生物医学工程在脓毒症治疗中的应用

探讨生物医学工程技术在治疗革兰氏阴性菌所致脓毒症中的作用。通过分析内毒素的化学结构和内毒素在导致脓毒性休克和多器官功能衰竭（MODS）中扮演的角色，并比较不同生物技术在治疗脓毒症中的效果。显示脓毒症与革兰氏阴性菌产生的内毒素有关，脓毒症的治疗缺乏有效药物。固定化多粘菌素B（PMX－B）血液灌流可特异地吸附血液中内毒素，能有效地降低血液中内毒素和细胞因子浓度，改善血液流变学。利用固定化亲和吸附剂去除血液中内毒素对治疗脓毒症有重要价值。     

高沸醇溶性木质素及其衍生物对内毒素的吸附性能研究

内毒素是革兰氏阴性菌细胞壁的外层脂多糖组分，可引起机体发热反应，重症将导致休克，而内毒索对热或有机溶剂都十分稳定，通常的灭菌操作难以使之除去．本文采用高沸醇木质素及其衍生物作为对内毒素的吸附剂，对它们与内毒素的吸附性能进行了研究．结果表明，在室温下，高沸醇木质素酚和高沸醇木质素胺都能吸附水溶液中的内毒素，加入有机溶剂后又可以释放出吸附的内毒素，解吸后的高沸醇木质素酚和高沸醇木质素胺可以再吸附内毒素，高沸醇木质素衍生物可成为清除内毒素的新型功能高分子材料．     

TREM-1 amplifies inflammation and is a crucial mediator of septic shock

Host innate responses to bacterial infections are primarily mediated by neutrophils and monocytes/macrophages. These cells express pattern recognition receptors (PRRs) that bind conserved molecular structures shared by groups of microorganisms. Stimulation of PRR signalling pathways initiates secretion of proinflammatory mediators, which promote the elimination of infectious agents and the induction of tissue repair. Excessive inflammation owing to bacterial infections can lead to tissue damage and septic shock. Here we show that inflammatory responses to microbial products are amplified by a pathway mediated by triggering receptor expressed on myeloid cells (TREM)-1. TREM-1 is an activating receptor expressed at high levels on neutrophils and monocytes that infiltrate human tissues infected with bacteria. Furthermore, it is upregulated on peritoneal neutrophils of patients with microbial sepsis and mice with experimental lipopolysaccaride (LPS)-induced shock. Notably, blockade of TREM-1 protects mice against LPS-induced shock, as well as microbial sepsis caused by live Escherichia coli or caecal ligation and puncture. These results demonstrate a critical function of TREM-1 in acute inflammatory responses to bacteria and implicate TREM-1 as a potential therapeutic target for septic shock.     

Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia

Bacterial infection of the mammalian bloodstream can lead to overwhelming sepsis, a potentially fatal syndrome of irreversible cardiovascular collapse (shock) and critical organ failure. Cachectin, also known as tumour necrosis factor, is a macrophage-derived peptide hormone released in response to bacterial lipopolysaccharide, and it has been implicated as a principal mediator of endotoxic shock, although its function in bacterial sepsis is not known. Anaesthetized baboons were passively immunized against endogenous cachectin and subsequently infused with an LD100 dose of live Escherichia coli. Control animals (not immunized against cachectin) developed hypotension followed by lethal renal and pulmonary failure. Neutralizing monoclonal anti-cachectin antibody fragments (F(ab')2) administered to baboons only one hour before bacterial challenge protected against shock, but did not prevent critical organ failure. Complete protection against shock, vital organ dysfunction, persistent stress hormone release and death was conferred by administration of antibodies 2 h before bacterial infusion. These results indicate that cachectin is a mediator of fatal bacteraemic shock, and suggest that antibodies against cachectin offer a potential therapy of life-threatening infection.     

Protective role of phospholipid oxidation products in endotoxin-induced tissue damage

Lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria, interacts with LPS-binding protein and CD14, which present LPS to toll-like receptor 4 (refs 1, 2), which activates inflammatory gene expression through nuclear factor kappa B (NF kappa B) and mitogen-activated protein-kinase signalling. Antibacterial defence involves activation of neutrophils that generate reactive oxygen species capable of killing bacteria; therefore host lipid peroxidation occurs, initiated by enzymes such as NADPH oxidase and myeloperoxidase. Oxidized phospholipids are pro-inflammatory agonists promoting chronic inflammation in atherosclerosis; however, recent data suggest that they can inhibit expression of inflammatory adhesion molecules. Here we show that oxidized phospholipids inhibit LPS-induced but not tumour-necrosis factor-alpha-induced or interleukin-1 beta-induced NF kappa B-mediated upregulation of inflammatory genes, by blocking the interaction of LPS with LPS-binding protein and CD14. Moreover, in LPS-injected mice, oxidized phospholipids inhibited inflammation and protected mice from lethal endotoxin shock. Thus, in severe Gram-negative bacterial infection, endogenously formed oxidized phospholipids may function as a negative feedback to blunt innate immune responses. Furthermore, identified chemical structures capable of inhibiting the effects of endotoxins such as LPS could be used for the development of new drugs for treatment of sepsis.     

Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage.

Inappropriate or prolonged inflammation is the main cause of many diseases; for this reason it is important to understand the physiological mechanisms that terminate inflammation in vivo. Agonists for several Gs-protein-coupled receptors, including cell-surface adenosine purinergic receptors, can increase levels of immunosuppressive cyclic AMP in immune cells; however, it was unknown whether any of these receptors regulates inflammation in vivo. Here we show that A2a adenosine receptors have a non-redundant role in the attenuation of inflammation and tissue damage in vivo. Sub-threshold doses of an inflammatory stimulus that caused minimal tissue damage in wild-type mice were sufficient to induce extensive tissue damage, more prolonged and higher levels of pro-inflammatory cytokines, and death of male animals deficient in the A2a adenosine receptor. Similar observations were made in studies of three different models of inflammation and liver damage as well as during bacterial endotoxin-induced septic shock. We suggest that A2a adenosine receptors are a critical part of the physiological negative feedback mechanism for limitation and termination of both tissue-specific and systemic inflammatory responses.     

Enhanced bacterial clearance and sepsis resistance in caspase-12-deficient mice

Caspases function in both apoptosis and inflammatory cytokine processing and thereby have a role in resistance to sepsis. Here we describe a novel role for a caspase in dampening responses to bacterial infection. We show that in mice, gene-targeted deletion of caspase-12 renders animals resistant to peritonitis and septic shock. The resulting survival advantage was conferred by the ability of the caspase-12-deficient mice to clear bacterial infection more efficiently than wild-type littermates. Caspase-12 dampened the production of the pro-inflammatory cytokines interleukin (IL)-1beta, IL-18 (interferon (IFN)-gamma inducing factor) and IFN-gamma, but not tumour-necrosis factor-alpha and IL-6, in response to various bacterial components that stimulate Toll-like receptor and NOD pathways. The IFN-gamma pathway was crucial in mediating survival of septic caspase-12-deficient mice, because administration of neutralizing antibodies to IFN-gamma receptors ablated the survival advantage that otherwise occurred in these animals. Mechanistically, caspase-12 associated with caspase-1 and inhibited its activity. Notably, the protease function of caspase-12 was not necessary for this effect, as the catalytically inactive caspase-12 mutant Cys299Ala also inhibited caspase-1 and IL-1beta production to the same extent as wild-type caspase-12. In this regard, caspase-12 seems to be the cFLIP counterpart for regulating the inflammatory branch of the caspase cascade. In mice, caspase-12 deficiency confers resistance to sepsis and its presence exerts a dominant-negative suppressive effect on caspase-1, resulting in enhanced vulnerability to bacterial infection and septic mortality.     

Recognition and plasma clearance of endotoxin by scavenger receptors.

Lipid A is the active moiety of lipopolysaccharide (LPS, also referred to as endotoxin), a surface component of Gram-negative bacteria that stimulates macrophage activation and causes endotoxic shock. Macrophages can bind, internalize and partially degrade LPS, lipid A and its bioactive precursor, lipid IVA. We report here that lipid IVA binding and subsequent metabolism to a less active form by macrophage-like RAW 264.7 cells is mediated by the macrophage scavenger receptor. Scavenger-receptor ligands inhibit lipid IVA binding to, and metabolism by, RAW cells, and lipid IVA binds to type I and type II bovine scavenger receptors on transfected Chinese hamster ovary cells. Although in vitro competition studies with RAW cells indicate that scavenger receptor binding is not involved in LPS or lipid IVA-induced stimulation of macrophages, in vivo studies show that scavenger-receptor ligands greatly inhibit hepatic uptake of lipid IVA in mice. Thus, scavenger receptors expressed on macrophages may have an important role in the clearance and detoxification of endotoxin in animals.     

