内毒素休克小鼠肝脏血管特异性结合肽的体内筛选及鉴定
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摘要
脓毒症(sepsis)是由各种致病微生物或其毒素引起的全身炎症反应综合征(systemic inflammatory response syndrome,SIRS),是严重感染、重度创伤和烧伤、大手术和休克等临床危重患者的严重并发症之一,其进一步发展可导致脓毒性休克(septic shock)、急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)和多器官功能障碍综合征(multiple organ dysfunction syndrome,MODS)等。
脓毒症及其并发症是危重病医学面临的棘手问题,一直是导致重症监护病房(intensive care unit,ICU)患者高发病率和高死亡率的主要原因,其中SIRS死亡率接近26%,脓毒症休克的死亡率更可高达82%。当前,脓毒症存在着患病率高、病死率高、治疗费用高的三高现象,已经构成对人类健康的严重威胁和经济发展的巨大负担。据统计,脓毒症的患病率约为总人口的0.3%,我国每年发生的总病例数超过400万例,并以每年约1.5%的比例增长,病死率平均为40%;美国每年约有75万人发生脓毒症休克,病死率可达50%以上。
近年来,脓毒症及发病机制的研究已成为十分活跃的前沿领域之一,日益受到国内、外广大临床医生和科研人员的高度关注。目前,在脓毒症发病机制与临床意义的研究上都取得了一定进展,但脓毒症、MODS治疗的Ⅲ期临床试验并未能取得预期的效果。尽管付出了巨大的努力,但由于脓毒症发病机制复杂,临床治疗困难,近40年来,其临床疗效和预后一直没有得到实质性地改善,已经构成对人类健康的严重威胁和经济社会的巨大负担。在生命科学得到巨大发展的今天,脓毒症仍然如此猖獗,无疑引起我们的高度重视。这些事实表明脓毒症的根本发病环节及作用机制尚有待深入研究,新的干预途径更值得探索。
流行病学分析显示,在细菌感染所致的脓毒症中,有45～60%为革兰阴性菌感染。内毒素是革兰阴性菌细胞壁外膜的结构成分之一,其化学本质为脂多糖(lipopolysaccharide,LPS),是引起脓毒症的主要因素。LPS进入血液循环后,可刺激单核巨噬细胞、内皮细胞等主要的炎症细胞,诱导其结构和功能发生改变,并导致相应的合成和分泌功能发生变化,导致细胞表面一些与炎症相关的分子表达增高或暴露其相应的活性结构部位。与正常的细胞相比,受刺激的细胞表面存在着数量和/或结构上具有差异的分子,这些分子可能与内毒素休克的发生发展密切相关,可作为内毒素休克治疗新的候选靶点。这些细胞表面差异分子有可能是炎症级联反应时某些重要炎症介质的结合位点,寻找其特异性结合肽,则有可能通过竞争性抑制相应炎症介质与效应细胞的结合,进而中和或阻断这些炎症介质的生物学效应,如进一步激活靶细胞释放细胞因子、介导细胞黏附等,从而达到阻断或减轻全身性炎症反应发生的目的。这种对特定细胞具有靶向治疗作用的生物活性肽的特点是更为高效和副作用小。其关键步骤为发现LPS刺激单核巨噬细胞或内皮细胞的特异性高亲和力结合肽。
噬菌体展示技术是20世纪90年代发展起来并得到广泛应用的新技术,其原理是将外源多肽或蛋白与噬菌体的一种衣壳蛋白融合表达,融合蛋白将展示在噬菌体的表面,而编码这个融合子的DNA则位于该噬菌体内。噬菌体展示技术的一个最基本的特征是将表现型和基因型有效联系起来,即噬菌体表面的特定表现型与噬菌体颗粒中的基因型信息相对应,如需得到某个特定的表现型,只需在噬菌体基因组中插入该表现型的相关基因即可。噬菌体展示技术使大量随机多肽与其DNA编码序列之间建立了直接联系,使各种靶分子(抗体、酶、细胞表面受体等)的多肽配体通过一种被称为淘选(panning)的体外选择程序得以快速鉴定。最简单的淘选程序,是将噬菌体展示肽库与包被有靶分子的平板(或磁珠)共温育,先洗去未结合的噬菌体,然后洗脱特异性结合的噬菌体。将被洗脱的噬菌体进行扩增,然后再进行下一轮的结合/扩增循环,以富集那些可结合序列。经3～4轮淘选后,通过DNA测序对每个可结合克隆进行定性。展示在噬菌体表面的随机肽库可应用于许多方面的研究,包括绘制抗原表位图谱、研究蛋白质-蛋白质相互作用和鉴定非肽配体的肽模拟物等。
Ph.D.-C7C噬菌体展示肽库是将随机七肽融合到M13噬菌体次要衣壳蛋白(pⅢ)上而构建成的一个组合文库。所展示的随机多肽两侧各有一个半胱氨酸(Cys)。在非还原条件下,这两个半胱氨酸自发地形成一个二硫键,使展示的多肽环化。受限于二硫键环内的7肽库已被证实能识别抗原表位结构、D-氨基酸靶分子的镜像配基及开发以多肽为基础的治疗药物等。
近年发展起来的体内噬菌体展示技术,是寻找组织、器官特异性结合多肽的有效手段。此方法可在受体分子尚不清楚的情况下,以受体天然存在的环境——组织器官为配基,利用噬菌体短肽的抗原特异性,寻找未知的靶分子,确定其结构域。本研究采用体内噬菌体展示技术,对内毒素休克小鼠肝脏血管内皮细胞进行了筛选,寻找其表面分子的特异性结合肽,以期为内毒素休克的治疗及其发生发展机制的研究提供新的思路与线索。
我们选取4只BALB/c小鼠,雄性,8周龄,以35 mg/kg的剂量腹腔注射LPS,颈动脉插管后,监测小鼠动脉血压,复制内毒素休克模型。我们对内毒素休克状态下小鼠的肝脏血管内皮细胞进行了四轮筛选,然后又用正常小鼠做了一次差减筛选,即得到了与休克小鼠肝脏血管特异性结合,而与正常小鼠肝血管不结合的噬菌体文库。我们对所筛得的噬菌体克隆进行了一个初步的体内回输实验,验证其在内毒素休克小鼠体内的靶向情况,一共验证了20个噬菌体克隆,发现有15个克隆为阳性克隆(单位重量肝脏中回输到的噬菌体是对照器官的三倍以上)。随机挑取噬菌体克隆,经过PCR扩增出其目的片段以后,进行DNA测序,并根据插入序列推导出外源七肽的氨基酸序列,一共得到正确的有插入片段的序列1,063条。
这些七肽序列中包含大量的三肽残基,其中一些三肽基序作为生化识别单元(biochemical recognition units)中的配基,往往是某些功能蛋白的模拟表位(mimotope),因此寻找这些模拟表位及其对应的功能蛋白具有重大意义。我们采用完全重排七肽的方法,应用混排(洗牌)算法(shuffling algorithm),统计大量噬菌体展示七肽中某特定三肽出现的次数(丰度)及其显著性,结果显示,1,063个阳性噬菌体展示七肽中包括有3,390个不同的三肽残基,其中丰度具有显著性意义的三肽有200个。然后,利用Clustal W软件对包含丰度具有显著性或丰度较高的三肽基序的七肽进行多序列比对(multiple sequence alignment)分析,并结合氨基酸的性质,在这些七肽中寻找特征性短肽基序(模拟表位),共得到62个特征性短肽基序;通过在线BLAST服务,将这些短肽基序同鼠蛋白质序列数据库(SWISS-PROT)进行相似性比对分析,获得所有已知的包含这些特征性短肽的鼠类蛋白;进一步应用SignalP和TMHMM预测软件筛选其中的分泌蛋白和跨膜蛋白,从中获得目的功能蛋白。
