脓毒症模型动物体内内毒素释放规律及对其干预的实验研究

发布时间：2018-04-28 23:12

本文选题：脓毒症 + 内毒素　；参考：《第三军医大学》2012年硕士论文

【摘要】：目的：脓毒症(sepsis)是由感染因素介导的全身性炎症反应综合征(systemic inflammatory response syndrome, SIRS),起病隐匿,进展迅速,死亡率高,是导致危重症患者死亡的首要因素。病原体携带的病原体相关分子(pathogen-associated molecular patterns, PAMPs)与机体相应的模式识别受体(pattern recognition receptors, PRRs)结合,是诱发脓毒症的始动因素。其中,构成革兰阴性菌(Gram-negative, G-)外膜的主要成分内毒素(lipopolysaccharide, LPS)是最主要的病原体相关分子,LPS在体内释放后,具有极强的诱导脓毒症发生的能力,其水平也与脓毒症病情密切相关。因此,基于脓毒症动物模型探讨LPS的释放规律并寻找相应的干预措施,有利于促进脓毒症的防治水平的提高。CLP模型是目前应用最为广泛的脓毒症动物模型。因与脓毒症病人病程较为相似,该模型一直被认作是脓毒症研究动物模型的“金标准”。如能在CLP动物模型中明确其体内LPS的变化规律,并在此基础上检验干预药物的相应疗效,将为临床脓毒症的防治提供可靠的实验支持。基于上述认识,首先,本课题将观察CLP模型大鼠体内内毒素的动态变化；其次,为更贴近临床治疗,在CLP模型中加入抗菌药物以清除病原体,明确给予抗生素后大鼠体内LPS的变化规律；然后,在抗生素治疗基础上加入抗内毒素物质苦柯胺B(Kukoamine B, KB)进行干预,监测大鼠体内内毒素的变化情况,并分别与CLP模型组和抗生素治疗组进行比较,研究KB体内对CLP模型大鼠内毒素的拮抗作用；最后,通过热灭活大肠埃希菌攻击小鼠脓毒症模型模拟内毒素攻击,观察KB对脓毒症小鼠保护作用,并在热灭活金黄色葡萄球菌攻击小鼠脓毒症模型中,观察KB对非G-菌感染引起的脓毒症作用情况,以进一步验证评价KB对脓毒症动物的保护作用及安全性。方法： 1.探索不同抗生素对大肠埃希菌体外诱导内毒素释放水平的影响,了解其内毒素释放的时效关系,量效关系,为CLP大鼠模型选择一种能够清除病原体同时产生较多内毒素的抗菌药物； 2.检测CLP模型大鼠各时相点内毒素水平,根据体外实验结果,加入抗菌药物干预,研究加入抗菌药物后内毒素的变化规律,在此基础上,使用KB进行治疗干预,检测动物体内毒素的动态变化,观察各组大鼠72h死亡率； 3.在致死剂量热灭活细菌攻击小鼠脓毒症动物模型中,观察KB对脓毒症小鼠的保护作用,同时与临床用药血必净和乌司他丁进行平行比较,以验证KB拮抗内毒素脓毒症小鼠的保护作用。结果： 1.三种抗生素抑／杀菌效果依次为头孢他啶亚胺培南舒氨西林,均能有效清除细菌,舒氨西林在杀灭细菌同时,释放较高水平内毒素,且在各浓度条件下,诱导释放的内毒素水平稳定； 2.CLP模型大鼠血液中细菌从4h开始大量繁殖,8h达到峰值,之后维持在2000CFU/ml水平,LPS水平从8h开始增加,12h-16h达到高峰,20h-72h均在较低水平；加入抗生素干预后,血液中较少检出细菌生长,各时相点内毒素水平高于CLP对照组；加入KB后,可明显降低各时相点大鼠体内LPS水平尤其是LPS峰值水平,并改善大鼠生存状态,提高大鼠生存率； 3.在热灭活大肠埃希菌攻击小鼠脓毒症模型中,KB可提高脓毒症小鼠生存率40%左右,在热灭活金黄色葡萄球菌攻击小鼠脓毒症模型中,可提高生存率15%左右,并呈一定的量效关系。结论： 1.舒氨西林可有效清除病原体,产生大量的内毒素,在CLP大鼠模型中使用,可使CLP大鼠模型的内毒素变化更接近临床实际； 2.明确了CLP模型大鼠体内LPS变化规律,加入抗生素干预后,CLP大鼠体内LPS释放增加,在使用抗生素基础上加入KB干预后,可降低各时相点脓毒症大鼠血液中内毒素水平,说明KB体内对内毒素有较强的拮抗作用,在控制感染的基础上使用KB降低LPS水平,可提高脓毒症动物的生存率； 3.在致死剂量热灭活细菌攻击小鼠脓毒症动物模型中,KB可延迟死亡脓毒症小鼠死亡时间,提高生存率,进一步验证了KB可通过拮抗内毒素发挥对脓毒症动物的保护作用,同时对非G-菌诱发的脓毒症也有一定效果,有很好的开发应用前景。
[Abstract]:Purpose :

