本文選題：膿毒癥 + 內(nèi)毒素　； 參考：《第三軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：目的： 膿毒癥(sepsis)是由感染因素介導(dǎo)的全身性炎癥反應(yīng)綜合征(systemic inflammatory response syndrome, SIRS),起病隱匿,進(jìn)展迅速,死亡率高,是導(dǎo)致危重癥患者死亡的首要因素。病原體攜帶的病原體相關(guān)分子(pathogen-associated molecular patterns, PAMPs)與機(jī)體相應(yīng)的模式識(shí)別受體(pattern recognition receptors, PRRs)結(jié)合,是誘發(fā)膿毒癥的始動(dòng)因素。其中,構(gòu)成革蘭陰性菌(Gram-negative, G-)外膜的主要成分內(nèi)毒素(lipopolysaccharide, LPS)是最主要的病原體相關(guān)分子,LPS在體內(nèi)釋放后,具有極強(qiáng)的誘導(dǎo)膿毒癥發(fā)生的能力,其水平也與膿毒癥病情密切相關(guān)。因此,基于膿毒癥動(dòng)物模型探討LPS的釋放規(guī)律并尋找相應(yīng)的干預(yù)措施,有利于促進(jìn)膿毒癥的防治水平的提高。CLP模型是目前應(yīng)用最為廣泛的膿毒癥動(dòng)物模型。因與膿毒癥病人病程較為相似,該模型一直被認(rèn)作是膿毒癥研究動(dòng)物模型的“金標(biāo)準(zhǔn)”。如能在CLP動(dòng)物模型中明確其體內(nèi)LPS的變化規(guī)律,并在此基礎(chǔ)上檢驗(yàn)干預(yù)藥物的相應(yīng)療效,將為臨床膿毒癥的防治提供可靠的實(shí)驗(yàn)支持。 基于上述認(rèn)識(shí),首先,本課題將觀察CLP模型大鼠體內(nèi)內(nèi)毒素的動(dòng)態(tài)變化；其次,為更貼近臨床治療,在CLP模型中加入抗菌藥物以清除病原體,明確給予抗生素后大鼠體內(nèi)LPS的變化規(guī)律；然后,在抗生素治療基礎(chǔ)上加入抗內(nèi)毒素物質(zhì)苦柯胺B(Kukoamine B, KB)進(jìn)行干預(yù),監(jiān)測(cè)大鼠體內(nèi)內(nèi)毒素的變化情況,并分別與CLP模型組和抗生素治療組進(jìn)行比較,研究KB體內(nèi)對(duì)CLP模型大鼠內(nèi)毒素的拮抗作用；最后,通過(guò)熱滅活大腸埃希菌攻擊小鼠膿毒癥模型模擬內(nèi)毒素攻擊,觀察KB對(duì)膿毒癥小鼠保護(hù)作用,并在熱滅活金黃色葡萄球菌攻擊小鼠膿毒癥模型中,觀察KB對(duì)非G-菌感染引起的膿毒癥作用情況,以進(jìn)一步驗(yàn)證評(píng)價(jià)KB對(duì)膿毒癥動(dòng)物的保護(hù)作用及安全性。 方法： 1.探索不同抗生素對(duì)大腸埃希菌體外誘導(dǎo)內(nèi)毒素釋放水平的影響,了解其內(nèi)毒素釋放的時(shí)效關(guān)系,量效關(guān)系,為CLP大鼠模型選擇一種能夠清除病原體同時(shí)產(chǎn)生較多內(nèi)毒素的抗菌藥物； 2.檢測(cè)CLP模型大鼠各時(shí)相點(diǎn)內(nèi)毒素水平,根據(jù)體外實(shí)驗(yàn)結(jié)果,加入抗菌藥物干預(yù),研究加入抗菌藥物后內(nèi)毒素的變化規(guī)律,在此基礎(chǔ)上,使用KB進(jìn)行治療干預(yù),檢測(cè)動(dòng)物體內(nèi)毒素的動(dòng)態(tài)變化,觀察各組大鼠72h死亡率； 3.在致死劑量熱滅活細(xì)菌攻擊小鼠膿毒癥動(dòng)物模型中,觀察KB對(duì)膿毒癥小鼠的保護(hù)作用,同時(shí)與臨床用藥血必凈和烏司他丁進(jìn)行平行比較,以驗(yàn)證KB拮抗內(nèi)毒素膿毒癥小鼠的保護(hù)作用。 結(jié)果： 1.三種抗生素抑／殺菌效果依次為頭孢他啶亞胺培南舒氨西林,均能有效清除細(xì)菌,舒氨西林在殺滅細(xì)菌同時(shí),釋放較高水平內(nèi)毒素,且在各濃度條件下,誘導(dǎo)釋放的內(nèi)毒素水平穩(wěn)定； 2.CLP模型大鼠血液中細(xì)菌從4h開始大量繁殖,8h達(dá)到峰值,之后維持在2000CFU/ml水平,LPS水平從8h開始增加,12h-16h達(dá)到高峰,20h-72h均在較低水平；加入抗生素干預(yù)后,血液中較少檢出細(xì)菌生長(zhǎng),各時(shí)相點(diǎn)內(nèi)毒素水平高于CLP對(duì)照組；加入KB后,可明顯降低各時(shí)相點(diǎn)大鼠體內(nèi)LPS水平尤其是LPS峰值水平,并改善大鼠生存狀態(tài),提高大鼠生存率； 3.在熱滅活大腸埃希菌攻擊小鼠膿毒癥模型中,KB可提高膿毒癥小鼠生存率40%左右,在熱滅活金黃色葡萄球菌攻擊小鼠膿毒癥模型中,可提高生存率15%左右,并呈一定的量效關(guān)系。 結(jié)論： 1.舒氨西林可有效清除病原體,產(chǎn)生大量的內(nèi)毒素,在CLP大鼠模型中使用,可使CLP大鼠模型的內(nèi)毒素變化更接近臨床實(shí)際； 2.明確了CLP模型大鼠體內(nèi)LPS變化規(guī)律,加入抗生素干預(yù)后,CLP大鼠體內(nèi)LPS釋放增加,在使用抗生素基礎(chǔ)上加入KB干預(yù)后,可降低各時(shí)相點(diǎn)膿毒癥大鼠血液中內(nèi)毒素水平,說(shuō)明KB體內(nèi)對(duì)內(nèi)毒素有較強(qiáng)的拮抗作用,在控制感染的基礎(chǔ)上使用KB降低LPS水平,可提高膿毒癥動(dòng)物的生存率； 3.在致死劑量熱滅活細(xì)菌攻擊小鼠膿毒癥動(dòng)物模型中,KB可延遲死亡膿毒癥小鼠死亡時(shí)間,提高生存率,進(jìn)一步驗(yàn)證了KB可通過(guò)拮抗內(nèi)毒素發(fā)揮對(duì)膿毒癥動(dòng)物的保護(hù)作用,同時(shí)對(duì)非G-菌誘發(fā)的膿毒癥也有一定效果,有很好的開發(fā)應(yīng)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 .

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R-332

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