我们对出现次数最多的噬菌体展示七肽(LTTWAPA)进行了体内导向效果鉴定和免疫组化染色分析。体内导向效果鉴定实验显示,呈现LTTWAPA的噬菌体在休克小鼠单位重量肝脏中的回收量是8.0×10~8/g,分别是肾脏的40倍(2.0×10~7/g),脑组织的80倍(1.0×10~7/g)。而在正常小鼠的验证中,目标噬菌体在单位重量肝脏中的回收量与脑组织和肾脏没有明显差异,均远低于在休克小鼠肝脏中的回收量。对照组的空噬菌体在休克小鼠各脏器的的回收量是基本保持一致的。初步认为,我们筛选到的该噬菌体克隆在内毒素休克小鼠肝脏有很好的导向效果。
免疫组化染色显示,表达LTTWAPA序列的噬菌体定位于内毒素休克小鼠肝脏的血管内皮细胞,而在休克小鼠的脑组织和肾脏以及正常小鼠的肝、肾、脑均未见明显染色。同时,我们设立了同等量的空噬菌体作为对照,对照组的空噬菌体在休克小鼠和正常小鼠的肝脏均未见染色。说明我们筛选到的该噬菌体能够有效地靶向于内毒素休克小鼠的肝脏血管,而不结合于休克小鼠的其它脏器,也不结合于正常小鼠的各脏器。
总之,本研究在靶分子未知的情况下,采用体内噬菌体展示技术,对内毒素休克小鼠肝脏血管内皮细胞进行4轮筛选,并与正常小鼠做了一次差减筛选,获得与内毒素休克小鼠肝脏血管内皮细胞特异性结合的短肽序列,应用生物信息学技术对这些短肽序列进行分析和预测,并进一步鉴定预测得到的功能蛋白和表位模拟肽的生物学活性。通过以上研究,我们得出以下几点结论:
1.成功复制了内毒素休克小鼠模型;
2.运用体内噬菌体展示技术,筛选到了内毒素休克小鼠肝脏血管特异性结合肽库。并且经过四轮筛选,噬菌体文库在内毒素休克小鼠肝脏得到了有效富集;
3.随机挑取20个噬菌体克隆进行了体内回输实验验证,有15个克隆为阳性噬菌体克隆;
4.通过挑斑和PCR扩增,得到了1,063条七肽序列,经过生物信息学分析,发现1,063条展示七肽中共包括有3,390个不同的三肽残基,其中具有显著性意义的三肽有200个;
5.应用Clustal W软件并结合氨基酸性质,分别对包含具有显著性意义三肽基序的所有七肽进行多序列比对分析,获得了62个具有表位模拟肽性质和功能的特征性短肽;
6.应用BLASTP程序与鼠蛋白质数据库进行同源性比对分析得到包含以上特征性短肽基序的蛋白2,922个,进一步利用SignalP和TMHMM预测软件获得分泌蛋白和跨膜蛋白,其中分泌蛋白98条,跨膜蛋白226条,既为分泌蛋白又为跨膜蛋白的序列20条;
7.对出现次数最多的噬菌体克隆(LTTWAPA)进行了体内回输实验验证,体内回输实验初步证实该噬菌体克隆能有效靶向于内毒素休克小鼠肝脏;
8.免疫组化实验进一步证实展示LTTWAPA七肽的噬菌体特异性结合于内毒素休克小鼠肝脏血管,并且定位于内皮细胞表面。
本研究应用分子生物学技术获得原始数据,运用生物信息学研究方法分析生物数据,预测出与疾病发生发展相关的特征性短肽,再进行生物实验验证,是一条高效的研究途经。
Sepsis is a systemic inflammatory response syndrome arising from infection caused by pathogenic microorganisms(usually bacteria) or their toxin invading the body.It is a kind of the critical complications related to serious infection,badly wound and bum,big surgery,shock and so on.Sepsis is part of a spectrum of conditions ranging from the acute respiratory distress syndrome(ARDS),systemic inflammatory response syndrome(SIRS) to septic shock and multiple organ dysfunction syndrome(MODS).
Sepsis and its complications continue to be the main causes leading to high morbidity and mortality in the intensive care unit(ICU).The mortality associated with these conditions ranges from around 26%in patients with SIRS to around 82% in patients with septic shock.Currently,septis is considered as high morbidity,high mortality and high treat expense,which has been a great burden to economic development and threaten to human health.As estimated,the morbidity of sepsis is about 0.3%.The total number of sepsis cases exceeds 4 million increasing by 1.5% each year.The mean mortality is near 40%.In the U.S.A.,about 750,000 people die of septic shock per year and the morbidity is over 50%.
Research about sepsis and its mechanism have been a popular area these years, which draws attention of clinical doctors and scientific researchers.Recently,some successes have been achieved in the study of mechanism and clinical signification, but the clinical therapy is still dissatisfactory.Although people have taken great efforts,the clinical therapy remains an area without significant improvement because of its complicated mechanism.In this modern society,many diseases have been cured effectively,while sepsis is still rampant.All the facts suggest that the basic mechanism of sepsis remains an area that needs intense research and the new method of therapy is worthy of exploring.
