【作者】 顾颈松；

【导师】 肖光夏；

【作者基本信息】 第三军医大学 ， 外科学， 2005， 博士

【摘要】 脓毒症(sepsis)和脓毒性休克是导致危重病患者死亡的主要原因之一,据统计,全球每年因脓毒症死亡的病人高达100 万以上,病死率高达40-60%。内毒素(Lipopolysaccharide,LPS)是引起脓毒症的主要启动子。LPS 通过血浆LPS 结合蛋白(LBP)转运,与单核/巨噬细胞膜上的受体CD14 结合,形成LPS-CD14复合物后再与跨膜受体TLRs 结合,将外源信号转入胞内,导致单核/巨噬细胞活化,释放TNF-α、IL-6 等细胞因子,介导脓毒症的发生。在脓毒症患者中,LPS 的检出率、检出水平均与病情及预后密切相关。因此,封闭LPS 的活性是从源头上防治内毒素血症的关键。国内外针对内毒素血症的防治策略目前主要有抑制LPS 的合成、阻断LPS 与其受体的结合、阻断LPS 的胞内信号转导、抗细胞因子疗法、糖皮质激素治疗等措施,但均收效甚微。多粘菌素B(PMB)虽能有效中和LPS,但其严重的毒副作用又限制了其临床应用。其他抗菌抗LPS 蛋白,如杀菌性/通透性增加蛋白、鲎抗内毒素因子及其衍化多肽和一些天然抗菌抗内毒素肽的研究大多处于实验阶段,至今仍无有效低毒的药物应用于临床,因此研制高亲和力、能中和细菌LPS,同时兼有杀菌活性的药物是当今世界医学领域研究的重点。如何能从源头上封闭LPS 的生物活性、阻止和切断其所致一系列炎症性“瀑布”反应呢?据文献报道,美洲鲎和东方鲎的鲎抗内毒素因子(LALF 及TALF)在体外和动物体内具有显著的抗G-杆菌及中和LPS 活性。LALF 高亲和性地以剂量依赖方式结合LPS 并封闭其生物学活性,对脓毒症小鼠模型具有显著保护作用,能有效延长其生命,降低死亡率,抑制LPS 诱导的TNFα分泌,避免脏器损伤。进一步采用LALF 的抗菌抗LPS 活性功能区即第31-52 氨基酸残基序列的研究发现,不论是预防性或是治疗性给予LALF31-52,均可显著保护暴发性腹膜脓毒症模型小鼠,改善全身性的TNFα反应,减少器官损害,增加生存率。迄今的研究还证实,TALF 41-53 是结合LPS 的最小序列,TALF29-59具有与LPS 结合最大潜能。前人的研究成果为我们进一步的研究奠定了坚实基础,为了能便捷获得大量有效更多还原