Potent ulcerogenic actions of platelet-activating factor on the stomach

Platelet-activating factor (PAF) is an endogenous phospholipid which has been implicated as a mediator of allergic and inflammatory processes. It is synthesized and released by neutrophils, platelets, macrophages, monocytes, basophils and endothelial cells, and is a potent platelet-aggregating agent, a vasodilator, increases vascular permeability, stimulates neutrophil aggregation and degranulation and induces release of lysosomal enzymes. A role for PAF in the hypotension associated with endotoxin shock and in necrotizing enterocolitis has recently been suggested. As there is an association between septic shock and acute gastric damage, we propose that PAF is an endogenous mediator of ulceration in the stomach. Indeed, as reported here, intravenous (i.v.) infusion of PAF to rats, at doses of 20-200 pmol per kg per min, resulted in the formation of extensive haemorrhagic erosions in the gastric mucosa. The ulcerogenic actions of PAF are not attributable solely to its hypotensive actions and were not mediated via effects on platelets or cyclooxygenase products, nor via histamine H1, H2 or alpha-adrenergic receptors. PAF is the most potent gastric ulcerogen yet described and its endogenous release may underlie or contribute to certain forms of gastric ulceration.     

细菌内毒素对基因免疫小鼠存活率和产仔率的影响

作为第三代疫苗,核酸疫苗具有易操作、低成本等优点,但是其内在危险性还有待进一步研究和排除,在其制备过程中可能掺入的细菌内毒素就是影响因素之一.细菌内毒素是革兰氏阴性菌细胞壁外膜结构中的脂多糖,体内释放后会引起发热、休克等多种病理变化.国外已有体外实验证明内毒素会对小鼠的生殖系统产生影响,本研究旨在通过在体实验探讨内毒素对免疫小鼠的存活率和产仔率的影响.采用常规方法(碱裂解法)、试剂盒2种方法,提取2种pCR3.1-Sry质粒(2种质粒的区别在于是否去除内毒素,其浓度和纯度均经过琼脂糖凝胶电泳和紫外分光光度法分析和控制).作为核酸疫苗对小鼠进行免疫:全程统计免疫小鼠的存活率和产仔率,同时注射灭菌ddH2O作为对照.分子生物学实验结果显示,所制备疫苗具有适宜的浓度和较高的纯度;免疫实验和数字统计结果显示,未去除内毒素实验组小鼠较去除内毒素组的存活率由93%下降到84%、产仔率由87.5%下降到35.7%,而去除内毒素组却与对照组没有明显差异.总之,核酸疫苗制备过程中掺入的细菌内毒素会对免疫小鼠造成较大不良影响,特别是生殖系统,体现在产仔率大幅降低.     

Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections

CD1d-restricted natural killer T (NKT) cells are innate-like lymphocytes that express a conserved T-cell receptor and contribute to host defence against various microbial pathogens. However, their target lipid antigens have remained elusive. Here we report evidence for microbial, antigen-specific activation of NKT cells against Gram-negative, lipopolysaccharide (LPS)-negative alpha-Proteobacteria such as Ehrlichia muris and Sphingomonas capsulata. We have identified glycosylceramides from the cell wall of Sphingomonas that serve as direct targets for mouse and human NKT cells, controlling both septic shock reaction and bacterial clearance in infected mice. In contrast, Gram-negative, LPS-positive Salmonella typhimurium activates NKT cells through the recognition of an endogenous lysosomal glycosphingolipid, iGb3, presented by LPS-activated dendritic cells. These findings identify two novel antigenic targets of NKT cells in antimicrobial defence, and show that glycosylceramides are an alternative to LPS for innate recognition of the Gram-negative, LPS-negative bacterial cell wall.     

基于神经网络和因子分解机的儿童脓毒症辅助诊断系统

脓毒症是感染引起的全身炎症反应综合征。是患者死亡的主要原因之一。由于严重脓毒症和脓毒性休克来势凶猛,病情发展迅速,给临床救治工作带来极大挑战。因此, 尽早明确脓毒症的诊断,及时治疗是改善其预后、降低病死率之关键。针对当前脓毒症临床诊断困难和对医生的临床经验有着较高要求,以及隐藏在大量电子病历中的信息没有得到充分利用的现状, 本文依据脓毒症临床数据特点提出了一种基于神经网络和因子分解机的数据挖掘算法结构,能够充分利用电子病历信息,辅助医生进行临床诊疗服务,快速准确地进行脓毒症诊断。     

外科感染性休克诊断与治疗

本文从理论到实践论述了外科感染性休克是一种常见的危及生命的综合症，阐述了休克的分类，提出早期诊断和及时有效的治疗是挽救病人生命的关键。由于感染性休克临床变化多端，治疗颇感棘手，所以掌握休克的预兆对尽早明确诊断，做好临床监护、进行积极有效地治疗，特别是控制感染，抗休克，改善微循环，预防并发症是至关重要的。凡有手术指征者，应毫不迟疑地进行手术。     

Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation

Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.     