As is revealed by the epidemiology,about 45%to 60%sepsis is caused by Gram-negative bacteria.Lippolysaccharide(LPS) or endtoxin,is the major component of Gram-negative bacteria's cellwall.LPS is a potent activator of the inflammatory cells such as monocytes and macrophages,which induces those cells to change their constructions and functions,accompanying with the change of producing and secreting.Those performances contribute to the systemic changes observed in gram-negative sepsis or septic shock.Compared with natural cells,LPS stimulated monocytes or endothelial cells expose their active sites of cell surface molecules more densely,which may be closely related to the development of sepsis and can act as potential therapeutic targets.As these changed molecules may play as binding sites on the cell surface for some critical cytokines in inflammation cascade reaction, specific binding peptides of these molecules which can competitively inhibit the cytokines binding to cells,may have potential abilities to block biological effects of the cytokines,such as the activation of the target cell to release more cytokine, accommodating the adhesion and so on.The biologically active peptides that target to specific cells show properties of high performance with low side effects.The key step is to find peptides that specifically bind to monocytes/eddothelial cell stimulated by LPS.
Phage-display technology is an effective molecular biologic tool that has been developed and widely used since the 1990s.It describes a selection technique in which a peptide or protein turns into fusion protein with a coat protein of a bacteriophage. The fused protein will be displayed on the surface of the bacteriophage,while the DNA encoding it resides within the virion.One of the most significant feature of phage-display technology is that it links phenotype with genotype successfully. According to this,the expression of a particular phenotype on phage is directly associated with the genetic information contained within the phage particle.The expression of the desired phenotype can be achieved by incorporating the relevant genetic material into the phage genome.Phage display has been used to create a directly physical linkage between a vast library of random peptide sequences to the DNA encoding each sequence,allowing rapid identification of peptide ligands for a variety of target molecules(antibodies,enzymes,cell-surface receptors,etc.) by an in vitro selection process,which is called panning.The simplest panning form is as follows:first,we incubate a library of phage-displayed peptides with plates(or beads) coated with targets,then wash away the unbound phage,and elute the specifically-bound phage.The eluted phage will be amplified before taken through additional binding/amplification cycles to enrich the binding sequences.After 3 to 4 rounds,individual clones will be characterized by DNA sequencing.Random peptide libraries displayed on phage can be used in a number of applications,including epitope mapping,mapping protein-protein contacts,and identification of peptide mimics of non-peptide ligands.
The Ph.D.-C7C Phage Display Peptide Library is based on a combinatorial library of random peptide 7-mers fused to a minor coat protein(pⅢ) of M13 phage. The randomized sequence is flanked by a pair of cysteine residues.Under nonreducing conditions the cysteines will form a disulfide cross-link spontaneously, and result in the cyclization of displayed peptides contrasted to the linear peptides. Disulfide-constrained peptide libraries have been proven to be useful in identification of structural epitopes,mirror-image ligands for D-amino acid targets,and development of peptide-based therapeutics.
The in vivo phage display technique,which is developed during recent years,is an effective method to fred specific peptide binding to organs or tissues.This method can help us to find the target and confirm the domain in the natural environment—tissues or organs,by using the different character of antigen of the phage.In order to provide a new method and clue for the therapy and mechanism of septic shock,we will screen the specific peptides binding to endothelial cell of liver vasculture of septic shock mouse using the in vivo phage display technique.
We injected LPS into four BALB/c mouse from abdomen with dosage of 35mg/kg.All of the mice are male and eight weeks old.After arteria carotis intubatton,,we monitord the blood pressure of the mouse.Septic shock mouse model was established by this way.Then we took four rounds of screening to endothelial cell of liver vasculture of septic shock mouse.The peptide library was injected into normal mouse,in order to subtract the peptides that bind to endothelial cells of liver vasculture of normal mouse.The phage clones were gathered and validated by vivo experiment.15 of 20 phage clones were checked to be positive clones.(The phage recovered from liver is three times higher than control organs).We selected the phage clones at random and amplified the objective segment by PCR method.All the segments were sequenced and transformed into amino acid sequence.In total,we have obtained 1063 right objective segments.
Some of the tripeptide fragments included in these heptapeptide sequences were mimotope of functional proteins,and may act as ligands of biochemical recognition units in protein-protein interactions.In this study,shuffling algorithm and completely rearranged peptides were used to calculate the abundance and significance of the tripeptides.After calculation and statistics analysis,we got 3,390 distinct tripeptides and for 200 of them,the appearance of frequency is significant.Then multiple sequence alignment of tripeptides with significance was conducted with Clustal W program,from which we got 62 mimetic peptides considering the properties of amino acid.The mimetic peptides were aligned to mice protein sequence library(SWISS-PROT) through online BLASTP service and mice proteins information containing these mimetic peptides were obtained.Signal peptide predicting program SignalP 3.0 and transmembrane helices predicting program TMHMM 2.0 were used to identify secreted proteins and transmembrane proteins respectively.With the bioinformatics analysis mentioned above,we could acquire objective protein.
We validated the phage(LTTWAPA) whose frequency is the highest in vivo experiment and immunohistochemical staining.The LTTWAPA phage recovered from liver was 8.0×10~8/g,which was 40 times higher than the amount recovered from kidney and 80 times higher than brain.While in normal mice,the objective phage recovered from liver was as many as recovered from kidney and brain,all of which were highly lower than the amount recovered from setic shock liver.The control phage recovered from liver,kidney,and brain were nearly the same among septic shock mice.So we drew an elementary conclution:the LTTWAPA phage which we got had a good effect on targeting to liver of septic shock mice.
As shown in immunohistochemical staining,the LTTWAPA phage bond to the vasculture endothelial cell of septic shock mice liver,while there was no apparent staining in kidney and brain.As for normal mice,there was no positive staining in all the organs,including liver,kidney and brain.Meanwhile,we set the same amount of M13 phage without inserted sequence as control.The control phage was stained in neither septic shock nor normal mice liver.These data proved that the LTTWAPA phage can effectively target to vasculture of septic shock mousee liver,while not to other organs.
On the whole,we have screened vasculture endothelial cell of septic mice livers for four rounds by in vivo phage display technique without knowing the target.Then we subtracted the peptide library in normal mice and got some phage-displayed peptides that specifically bind to vasculture endothelial cells of septic mice livers. Functional proteins and their mimetic peptides were acquired by bioinformatics analysis and prediction and identified to be biologic activity.Taken together,we drew the following conclusions:
1.We successfully established septic shock mice model.
