MPV和PDW組間差異無(wú)統(tǒng)計(jì)學(xué)意義。而對(duì)24h內(nèi)所有LPS劑量組,包括致死劑量組進(jìn)行分析發(fā)現(xiàn),PLT、MPV和PDW在不同處理組之間差異均有統(tǒng)計(jì)學(xué)意義。第二部分：與對(duì)照組比較IL-6和TNF-α在不同劑量LPS處理組均明顯下降,而不同劑量LPS組之間差異無(wú)統(tǒng)計(jì)學(xué)意義,這表明在指定的觀察時(shí)點(diǎn)小鼠機(jī)體并非處于過(guò)度炎癥反應(yīng)狀態(tài),且在相同時(shí)點(diǎn)不同劑量LPS處理組動(dòng)物的炎癥反應(yīng)狀態(tài)無(wú)差異。與對(duì)照組比較TAT水平在LPS處理組明顯下降,而不同劑量LPS處理組間無(wú)差異,這表明,雖然LPS可引起TAT的明顯下降,但在不同劑量LPS處理組之間,凝血酶活化狀態(tài)沒(méi)有差別。同對(duì)照組比較,LPS處理組D-Dimer和FDP濃度無(wú)明顯變化,且不同劑量LPS處理組之間無(wú)差異,這表明,小鼠凝血系統(tǒng)并未發(fā)生過(guò)度活化及纖溶亢進(jìn)。血漿TPO濃度及腎TPOmRNA表達(dá)水平組間無(wú)差異,表明在本實(shí)驗(yàn)條件下,機(jī)體并未通過(guò)增加TPO的表達(dá)發(fā)揮代償作用。第三部分：以對(duì)照組為陰性對(duì)照,以凝血酶為陽(yáng)性對(duì)照觀察LPS與血小板孵育后,可引起血小板總ATP量升高、PS外翻、△Ψm去極化、Caspase-3活性增高、Caspase-8、-9、 Bak、Bax和Bcl-2的表達(dá)增高。結(jié)論第一部分：?jiǎn)未胃骨蛔⑸銵PS可誘導(dǎo)穩(wěn)定的小鼠TCP模型,TCP程度及持續(xù)時(shí)間與LPS呈劑量依賴性。同時(shí),機(jī)體可啟動(dòng)快速的代償性機(jī)制增加血小板的生成。第二部分：?jiǎn)未胃骨蛔⑸銵PS誘導(dǎo)TCP并不伴有炎癥反應(yīng)和凝血系統(tǒng)活化過(guò)度狀態(tài),這提示LPS可通過(guò)不依賴于炎癥反應(yīng)和凝血系統(tǒng)過(guò)度活化的途徑誘導(dǎo)TCP。機(jī)體的快速代償性血小板釋放反應(yīng)是通過(guò)非TPO依賴的途徑介導(dǎo)的。第三部分：體外條件下,LPS可誘導(dǎo)血小板發(fā)生以AΨm去極化、Caspase-3活性增高、Caspase-8、-9、Bak、 Bax口Bcl-2的表達(dá)增高為特點(diǎn)的凋亡性改變,這種凋亡性改變可能繼發(fā)于LPS的血小板過(guò)度活化。
[Abstract]:Objective thrombocytopenia (thrombocytopenia, TCP) is a serious complication of sepsis, often in patients with increased bleeding tendency. At the same time it is also predictors of the severity of sepsis evaluation and judgment of sepsis patients. But the mechanism of sepsis with TCP did not completely clear the existing research. The results showed that TCP in sepsis induced by multiple factors, may be involved in inflammation, coagulation system and immune response. With lipopolysaccharide (lipopolysaccharide, LPS) against mice can induce rapid TCP, but this kind of TCP is LPS mediated inflammation or caused by excessive activation of the coagulation system still, platelet LPS and platelet Toll like receptor 4 interaction directly after the damage caused by is still not clear. Therefore, we established a single intraperitoneal injection of different doses of LPS induced TCP mice model, observation LP The change trend of fast TCP S induced the effect of detection of plasma inflammatory reaction and coagulation system activation markers and LPS on separation of platelet apoptosis index, and then determine the relationship between TCP and LPS induced rapid TCP inflammatory and coagulation status, platelet survival state induced by LPS and platelet and direct effect of change the role in TCP. The content of 1, establishment and evaluation of a single intraperitoneal injection of LPS induced mouse model of TCP.2, LPS TCP in mice induced by inflammatory reaction and coagulation status of.3 and the relationship with the occurrence of TCP, in vitro, LPS induced platelet apoptosis. Methods: in the first part, the establishment of a single intraperitoneal injection of LPS mice induced by TCP model, the effects of different doses of LPS after intraperitoneal injection of PLT mice at different time points, the changes of MPV and PDW, the second part and mortality were observed: transabdominal animal Cavity injection of non lethal dose (0.5mg/kg) and lethal dose (50mg/kg) of LPS, 4H and 24h after administration were collected, ELISA was used to detect the plasma IL-6, TNF- alpha, TAT, FDP, D-Dimer and TPO concentration, changes of pathological morphology in lung, the expression of real-time PCR TPO mRNA application. The third part renal preparation of washed platelets, 3Omin