鼠奇异变形杆菌的鉴别诊断

目的对一株小鼠奇异变形杆菌进行了鉴定,为实验动物细菌检测、鉴别诊断提供参考依据。方法通过培养特性、菌落形态、染色和系列生化试验等检查,对疑似沙门氏菌的小鼠分离菌株进行初步鉴定,并采用血清凝集试验对小鼠分离菌株作进一步鉴定。结果通过表型生物学特性鉴定,并结合血清学诊断鉴别方法,确证该小鼠分离菌株为奇异变形杆菌。结论尽管检出的奇异变形杆菌不是SPF动物所必须排除的病原菌,但仍需指出:该菌为条件致病菌,对实验动物和研究人员均有潜在的危害,对科学实验也可能造成不利影响,故应引起高度重视。     

参附注射液对内毒素休克大鼠肺组织表达PPARγ和TNFα的干预作用

摘要:目的 观察内毒素休克大鼠肺组织中过氧化物酶体增殖物激活受体γ(PPARγ)和肿瘤坏死因子α(TNFα)的表达情况及参附注射液的干预作用。方法 经腹腔注射脂多糖(LPS)建立内毒素休克SD大鼠模型,用低、中、高剂量参附注射液进行治疗,观察肺组织病L改变,检测肺组织中PPARγ和TNFα的表达量,同时检测血浆中TNFα和IL-1β的水平。结果 模型组大鼠具有典型的急性肺损伤表现;肺组织中PPARγ的转录和表达(P<0.01)量均显著下调,TNFα的表达明显增加(P<0.01);血浆中TNFα和IL-1β水平明显上升(P<0.01)。与模型组比较,参附注射液改善肺组织充血、水肿及炎性细胞浸润;呈剂量依赖性上调肺组织中PPARγ的转录和表达,下调TNFα的表达;同时抑制血浆中炎症介质TNFα和IL-1β水平(P<0.01)。结论 参附注射液保护内毒素休克大鼠肺组织,其作用机制可能与上调PPARγ从而抑制炎症介质的产生有关。     

Tumour necrosis factor alpha stimulates resorption and inhibits synthesis of proteoglycan in cartilage

During inflammatory reactions, activated leukocytes are thought to produce a variety of small proteins (cytokines) that influence the behaviour of other cells (including other leukocytes). Of these substances, which include the interleukins, interferons and tumour necrosis factors (TNFs), interleukin-1 (IL-1) has been considered potentially a most important inflammatory mediator because of its wide range of effects. In vivo it is pyrogenic and promotes the acute phase response; in vitro it activates lymphocytes and stimulates resorption of cartilage and bone. Cartilage resorption is a major feature of inflammatory diseases such as rheumatoid arthritis, and IL-1 is the only cytokine hitherto known to promote it. TNFs are characterized by their effects on tumours and cytotoxicity to transformed cells, but share some actions with IL-1. I report here that recombinant human TNF alpha stimulates resorption and inhibits synthesis of proteoglycan in explants of cartilage. Its action is similar to and additive with IL-1, and it is a second macrophage-derived cytokine whose production in rheumatoid arthritis, or inflammation generally, could contribute to tissue destruction.     

水产动物细胞培养方法及前景

综述了鱼虾贝类等水产动物细胞培养的方法,分析了动物细胞大规模培养过程中的问题与对策,展望了动物细胞培养的广阔前景.     

白介素24融合蛋白的表达、纯化和促伤口愈合功能的鉴定

本研究通过优化小鼠白介素24(mIL-24)基因序列,构建以Fc-tag为标签的mIL-24-Fc融合蛋白.对CHO进行MTX分级加压筛选,获得了稳定、高表达mIL-24-Fc蛋白的阳性克隆,同时优化了CHO细胞无血清驯化与稳定表达蛋白体系.经protein A亲和层析纯化mIL-24-Fc蛋白并利用AP binding实验检测生物学活性后,小鼠皮内注射mIL-24-Fc蛋白和伤口愈合实验证明:注射mIL-24-Fc蛋白后,小鼠表皮产生2~3层细胞增生,伤口愈合速度加快.