2.We screened the peptide library specifically binding to vasculture endothelial cell of septic mice liver.After four rounds of screening,the phage libraries successfully gathered in septic shock mice liver.
3.We scraped the phage clones at random and found 15 of 20 clones were positive clones in the vivo experiment.
4.We obtained 1,063 peptide sequence by PCR method.We got 3,390 distinct tripeptides,among 200 of which the appearance of frequency is significant through bioinformatics analysis.
5.Multiple sequence alignment of tripeptides with significance were conducted with Clustal W program,considering the properties of amino acid,we got 62 mimetic peptides.
6.The mimetic peptides were aligned to mice protein sequence library(SWISS-PROT) by BLASTP program and 2,922 mice proteins containing these mimetic peptides were obtained.Signal peptide predicting program SignalP 3.0 and transmembrane helices predicting program TMHMM 2.0 were used to identify secreted proteins and transmembrane proteins respectively.After calculation,98 of them were secreted protein and 226 of them were transmembrane protein.
7.We validated the LTTWAPA phage whose frequency is the highest in the vivo experiment and found this phage clone can target to septic shock mice liver effectively.
8.Immunohistochemical staining result also proved that the LTTWAPA phage can specifically bind to the vasculture endothelial cell of septic shock mice liver.
In summary,we have successfully established a high-performance method to screen peptides that bind specifically to the membrane of endothelial cells.The method includes raw data acquiring through biological experiment,computational analysis of biological data and biology experiment identification of results predicted by bioinformatics.
脓毒症及其并发症是危重病医学面临的棘手问题,一直是导致重症监护病房(intensive care unit,ICU)患者高发病率和高死亡率的主要原因,其中SIRS死亡率接近26%,脓毒症休克的死亡率更可高达82%。当前,脓毒症存在着患病率高、病死率高、治疗费用高的三高现象,已经构成对人类健康的严重威胁和经济发展的巨大负担。据统计,脓毒症的患病率约为总人口的0.3%,我国每年发生的总病例数超过400万例,并以每年约1.5%的比例增长,病死率平均为40%;美国每年约有75万人发生脓毒症休克,病死率可达50%以上。
近年来,脓毒症及发病机制的研究已成为十分活跃的前沿领域之一,日益受到国内、外广大临床医生和科研人员的高度关注。目前,在脓毒症发病机制与临床意义的研究上都取得了一定进展,但脓毒症、MODS治疗的Ⅲ期临床试验并未能取得预期的效果。尽管付出了巨大的努力,但由于脓毒症发病机制复杂,临床治疗困难,近40年来,其临床疗效和预后一直没有得到实质性地改善,已经构成对人类健康的严重威胁和经济社会的巨大负担。在生命科学得到巨大发展的今天,脓毒症仍然如此猖獗,无疑引起我们的高度重视。这些事实表明脓毒症的根本发病环节及作用机制尚有待深入研究,新的干预途径更值得探索。
流行病学分析显示,在细菌感染所致的脓毒症中,有45～60%为革兰阴性菌感染。内毒素是革兰阴性菌细胞壁外膜的结构成分之一,其化学本质为脂多糖(lipopolysaccharide,LPS),是引起脓毒症的主要因素。LPS进入血液循环后,可刺激单核巨噬细胞、内皮细胞等主要的炎症细胞,诱导其结构和功能发生改变,并导致相应的合成和分泌功能发生变化,导致细胞表面一些与炎症相关的分子表达增高或暴露其相应的活性结构部位。与正常的细胞相比,受刺激的细胞表面存在着数量和/或结构上具有差异的分子,这些分子可能与内毒素休克的发生发展密切相关,可作为内毒素休克治疗新的候选靶点。这些细胞表面差异分子有可能是炎症级联反应时某些重要炎症介质的结合位点,寻找其特异性结合肽,则有可能通过竞争性抑制相应炎症介质与效应细胞的结合,进而中和或阻断这些炎症介质的生物学效应,如进一步激活靶细胞释放细胞因子、介导细胞黏附等,从而达到阻断或减轻全身性炎症反应发生的目的。这种对特定细胞具有靶向治疗作用的生物活性肽的特点是更为高效和副作用小。其关键步骤为发现LPS刺激单核巨噬细胞或内皮细胞的特异性高亲和力结合肽。
噬菌体展示技术是20世纪90年代发展起来并得到广泛应用的新技术,其原理是将外源多肽或蛋白与噬菌体的一种衣壳蛋白融合表达,融合蛋白将展示在噬菌体的表面,而编码这个融合子的DNA则位于该噬菌体内。噬菌体展示技术的一个最基本的特征是将表现型和基因型有效联系起来,即噬菌体表面的特定表现型与噬菌体颗粒中的基因型信息相对应,如需得到某个特定的表现型,只需在噬菌体基因组中插入该表现型的相关基因即可。噬菌体展示技术使大量随机多肽与其DNA编码序列之间建立了直接联系,使各种靶分子(抗体、酶、细胞表面受体等)的多肽配体通过一种被称为淘选(panning)的体外选择程序得以快速鉴定。最简单的淘选程序,是将噬菌体展示肽库与包被有靶分子的平板(或磁珠)共温育,先洗去未结合的噬菌体,然后洗脱特异性结合的噬菌体。将被洗脱的噬菌体进行扩增,然后再进行下一轮的结合/扩增循环,以富集那些可结合序列。经3～4轮淘选后,通过DNA测序对每个可结合克隆进行定性。展示在噬菌体表面的随机肽库可应用于许多方面的研究,包括绘制抗原表位图谱、研究蛋白质-蛋白质相互作用和鉴定非肽配体的肽模拟物等。