This model is the most widely used animal model for sepsis , and it has been recognized as the " gold standard " in the animal model of sepsis .

Based on the above - mentioned recognition , first , we will observe the dynamic changes of endotoxin in rats with CLP model .
Secondly , in order to get closer to clinical treatment , antibacterial drugs are added to CLP model to remove pathogens , and the changes of LPS in rats after administration of antibiotics are clearly defined .
Then , the anti - endotoxin ( Kukoamine B , KB ) was added on the basis of antibiotic therapy , and the changes of endotoxin in rats were monitored and compared with CLP model group and antibiotic treatment group respectively .
Finally , the effect of KB on sepsis induced by non - G - bacteria infection was observed by simulating endotoxin attack by heat - inactivated large intestine Escherichia coli in mice sepsis model , and the effect of KB on sepsis caused by non - G - bacteria infection was observed in the model of heat - inactivated staphylococcus aureus attacking mice sepsis , so as to further verify the protective effect and safety of KB on sepsis .

Method :

1 . To explore the effect of different antibiotics on endotoxin release level in vitro of Escherichia coli , to understand the time - effect relationship and dose - effect relationship of endotoxin release , and to select a kind of antibacterial drug which can eliminate pathogens and produce more endotoxin at the same time .

2 . To detect the endotoxin level at each time point of CLP model rats . According to the results of in vitro experiment , the changes of endotoxin in rats were studied . On the basis of this , the changes of endotoxin in rats were studied . On the basis of this , the dynamic changes of toxin in animals were detected by using KB , and the mortality rate of 72 hours in each group was observed .

3 . The protective effect of KB on septic mice was observed in animal model of sepsis induced by lethal dose of heat - killing bacteria .

Results :

1 . Three kinds of antibiotic - inhibiting / sterilizing effects are cefalotidin - mipenem and ampicillin , which can effectively remove bacteria , and can release high - level endotoxin at the same time of killing bacteria , and the endotoxin level induced by induced release is stable under various concentration conditions ;

2 . The bacteria in the blood of CLP model began to propagate in mass from 4 h , peaked at 8 h , then maintained at 2000 CFU / ml level , LPS level increased from 8 h , peaked at 12 h - 16 h , and at 20 h - 72h were at a lower level ;
After antibiotic intervention , the bacterial growth was less detected in the blood , and the endotoxin level in each phase was higher than that of the CLP control group .
After addition of KB , the level of LPS in rats , especially the LPS peak level , was significantly decreased , and the survival status of rats was improved and the survival rate of rats was improved ;

3 . In the model of septic shock mice , KB could improve the survival rate of septic mice by about 40 % , and the survival rate could be improved by about 15 % in the model of heat - inactivated Staphylococcus aureus attack mice sepsis .

Conclusion :

1 . sulfasin can effectively remove pathogens , generate a large amount of endotoxin , and can be used in the CLP rat model , so that the endotoxin change in the CLP rat model can be closer to clinical practice ;

2 . The changes of LPS in rats with CLP model were defined . After the intervention of antibiotics , the release of LPS increased in CLP rats . After addition of KB intervention on the basis of antibiotics , the levels of endotoxin in blood of each time - phase - point septic rats could be decreased , indicating that KB had stronger antagonism to endotoxin , and the level of LPS was reduced on the basis of control infection , which could improve survival rate of sepsis animals .

3 . In the animal model of mice sepsis induced by lethal dose of heat - killing bacteria , KB can delay the dead time of the dead sepsis mice , improve the survival rate , and further verify that KB can play a role in protecting the sepsis animal by antagonistic endotoxin , and also has a certain effect on non - G - bacteria - induced sepsis , and has good development application prospect .

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332

【参考文献】