蔣建新
摘 要
關于感染,尤其是內源性感染的研究近年來在外科領域內備受人們關注。目前研究表明，細菌內毒素(ET)移位可能是臨床上感染發生率難以降低的主要原因。為了進一步闡明內源性ET移位在失血性休克中的作用地位及其機制，本課題從以下三方面進行研究：1.探討休克后內源性ET在組織內的分布及其在休克中的作用，包括：⑴內源性ET在肝、肺、腎組織以及循環內的分布規律；⑵組織ET水平與器官功能損害的關系；⑶ET移位與休克時間的關系；⑷拮抗ET(殺菌/通透性增強蛋白，BPI)對失血性休克轉歸的影響。2. 探討休克時體內細胞因子反應及其與ET移位的關系，包括：⑴休克時肝、肺、腎、腸組織內TNFα、IL-1(、IL-6 mRNA的表達；⑵以TNFα為代表，探討休克對組織器官內細胞因子釋放的影響，及其與ET移位的關系。3. 探討休克時低劑量內毒素能否發揮其損傷作用及其可能機制，包括：⑴休克家兔對低劑量LPS的反應；⑵休克時LBP/CD14 mRNA的表達及其意義。通過上述研究，以期進一步揭示內源性ET移位與失血性休克的內在聯系。主要結果與結論如下:
1、根據血漿鱟試劑測定法，首次建立了組織勻漿內內毒素水平的定量測定方法。
2、本研究選用失血性休克動物模型，首次較系統地觀察了休克后內源性ET在組織器官內的分布規律 及其與休克后相應器官功能損害和休克預后的關系。結果表明，失血性休克能引起明顯的內源性ET移位,且與休克持續時間密切相關；容量復蘇,雖可恢復有效循環血量,改善組織灌流,但復蘇后ET移位仍持續存在,并較休克時明顯；休克時進入體內的ET主要分布于肝、肺、腎等組織內,其中肝臟是休克時ET最早、最主要的聚積場所,ET在組織內能較長時間地保留其生物活性；休克后內源性ET的移位與休克后器官功能損害及其預后有著一定的內在聯系,其中分布于組織中的ET可能起著關鍵作用。
3、本研究采用RT-PCR技術，首次較系統地觀察了失血性休克后重要器官內細胞因子的基因表達。結果表明，失血性休克后，肝、肺、腎、腸器官內TNFα、IL-1?、IL-6 等細胞因子基因表達可相繼增加，其中，TNFα是休克后最先表達的細胞因子。
4、本研究還較系統地探討了失血性休克后組織及循環內TNFα水平的動態變化。結果顯示，失血性休克后體內產生的細胞因子主要分布在各組織器官內,并且在組織內存在的時間要長于在循環中存在的時間,這說明與其內分泌活性相比,細胞因子的旁分泌、自分泌活性在休克后器官功能損害中更具有重要的病理意義。休克后組織器官內細胞因子的基因表達和釋放增加與休克后并發的ET移位有關。
5、本研究進一步在家兔失血性休克模型上證實失血性休克具有增敏門靜脈源性ET的作用。
6、本研究采用RT-PCR和細胞原位雜交技術，首次發現失血性休克及復蘇可上調LBP/CD14的基因表達，并率先提出了休克增敏ET作用的可能機制。
關鍵詞:失血性休克,細菌內毒素,RT-PCR, 細胞原位雜交，TNF, IL-1, IL-6，細胞因子 mRNA, CD14 mRNA, RT-PCR，細胞原位雜交
Role of endogenous endotoxin translocation in hemorrhagic
shock and its molecular mechanisms
Abstract
Much attention has been still focused on the studies in infection, especially endogenous infection in the field of surgery research. Recently bacterial endotoxin has been shown to be a major cause for the increasing morbidity of infection in human. In order to further elucidate the role of endogenous endotoxin(ET) translocation in hemorrhagic shock and its mechanisms, the present study was performed as the following three aspects: 1. To explore the distribution of endogenous ET into organ tissues and its role in shock, including: (1) the kinetics of endogenous ET distributed into the hepatic, pulmonary and renal tissues and into the circulation, (2) correlation of ET levels in tissues with organ damage, (3) relationship of ET translocation with shock duration, and (4) effect of ET neutralization (bactericidal/permeability-increasing protein, BPI) on the outcome of hemorrhagic shock. 2. To explore cytokine response and its relationship with ET translocation following hemorrhagic shock, including: (1) expression of TNFα,IL-1β,IL-6 mRNA in the liver, lungs, kidneys, and intestine after shock, (2) release of TNFα in hepatic, pulmonary, and renal tissues and its relationship with ET translocation. 3. To explore the effect of hemorrhagic shock on low-dose endotoxin and its possible mechanism, including: (1) response of shocked rabbits to low-dose lipopolysaccharide, (2) expression of lipopolysaccharide-binding protein(LBP) and CD14 mRNA following shock and their significance. The following main results and conclusins can be obtained from the present study:
1. Assay of ET levels in tissue homogenates was firstly established according to limulus amebocyte lysate(LAL) chromogenic assay for plasma ET determination. The results showed that the modified LAL chromogenic assay can be used to accurately quantitize the content of ET in tissue homogenates.
2. The present study, for the first time, examined the kinetics of endogenous ET distributed into tissue organs and its relationship with organ damage and outcome of shock in an animal model of hemorrhagic shock. It was found that hemorrhagic shock could cause marked ET translocation, which was closely related to the duration of shock. Although resuscitation could restore the effective circulating volume and improve the tissue perfusion, ET translocation still existed after resuscitation, which, to some extent, was more severe. The translocated ET following shock mainly distributed into the liver, lung, kidney and so on, in which the liver was the organ for ET to earliest and mainliest accumulate following shock. The ET bio-activity in tissues could remain for long time after shock. There was certain intrinsic relationship between ET translocation and post-shock organ damage and outcome of shock, in which ET accumulated in tissues might play a key role.
3. The present study firstly systemically examined expression of cytokine mRNA in several vital organs following hemorrhagic shock with RT-PCR. It was found that the expression of TNFα, IL-1βand IL-6 mRNA in the liver, lung, kidney and intestine could successively increased following hemorrhagic shock, in which TNFα might be the first cytokine to be expressed after shock.
4. The present study also systemically explored the kinetics of TNFαlevels both in tissues and circulation. The results indicated that the cytokines produced after shock were mainly distributed in the tissue organs, and that the duration for TNFα present in tissues was longer than that in the circulation. It suggests that paracrine and autocrine activities of cytokines were more important for organ damage after shock when compared with their endocrine ones. Increases in mRNA expression and release of cytokines following shock might be related to ET translocation.
5. The present study further confirmed that hemorrhagic shock could sensitize the effects of portal-derived ET on a rabbit model of hemorrhagic shock.
6. The present study firstly discovered that hemorrhagic shock and resuscitation could up-regulate the expression of LBP and CD14 mRNA with RT-PCR and cell in situ hybridization, which might be an important mechanism for hemorrhagic shock to sensitize the effects of ET.
Keywords: Hemorrhagic shock, RT-PCR, Cell in situ hybridization, Bacterial endotoxin, TNF , IL-1, IL-6, Cytokine mRNA, LBP, CD14 |