Ph.D.-C7C噬菌体展示肽库是将随机七肽融合到M13噬菌体次要衣壳蛋白(pⅢ)上而构建成的一个组合文库。所展示的随机多肽两侧各有一个半胱氨酸(Cys)。在非还原条件下,这两个半胱氨酸自发地形成一个二硫键,使展示的多肽环化。受限于二硫键环内的7肽库已被证实能识别抗原表位结构、D-氨基酸靶分子的镜像配基及开发以多肽为基础的治疗药物等。
近年发展起来的体内噬菌体展示技术,是寻找组织、器官特异性结合多肽的有效手段。此方法可在受体分子尚不清楚的情况下,以受体天然存在的环境——组织器官为配基,利用噬菌体短肽的抗原特异性,寻找未知的靶分子,确定其结构域。本研究采用体内噬菌体展示技术,对内毒素休克小鼠肝脏血管内皮细胞进行了筛选,寻找其表面分子的特异性结合肽,以期为内毒素休克的治疗及其发生发展机制的研究提供新的思路与线索。
我们选取4只BALB/c小鼠,雄性,8周龄,以35 mg/kg的剂量腹腔注射LPS,颈动脉插管后,监测小鼠动脉血压,复制内毒素休克模型。我们对内毒素休克状态下小鼠的肝脏血管内皮细胞进行了四轮筛选,然后又用正常小鼠做了一次差减筛选,即得到了与休克小鼠肝脏血管特异性结合,而与正常小鼠肝血管不结合的噬菌体文库。我们对所筛得的噬菌体克隆进行了一个初步的体内回输实验,验证其在内毒素休克小鼠体内的靶向情况,一共验证了20个噬菌体克隆,发现有15个克隆为阳性克隆(单位重量肝脏中回输到的噬菌体是对照器官的三倍以上)。随机挑取噬菌体克隆,经过PCR扩增出其目的片段以后,进行DNA测序,并根据插入序列推导出外源七肽的氨基酸序列,一共得到正确的有插入片段的序列1,063条。
这些七肽序列中包含大量的三肽残基,其中一些三肽基序作为生化识别单元(biochemical recognition units)中的配基,往往是某些功能蛋白的模拟表位(mimotope),因此寻找这些模拟表位及其对应的功能蛋白具有重大意义。我们采用完全重排七肽的方法,应用混排(洗牌)算法(shuffling algorithm),统计大量噬菌体展示七肽中某特定三肽出现的次数(丰度)及其显著性,结果显示,1,063个阳性噬菌体展示七肽中包括有3,390个不同的三肽残基,其中丰度具有显著性意义的三肽有200个。然后,利用Clustal W软件对包含丰度具有显著性或丰度较高的三肽基序的七肽进行多序列比对(multiple sequence alignment)分析,并结合氨基酸的性质,在这些七肽中寻找特征性短肽基序(模拟表位),共得到62个特征性短肽基序;通过在线BLAST服务,将这些短肽基序同鼠蛋白质序列数据库(SWISS-PROT)进行相似性比对分析,获得所有已知的包含这些特征性短肽的鼠类蛋白;进一步应用SignalP和TMHMM预测软件筛选其中的分泌蛋白和跨膜蛋白,从中获得目的功能蛋白。
我们对出现次数最多的噬菌体展示七肽(LTTWAPA)进行了体内导向效果鉴定和免疫组化染色分析。体内导向效果鉴定实验显示,呈现LTTWAPA的噬菌体在休克小鼠单位重量肝脏中的回收量是8.0×10~8/g,分别是肾脏的40倍(2.0×10~7/g),脑组织的80倍(1.0×10~7/g)。而在正常小鼠的验证中,目标噬菌体在单位重量肝脏中的回收量与脑组织和肾脏没有明显差异,均远低于在休克小鼠肝脏中的回收量。对照组的空噬菌体在休克小鼠各脏器的的回收量是基本保持一致的。初步认为,我们筛选到的该噬菌体克隆在内毒素休克小鼠肝脏有很好的导向效果。
免疫组化染色显示,表达LTTWAPA序列的噬菌体定位于内毒素休克小鼠肝脏的血管内皮细胞,而在休克小鼠的脑组织和肾脏以及正常小鼠的肝、肾、脑均未见明显染色。同时,我们设立了同等量的空噬菌体作为对照,对照组的空噬菌体在休克小鼠和正常小鼠的肝脏均未见染色。说明我们筛选到的该噬菌体能够有效地靶向于内毒素休克小鼠的肝脏血管,而不结合于休克小鼠的其它脏器,也不结合于正常小鼠的各脏器。
总之,本研究在靶分子未知的情况下,采用体内噬菌体展示技术,对内毒素休克小鼠肝脏血管内皮细胞进行4轮筛选,并与正常小鼠做了一次差减筛选,获得与内毒素休克小鼠肝脏血管内皮细胞特异性结合的短肽序列,应用生物信息学技术对这些短肽序列进行分析和预测,并进一步鉴定预测得到的功能蛋白和表位模拟肽的生物学活性。通过以上研究,我们得出以下几点结论:
1.成功复制了内毒素休克小鼠模型;
2.运用体内噬菌体展示技术,筛选到了内毒素休克小鼠肝脏血管特异性结合肽库。并且经过四轮筛选,噬菌体文库在内毒素休克小鼠肝脏得到了有效富集;
3.随机挑取20个噬菌体克隆进行了体内回输实验验证,有15个克隆为阳性噬菌体克隆;
4.通过挑斑和PCR扩增,得到了1,063条七肽序列,经过生物信息学分析,发现1,063条展示七肽中共包括有3,390个不同的三肽残基,其中具有显著性意义的三肽有200个;
5.应用Clustal W软件并结合氨基酸性质,分别对包含具有显著性意义三肽基序的所有七肽进行多序列比对分析,获得了62个具有表位模拟肽性质和功能的特征性短肽;
6.应用BLASTP程序与鼠蛋白质数据库进行同源性比对分析得到包含以上特征性短肽基序的蛋白2,922个,进一步利用SignalP和TMHMM预测软件获得分泌蛋白和跨膜蛋白,其中分泌蛋白98条,跨膜蛋白226条,既为分泌蛋白又为跨膜蛋白的序列20条;
7.对出现次数最多的噬菌体克隆(LTTWAPA)进行了体内回输实验验证,体内回输实验初步证实该噬菌体克隆能有效靶向于内毒素休克小鼠肝脏;
8.免疫组化实验进一步证实展示LTTWAPA七肽的噬菌体特异性结合于内毒素休克小鼠肝脏血管,并且定位于内皮细胞表面。
本研究应用分子生物学技术获得原始数据,运用生物信息学研究方法分析生物数据,预测出与疾病发生发展相关的特征性短肽,再进行生物实验验证,是一条高效的研究途经。
Sepsis is a systemic inflammatory response syndrome arising from infection caused by pathogenic microorganisms(usually bacteria) or their toxin invading the body.It is a kind of the critical complications related to serious infection,badly wound and bum,big surgery,shock and so on.Sepsis is part of a spectrum of conditions ranging from the acute respiratory distress syndrome(ARDS),systemic inflammatory response syndrome(SIRS) to septic shock and multiple organ dysfunction syndrome(MODS).
