蒋建新
摘 要
关于感染,尤其是内源性感染的研究近年来在外科领域内备受人们关注。目前研究表明，细菌内毒素(ET)移位可能是临床上感染发生率难以降低的主要原因。为了进一步阐明内源性ET移位在失血性休克中的作用地位及其机制，本课题从以下三方面进行研究：1.探讨休克后内源性ET在组织内的分布及其在休克中的作用，包括：⑴内源性ET在肝、肺、肾组织以及循环内的分布规律；⑵组织ET水平与器官功能损害的关系；⑶ET移位与休克时间的关系；⑷拮抗ET(杀菌/通透性增强蛋白，BPI)对失血性休克转归的影响。2. 探讨休克时体内细胞因子反应及其与ET移位的关系，包括：⑴休克时肝、肺、肾、肠组织内TNFα、IL-1(、IL-6 mRNA的表达；⑵以TNFα为代表，探讨休克对组织器官内细胞因子释放的影响，及其与ET移位的关系。3. 探讨休克时低剂量内毒素能否发挥其损伤作用及其可能机制，包括：⑴休克家兔对低剂量LPS的反应；⑵休克时LBP/CD14 mRNA的表达及其意义。通过上述研究，以期进一步揭示内源性ET移位与失血性休克的内在联系。主要结果与结论如下:
1、根据血浆鲎试剂测定法，首次建立了组织匀浆内内毒素水平的定量测定方法。
2、本研究选用失血性休克动物模型，首次较系统地观察了休克后内源性ET在组织器官内的分布规律 及其与休克后相应器官功能损害和休克预后的关系。结果表明，失血性休克能引起明显的内源性ET移位,且与休克持续时间密切相关；容量复苏,虽可恢复有效循环血量,改善组织灌流,但复苏后ET移位仍持续存在,并较休克时明显；休克时进入体内的ET主要分布于肝、肺、肾等组织内,其中肝脏是休克时ET最早、最主要的聚积场所,ET在组织内能较长时间地保留其生物活性；休克后内源性ET的移位与休克后器官功能损害及其预后有着一定的内在联系,其中分布于组织中的ET可能起着关键作用。
3、本研究采用RT-PCR技术，首次较系统地观察了失血性休克后重要器官内细胞因子的基因表达。结果表明，失血性休克后，肝、肺、肾、肠器官内TNFα、IL-1?、IL-6 等细胞因子基因表达可相继增加，其中，TNFα是休克后最先表达的细胞因子。
4、本研究还较系统地探讨了失血性休克后组织及循环内TNFα水平的动态变化。结果显示，失血性休克后体内产生的细胞因子主要分布在各组织器官内,并且在组织内存在的时间要长于在循环中存在的时间,这说明与其内分泌活性相比,细胞因子的旁分泌、自分泌活性在休克后器官功能损害中更具有重要的病理意义。休克后组织器官内细胞因子的基因表达和释放增加与休克后并发的ET移位有关。
5、本研究进一步在家兔失血性休克模型上证实失血性休克具有增敏门静脉源性ET的作用。
6、本研究采用RT-PCR和细胞原位杂交技术，首次发现失血性休克及复苏可上调LBP/CD14的基因表达，并率先提出了休克增敏ET作用的可能机制。
关键词:失血性休克,细菌内毒素,RT-PCR, 细胞原位杂交，TNF, IL-1, IL-6，细胞因子 mRNA, CD14 mRNA, RT-PCR，细胞原位杂交
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 |