【Abstract】 Sepsis and septic shock is the most frequent cause of death in critical ill patients with a high mortality about 40-60%. Lipopolysaccharide(LPS) usually called endotoxin, is the main initiator of sepsis. By binding with lipopolysaccharide-binding protein(LBP), a LPS-CD14 compound is formed, which together with the Toll-like receptors on the cytoplasmic membrane of mononuclear/ macrophage, thus activates the signal transduction pathway, induces cytokines such as TNF-α, IL-6 etc release, which at last results in sepsis, septic shock or even death. In patients with severe sepsis, the endotoxin level had strong correlation with prognosis, therefore neutralizing LPS would be the key step in the treatment of sepsis. The promising strategies targeting sepsis are preventing LPS from binding to its receptors, blockage of LPS intracellular signal transduction, antagonism of cytokine and application of glucocorticoids,but unfortunately all these therapies got little clinical benefits. Although polymyxin B has the effectiveness of neutralizing LPS, but the toxical and side effects forbid its wide clinical utilization. Other anti-LPS proteins such as Bactericidal/permeability increasing protein(BPI), Limulus Anti-lipopolysaccharide factor(LALF) and other peptides derived from them as well as other natural antibacterial/LPS peptides are still under experimental trials. There is no effective and safe drugs available till now. The ideal drugs with high affinity to LPS along with bactericidal activity is at the front line in contemporary medical study. How to block the LPS bioactivity at the very beginning thus to prevent the inflammatory cascade reaction from the LPS activation? The LALF and TALF from both limulus polyphemus and Tachypleus tridentatus Leach have been proved striking anti-gram negative bacillus as well as anti-LPS effects in vitro and in vivo. The LALF can bind to LPS with high affinity in a dose dependent manner and thereafter block the bioactivity of LPS. LALF demonstrated conspicuous protective effect in the sepsis mice model manifested by prolonged life span and decreased mortality. Hoess’s work verify that the 31st to 52nd amino-acid residue (LALF 31-52) is the functional domain of LALF. In a model in mice with peritoneal fulminating sepsis, LALF31-52 showed both preventive and therapeutical effects which can obviously increase the survival rate of mice under LPS attack. Above studies also show that LALF31-52 is the minimum sequence .The another study proves that TALF 41-53 is also the minimum sequence and the TALF29-59 has the maximum binding potential to LPS. Previous work has laid a solid foundation for further study but unfortunately, taking advantage of nature resources have been confined by the fact that Tachypleus tridentatus Leach being on the verge of extinction. In order to circumvent this dilemma, based on the analysis of LALF and TALF structural features as well as their functional domains by way of bioinformatics methods, we designed a series of MPLALFS ( modeling peptides from Limulus anti-lipopolysaccharide factor ) by computational molecular modeling and then subjected them to chemosynthesis as well as in vitro and in vivo bioactivity studies. Two MPLALFS, the RREMP and TYS1 with optimal anti-LPS and anti-bacterial effects were obtained. Results: 1. The molecular structure, physico-chemical property and molecular dynamic parameters of LALF and TALF and their functional domains were obtained by analyzing with online PHD and GOR in combination with Antheprot 5.0 and APD, which gave abundant information for molecular modeling .Our experience indicated that PHD method was superior to GOR method. 2. The sequences of MPLALFS were successfully got by computational simulation with appropriate allostery and modification. 3. Sixteen MPLALFS were successfully synthesized by the automatic and manual combinated approach . 4. The anti-LPS efficiency of these modeling peptides were screened and tested by limulus Amebocyte Lysate test, biosense, cytokine stimulating test, MTT test, anti-bacterial test, LPS-neutralizing test and protecting test in vitro or vivo. There were three peptides, the S4, RREMP and TYS1 with ideal anti-LPS and anti-bacterial effects were obtained from 16 MPLALFS. 5. RREMP and TYS1 could effectively inhibit the TNF-αrelease induced by LPS and had a potent action against lethal dose LPS attack in mice. Both peptides at theminimum concentration of 5μM could make a averagely 60% of mice survive in seven days after LPS attack. The survival time was increased obviously. 6. Both RREMP and TYS1 showed a potent and stable anti-LPS effects both in vitro and in vivo in comparison with that of peptides reported in literatures would make them become very promising pro-drugs. 7. After changing the salt from trifluoroacetic acid to acetate in the synthesis process, S4 and S1 showed a dramatic changes in their bioactivities. S4 lost its good anti-LPS property in vitro but S1 got a wider anti-bacterial spectrum and more powerful anti-bacterial ability. This suggested that special salts could change the peptides’bioactivity. Conclusions 1. Without maxicomputer and expensive commercial software, we had successfully completed the molecular design of 15 sequences of MPLALFs by using economic, practical and timely minicomputer with common software and net servers. Of which there were 2 MPLALFs with ideal anti-LPS and anti-bacterial effects had been obtained. The methods we adopted in the study saved us a lot of time and experiment funds. 2. That the change of salt treatment in the peptide synthesis could radically affect the bioactivity of peptide is so far the first report, which suggested that the salt change may be a key factor to the maintenance of peptides’bioactivity. 3. RREMP and TYS1 had ideal and stable anti-LPS effects both in vitro and in vivo. Both peptides could effectively inhibit the TNF-αrelease induced by LPS and showed not only a powerful efficiency against LPS lethal dose attack in mice but also the bioactivity against G-and G+ bacteria in vitro. Otherwise, another peptide S4 also had a stronger anti-LPS effect in vitro. These three peptides are all the new molecules designed by ourselves and have the full intellectual property rights. The patent protection of them has been requesting. 4. As for the second structure analyzing method of protein or peptide, the PHD based on the neural network algorithm was superior to GOR based on the information theory algorithm. In the molecular design of modeling peptide, the minimum domain conservation and precise sequence arrangement of a protein or a peptide, as well as molecular cyclization may be the key to the ideal bioactivity.更多还原