Sepsis and its complications continue to be the main causes leading to high morbidity and mortality in the intensive care unit(ICU).The mortality associated with these conditions ranges from around 26%in patients with SIRS to around 82% in patients with septic shock.Currently,septis is considered as high morbidity,high mortality and high treat expense,which has been a great burden to economic development and threaten to human health.As estimated,the morbidity of sepsis is about 0.3%.The total number of sepsis cases exceeds 4 million increasing by 1.5% each year.The mean mortality is near 40%.In the U.S.A.,about 750,000 people die of septic shock per year and the morbidity is over 50%.
Research about sepsis and its mechanism have been a popular area these years, which draws attention of clinical doctors and scientific researchers.Recently,some successes have been achieved in the study of mechanism and clinical signification, but the clinical therapy is still dissatisfactory.Although people have taken great efforts,the clinical therapy remains an area without significant improvement because of its complicated mechanism.In this modern society,many diseases have been cured effectively,while sepsis is still rampant.All the facts suggest that the basic mechanism of sepsis remains an area that needs intense research and the new method of therapy is worthy of exploring.
As is revealed by the epidemiology,about 45%to 60%sepsis is caused by Gram-negative bacteria.Lippolysaccharide(LPS) or endtoxin,is the major component of Gram-negative bacteria's cellwall.LPS is a potent activator of the inflammatory cells such as monocytes and macrophages,which induces those cells to change their constructions and functions,accompanying with the change of producing and secreting.Those performances contribute to the systemic changes observed in gram-negative sepsis or septic shock.Compared with natural cells,LPS stimulated monocytes or endothelial cells expose their active sites of cell surface molecules more densely,which may be closely related to the development of sepsis and can act as potential therapeutic targets.As these changed molecules may play as binding sites on the cell surface for some critical cytokines in inflammation cascade reaction, specific binding peptides of these molecules which can competitively inhibit the cytokines binding to cells,may have potential abilities to block biological effects of the cytokines,such as the activation of the target cell to release more cytokine, accommodating the adhesion and so on.The biologically active peptides that target to specific cells show properties of high performance with low side effects.The key step is to find peptides that specifically bind to monocytes/eddothelial cell stimulated by LPS.
Phage-display technology is an effective molecular biologic tool that has been developed and widely used since the 1990s.It describes a selection technique in which a peptide or protein turns into fusion protein with a coat protein of a bacteriophage. The fused protein will be displayed on the surface of the bacteriophage,while the DNA encoding it resides within the virion.One of the most significant feature of phage-display technology is that it links phenotype with genotype successfully. According to this,the expression of a particular phenotype on phage is directly associated with the genetic information contained within the phage particle.The expression of the desired phenotype can be achieved by incorporating the relevant genetic material into the phage genome.Phage display has been used to create a directly physical linkage between a vast library of random peptide sequences to the DNA encoding each sequence,allowing rapid identification of peptide ligands for a variety of target molecules(antibodies,enzymes,cell-surface receptors,etc.) by an in vitro selection process,which is called panning.The simplest panning form is as follows:first,we incubate a library of phage-displayed peptides with plates(or beads) coated with targets,then wash away the unbound phage,and elute the specifically-bound phage.The eluted phage will be amplified before taken through additional binding/amplification cycles to enrich the binding sequences.After 3 to 4 rounds,individual clones will be characterized by DNA sequencing.Random peptide libraries displayed on phage can be used in a number of applications,including epitope mapping,mapping protein-protein contacts,and identification of peptide mimics of non-peptide ligands.
The Ph.D.-C7C Phage Display Peptide Library is based on a combinatorial library of random peptide 7-mers fused to a minor coat protein(pⅢ) of M13 phage. The randomized sequence is flanked by a pair of cysteine residues.Under nonreducing conditions the cysteines will form a disulfide cross-link spontaneously, and result in the cyclization of displayed peptides contrasted to the linear peptides. Disulfide-constrained peptide libraries have been proven to be useful in identification of structural epitopes,mirror-image ligands for D-amino acid targets,and development of peptide-based therapeutics.
The in vivo phage display technique,which is developed during recent years,is an effective method to fred specific peptide binding to organs or tissues.This method can help us to find the target and confirm the domain in the natural environment—tissues or organs,by using the different character of antigen of the phage.In order to provide a new method and clue for the therapy and mechanism of septic shock,we will screen the specific peptides binding to endothelial cell of liver vasculture of septic shock mouse using the in vivo phage display technique.
We injected LPS into four BALB/c mouse from abdomen with dosage of 35mg/kg.All of the mice are male and eight weeks old.After arteria carotis intubatton,,we monitord the blood pressure of the mouse.Septic shock mouse model was established by this way.Then we took four rounds of screening to endothelial cell of liver vasculture of septic shock mouse.The peptide library was injected into normal mouse,in order to subtract the peptides that bind to endothelial cells of liver vasculture of normal mouse.The phage clones were gathered and validated by vivo experiment.15 of 20 phage clones were checked to be positive clones.(The phage recovered from liver is three times higher than control organs).We selected the phage clones at random and amplified the objective segment by PCR method.All the segments were sequenced and transformed into amino acid sequence.In total,we have obtained 1063 right objective segments.
Some of the tripeptide fragments included in these heptapeptide sequences were mimotope of functional proteins,and may act as ligands of biochemical recognition units in protein-protein interactions.In this study,shuffling algorithm and completely rearranged peptides were used to calculate the abundance and significance of the tripeptides.After calculation and statistics analysis,we got 3,390 distinct tripeptides and for 200 of them,the appearance of frequency is significant.Then multiple sequence alignment of tripeptides with significance was conducted with Clustal W program,from which we got 62 mimetic peptides considering the properties of amino acid.The mimetic peptides were aligned to mice protein sequence library(SWISS-PROT) through online BLASTP service and mice proteins information containing these mimetic peptides were obtained.Signal peptide predicting program SignalP 3.0 and transmembrane helices predicting program TMHMM 2.0 were used to identify secreted proteins and transmembrane proteins respectively.With the bioinformatics analysis mentioned above,we could acquire objective protein.
We validated the phage(LTTWAPA) whose frequency is the highest in vivo experiment and immunohistochemical staining.The LTTWAPA phage recovered from liver was 8.0×10~8/g,which was 40 times higher than the amount recovered from kidney and 80 times higher than brain.While in normal mice,the objective phage recovered from liver was as many as recovered from kidney and brain,all of which were highly lower than the amount recovered from setic shock liver.The control phage recovered from liver,kidney,and brain were nearly the same among septic shock mice.So we drew an elementary conclution:the LTTWAPA phage which we got had a good effect on targeting to liver of septic shock mice.