and LPS were incubated at room temperature, the determination of ATP level and Caspase-3 activity by chemiluminescence determination, depolarized PS and A while m eversion using flow cytometry, the application of Western Blot -9, AK, Caspase-8 assay, the expression of Bax and Bcl-2. The results of the first part: single intraperitoneal injection of LPS can be stably induced by TCP, TCP depends on the dose of LPS. According to the effect on mortality, the dosage of LPS is divided into non lethal dose (0.05mg/kg-5mg/kg) and lethal dose (50mg/kg). Non lethal dose groups with the dose of LPS Different, the difference was statistically significant between group PLT, and there was no significant difference between groups MPV and PDW. The 24h in all LPS dose groups, including lethal dose group analysis showed that PLT, MPV and PDW in different treatment groups were statistically significant. The second part: compared with the control group IL-6 and TNF- alpha in different doses of LPS treatment groups were significantly decreased, while there was no significant difference between different dose LPS groups, suggesting that the excessive inflammatory reaction in the specified state at the observation point in mice is not in the same time, and no difference in different doses of LPS treatment group animal inflammation. Compared with the control group the level of TAT in the LPS group different doses of LPS significantly decreased, and the treatment group had no difference, which shows that, although significantly decreased LPS can cause TAT, but between different doses of LPS treatment group, thrombin activation did not differ with the control group. Compared with group D-Dimer and FDP concentration of LPS treatment had no obvious change, no difference between the treatment group and different dose of LPS showed that the blood coagulation system in mice did not have excessive activation and fibrinolysis. Plasma TPO concentration and renal TPOmRNA expression was no significant difference between group level, showed that under the experimental conditions, the body is not through increased TPO expression play a compensatory role. The third part: in the control group as negative control, positive control by thrombin and platelet LPS was observed after incubation, can cause platelet total ATP increased, PS valgus, lpli m depolarization, Caspase-3 activity increased, Caspase-8, -9, Bak, the expression of Bax and Bcl-2. Conclusion the first part: the mouse model of TCP single intraperitoneal injection of LPS can induce stable, TCP severity and duration and LPS in a dose-dependent manner. At the same time, the body can activate the rapid compensatory mechanism of increased platelet production. The second part: single Intraperitoneal injection of LPS induced by TCP was not accompanied by inflammation and excessive activation of coagulation system, suggesting that LPS can not dependent pathway on the inflammatory response and blood coagulation system activation induced by TCP. body rapid compensatory platelet release reaction is mediated by means of non TPO dependent guide. The third part: in vitro, LPS can induce the occurrence of platelet with A while m depolarization, Caspase-3 activity increased, Caspase-8, -9, Bak, Bax and Bcl-2 increased the expression of apoptotic characteristics change, the change of apoptosis may be secondary to LPS excessive platelet activation.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R459.7
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本文編號(hào)：1592789