LPS诱导小鼠血小板减少症机制的研究

发布时间：2018-03-10 09:42

  本文选题：脂多糖　切入点：内毒素　出处：《天津医科大学》2013年博士论文　论文类型：学位论文

【摘要】：目的血小板减少症(thrombocytopenia, TCP)是脓毒症的严重并发症之一,常会增加患者的出血倾向。同时它也是脓毒症严重程度的评价指标及判断脓毒症患者预后的预测指标。但脓毒症合并TCP的发生机制并未完全明确。已有的研究结果表明,脓毒症时TCP的发生是多因素的,可能涉及到炎症反应、凝血系统及免疫反应。用大肠杆菌脂多糖(lipopolysaccharide, LPS)攻击小鼠可诱导出快速的TCP,但这种TCP的发生是LPS介导的炎症反应或凝血系统过度活化所致,还是LPS与血小板Toll样受体4相互作用后直接引起的血小板破坏所致仍不明确。因此,我们通过建立单次腹腔注射不同剂量LPS诱导小鼠TCP模型,观察LPS诱导的快速TCP的变化趋势、检测血浆炎症反应及凝血系统活化的标志物水平以及LPS对分离血小板凋亡指标的影响,进而判断TCP与LPS诱导快速TCP时机体的炎症反应和凝血系统状态的关系,及LPS与血小板直接作用引起的血小板生存状态的改变在TCP发生中的作用。内容1、单次腹腔注射LPS诱导小鼠TCP模型的建立及评价。2、LPS诱导小鼠TCP时机体炎症反应和凝血状态,及与TCP发生的关系的研究。3、体外条件下,LPS诱导血小板凋亡的研究。方法第一部分：建立单次腹腔注射LPS诱导小鼠TCP模型,观察不同剂量LPS腹腔注射后不同时间点小鼠PLT、MPV及PDW的变化,并观察各组动物的死亡率第二部分：经腹腔注射非致死剂量(0.5mg/kg)和致死剂量(50mg/kg) LPS,于给药后4h和24h采集标本,应用ELISA方法检测血浆IL-6、TNF-α、TAT、 FDP、D-Dimer和TPO浓度,观察肺组织病理形态学改变,应用real-time PCR测定肾TPO mRNA表达量。第三部分：制备洗涤血小板,室温条件下与LPS共同孵育3Omin,应用化学发光法测定ATP水平和Caspase-3活性,应用流式细胞技术测定PS外翻和AΨm去极化,应用Western Blot测定Caspase-8、-9、 ak、Bax和 Bcl-2的表达。结果第一部分：单次腹腔注射LPS,可稳定地诱导小鼠TCP的发生,TCP的程度取决于LPS的剂量。根据对死亡率的影响,将LPS剂量分为非致死剂量(0.05mg/kg-5mg/kg)和致死剂量(50mg/kg)。非致死剂量各组随LPS剂量不同,PLT组间差异有统计学意义,而MPV和PDW组间差异无统计学意义。而对24h内所有LPS剂量组,包括致死剂量组进行分析发现,PLT、MPV和PDW在不同处理组之间差异均有统计学意义。第二部分：与对照组比较IL-6和TNF-α在不同剂量LPS处理组均明显下降,而不同剂量LPS组之间差异无统计学意义,这表明在指定的观察时点小鼠机体并非处于过度炎症反应状态,且在相同时点不同剂量LPS处理组动物的炎症反应状态无差异。与对照组比较TAT水平在LPS处理组明显下降,而不同剂量LPS处理组间无差异,这表明,虽然LPS可引起TAT的明显下降,但在不同剂量LPS处理组之间,凝血酶活化状态没有差别。同对照组比较,LPS处理组D-Dimer和FDP浓度无明显变化,且不同剂量LPS处理组之间无差异,这表明,小鼠凝血系统并未发生过度活化及纤溶亢进。血浆TPO浓度及肾TPOmRNA表达水平组间无差异,表明在本实验条件下,机体并未通过增加TPO的表达发挥代偿作用。第三部分：以对照组为阴性对照,以凝血酶为阳性对照观察LPS与血小板孵育后,可引起血小板总ATP量升高、PS外翻、△Ψm去极化、Caspase-3活性增高、Caspase-8、-9、 Bak、Bax和Bcl-2的表达增高。结论第一部分：单次腹腔注射LPS可诱导稳定的小鼠TCP模型,TCP程度及持续时间与LPS呈剂量依赖性。同时,机体可启动快速的代偿性机制增加血小板的生成。第二部分：单次腹腔注射LPS诱导TCP并不伴有炎症反应和凝血系统活化过度状态,这提示LPS可通过不依赖于炎症反应和凝血系统过度活化的途径诱导TCP。机体的快速代偿性血小板释放反应是通过非TPO依赖的途径介导的。第三部分：体外条件下,LPS可诱导血小板发生以AΨm去极化、Caspase-3活性增高、Caspase-8、-9、Bak、 Bax口Bcl-2的表达增高为特点的凋亡性改变,这种凋亡性改变可能继发于LPS的血小板过度活化。
[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.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R459.7
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本文编号：1592789