As shown in immunohistochemical staining,the LTTWAPA phage bond to the vasculture endothelial cell of septic shock mice liver,while there was no apparent staining in kidney and brain.As for normal mice,there was no positive staining in all the organs,including liver,kidney and brain.Meanwhile,we set the same amount of M13 phage without inserted sequence as control.The control phage was stained in neither septic shock nor normal mice liver.These data proved that the LTTWAPA phage can effectively target to vasculture of septic shock mousee liver,while not to other organs.
On the whole,we have screened vasculture endothelial cell of septic mice livers for four rounds by in vivo phage display technique without knowing the target.Then we subtracted the peptide library in normal mice and got some phage-displayed peptides that specifically bind to vasculture endothelial cells of septic mice livers. Functional proteins and their mimetic peptides were acquired by bioinformatics analysis and prediction and identified to be biologic activity.Taken together,we drew the following conclusions:
1.We successfully established septic shock mice model.
2.We screened the peptide library specifically binding to vasculture endothelial cell of septic mice liver.After four rounds of screening,the phage libraries successfully gathered in septic shock mice liver.
3.We scraped the phage clones at random and found 15 of 20 clones were positive clones in the vivo experiment.
4.We obtained 1,063 peptide sequence by PCR method.We got 3,390 distinct tripeptides,among 200 of which the appearance of frequency is significant through bioinformatics analysis.
5.Multiple sequence alignment of tripeptides with significance were conducted with Clustal W program,considering the properties of amino acid,we got 62 mimetic peptides.
6.The mimetic peptides were aligned to mice protein sequence library(SWISS-PROT) by BLASTP program and 2,922 mice proteins containing these mimetic peptides were obtained.Signal peptide predicting program SignalP 3.0 and transmembrane helices predicting program TMHMM 2.0 were used to identify secreted proteins and transmembrane proteins respectively.After calculation,98 of them were secreted protein and 226 of them were transmembrane protein.
7.We validated the LTTWAPA phage whose frequency is the highest in the vivo experiment and found this phage clone can target to septic shock mice liver effectively.
8.Immunohistochemical staining result also proved that the LTTWAPA phage can specifically bind to the vasculture endothelial cell of septic shock mice liver.
In summary,we have successfully established a high-performance method to screen peptides that bind specifically to the membrane of endothelial cells.The method includes raw data acquiring through biological experiment,computational analysis of biological data and biology experiment identification of results predicted by bioinformatics.
引文
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13.Pasqualini R,Ruoslahti E.Organ targeting in vivo using phage display peptide libraries.Nature,1996,380(6572):364-366.
14.田媛,胡宝成.体内筛选技术在靶向治疗中的新进展.中国生物工程杂志,2004,24(11):117-120.
15.阚文宏,闫文生,姜勇等.P38 MAPK在内毒素休克小鼠肺组织诱导型一氧化氮合酶表达中的作用.中华老年多器官疾病杂,2002,1(1):41-47.
16.Kuzu I,Bicknell R,Fletcher CD,et al.Expression of adhesion molecules on the endothelium of normal tissue vessels and vascular tumors.Lab Invest,1993,69(12):322-328.
17.Dale JC,Elizabeth BG,Benson RE,et al.Phage display for target-based antibacterial drug discovery.Drug Discovery Today,2001,6(7):721-727.
18.Lu D,Jimenez X,Zhang H,et al.Selection of high affinity human neutralizing antibodies to VEGFR2 from a large antibody phage display library for antiangiogenesis therapy. Int J Cancer, 2002,97(3): 393-399.
19. Pinilla C, Rubio-Godoy V, Dutoit V, et al. Combinatorial peptide libraries as an alternative approach to the identification of ligands for tumor-reactive cytolytic T lymphocytes. Cancer Res, 2001, 61(13): 5153-5160.
20. Keresztessy Z, Csosz E, Harsfalvi J, et al. Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase. Protein Sci, 2006, 15(11):2466-2480.
21. Nixon AE. Phage display as a tool for protease ligand discovery. Curr Pharm Biotechnol,2002,3(1): 1-12.
22. Fredericks ZL, Forte C, Capuano IV, et al. Identification of potent human anti-IL-1RI antagonist antibodies. Protein Eng Des Sel, 2004, 17(1): 95-106.
23. Venkatesh N, Zaltsman Y, Somjen D, et al. A synthetic peptide with estrogen-like activity derived from a phage-display peptide library. Peptides, 2002,23(3): 573-580.
24. Lohse PA, Wright MC. In vitro protein display in drug discovery. Curr Opin Drug Discov Devel,2001,4(2): 198-204.
25. Arap W, Pasqualini R, Ruoslahti E, et al. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse mode. Science, 1998, 279(5349): 377-380.
26. Essler M, Ruoslahti E. Molecular specialization of breast vasculature: a breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature. Proc Natl Acad Sci USA, 2002, 99(4): 2252-2257.
27. Liu C, Bhattacharjee G, Boisvert W, et al. In vivo interrogation of the molecular display of atherosclerotic lesion surfaces. Am J Pathol, 2003, 163(5): 1859-1871.
28. Hong HY, Lee HY, Kwak WJ, et al. Phage display selection of peptides that home to atherosclerotic plaques: IL-4 receptor as a candidate target in atherosclerosis. J Cell Mol Med,2007, 12(10):180-186.
29. Arap W, Kolonin MG, Trepel M, et al. Steps toward mapping the human vasculture by phage display. Nat Med, 2002, 8(2): 121-127.
30. Ladner RC. Constrained peptides as binding entities. Trends Biotechnol, 1995, 13 (10):426-430.
31. Trepel M, Arap W, Pasqualini R. In vivo phage display and vascular heterogeneity:implications for targeted medicine. Curr Opin Chem Biol, 2002,6(3): 399-404.
32. Mizutani Y, Kihara A, Iqarashi Y, et al. Mammalian Lass6 and its related family members regulate synthesis of specific ceramides. Biochem J. 2005,390(1): 263-271.
33. Li W, Godzik A. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics, 2006, 22(13): 1658-1659.
34. Higgins D, Thompson T, Gibson JD, et al. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res, 1994,22(22): 4673-4680.
35. Altschul SF, Gish W, Miller EW, et al. Basic local alignment search tool. J Mol Biol, 1990,215(3): 403-410.
36. Henrik N, Jacob E, Sren B, et al. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering, 1997,10(1): 1-6.
37. Krogh A, Larsson B, Heijne B, et al. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mo Biol, 2001, 305(3): 567-80.
38. Samuel AL, Steven W, Sophien K, et al. An analysis of the Candida albicans genome database for soluble secreted proteins using computer-based prediction algorithms. Yeast,2003,20(7): 595-610.
39. Feng D, Doolittle R. Progressive sequence alignment as prerequisite to correct phylogenetic trees. J Mol Evol, 1987,25(4): 351-360.
40. Klein RD, Gu Q, Goddard A, et al. Selection for genes encoding secreted proteins and receptors. Proc Natl Acad Sci USA, 1996, 93(14): 7108-7113.
41. Grabley S, Thiericke R. Bioactive agents from natural sources: trends in discovery and application. Adv Biochem Eng Biotechnol, 1999, 64(1): 101-154.
42. Sonnhammer EL, Heijne YG, Krogh A, et al. A hidden markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol, 1998, 6(2):175-182.
1. Triantafilou M, Triantafilou K. Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends Immunol, 2002, 23(6): 301-304.
2. Nachman RL, Jaffe. EA. Endothelial cell culture: beginnings of modern vascular biology. J Clin Invest, 2004, 114(8): 1037-1040.
3. Shen Q. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells.Science, 2004, 304(5675): 1338-1340.
4. Kuzu I, Bicknell R, Fletcher CD, et al. Expression of adhesion molecules on the endothelium of normal tissue vessels and vascular tumors Lab Invest, 1993, 69(12): 322
5. Tedder TF , Steeber DA, Chen A, et al. The selectins: Vascular adhesion molecules. FASEB J,1995;9(2): 816-821.
6. Essani NA, Meguire GM, Manning AM, et al. Differential induction of mRNA for ICAM - 1 and selectins in hepatocytes kupper cells and endothelia cell during endotoxemia. Biochem Biophys Res Commun, 1995, 211(1): 74-82.
7. Dustin ML, Rothlein R, Bhan AK, et al. Introduction by IL-1 and interferon tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J Immunol, 1986, 137(2): 245-249.
8. Lee JH, Sorbo LD. Intercellular adhesion molecule-1 mediates cellular cross-talk between parenchymal and immune cells after lipopolysaccharide neutralization. J Immunol, 2004,172(1): 608-616.
9. Yoko S, Yoshinori N. Effects of continuous low-dose infusion lipopolysaccharide on expression of E-selectin and intercellular adhesion molecule-1 messenger RNA and neutrophil accumulation in specific organs in dogs. Am J Veterin Res, 2005, 66(7):1259-1266.
10. Duk CJ , Shimaoka M, Carman CV, et al. Immunology dime rizat ion and the effectiveness of ICAM-1 in mediati ng LFA-1 -dependent adhesion. Natl Acad Sci USA, 2001, 98(12):6830-6835.
11. Lobb RR, Antognetti G. A direct binding assay for the vascular cell adhesion molecule-1(VACM-1) interaction with alpha 4 integrins. Cell Adhe Communi, 1995, 3(5):385-397.
12. Chesnutt BC. Induction of LFA-1-dependent neutrophil rolling on ICAM-1 by engagement of E-Selectin. Microcirculation, 2006 , 13(2): 99-109.
13. Iba T, Yagi Y, Kidoko R, et al. Increased plasma levels of soluble thrombomodulin in patients with sepsis and organ failure. Surg Today, 1995,25(7): 585-590.
14. Vallet B. Bench-to-beside review: Endothelial cell dysfunction in severe sepsis: a role in organ dysfunction. Crit Care, 2003, 7(2): 130-138.
15. Tseng Y, Yuan P. Catalytic efficiency of a thrombomodulin-functionalized membrane-mimetic film in a flow model.Biomaterials, 2006,27(13): 2768-2775.
16. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelail cell. Nature, 1988; 332(6163): 411-415.
17. Anggard E, Bottine RM, Vane IR, et al. Endothelin-derived vasoconstricting factors.lancet. 1990, 30(2): 7-20.
18. Lassala P, Molet S,Ianin A,et al. ESM-1 is a novel human endothelial cell-specific molecule expressed in lung and regulated by cytokines. J Biol Chem, 1996, 271(34): 20458-20464.
19. Tsai JC, Zhang J,Voland C, et al. Cloning and characterization of thehuman lung endothelial-cell-specific-molecule promoter. J Vase Res, 2002, 39(2): 148-159.
20. Bechard D, Scherpereel A, Hammad H, et al. Human endothelial-cell specific molecule-1 binds directly to the integrin CD11a/CD18(LFA-1) and blocks binding to intercellular adhesion molecule-1. J Immunol, 2001,167(6): 3099-106.
21. Scherpereel A. Endocan, a new endothelial marker in human sepsis. Criticl Care Med, 2006,34(2): 532-537.
22. Filep JG. Endocan or endothelial cell-specific molecule-1: a novel prognostic marker of sepsis? Crit Care Med, 2006,34(2): 574-575.
23. Harlan JM, Winn RK. Leukocyte-endothelial interactionsxlinical trials of anti-adhesion therapy. Crit Care Med, 2002,30(2): 214-219.
24. Aird WC. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood, 2003,101(10): 3763-3777.
25. Alberio L, Lammle B, Esmon CT. Protein C replacement in severe meningococcemia:rationale and clinical experience. Clin Infect Dis, 2001, 32(9): 1338-1342.
26. Scholzen TE, Kotter SC, et al. a-Melanocyte stimulating hormone prevents lipopolysaccharide-Induced vasculitis by down-regulating endothelial cell adhesion molecule expression. Endocrinology, 2003,144(1): 360-370.
2.Angus DC,Linde-Zwirble WT,Lidicker J,et al.Epidemiology of severe sepsis in the Unite States:analysis of incidence,outcome,and associated costs of care.Crit Care Med,2001,29(10):1303-1310.
3.Reilly OM,Newcomb DE,Remick D,et al.Endotoxin,sepsis,and the primrose path.Shock,1999 12(6):411-419.
4.Triantafilou M,Triantafilou K.Lipopolysaccharide recognition:CD14,TLRs and the LPS-activation cluster.Trends Immunol,2002,23(6):301-304.
5.Chang-Duk J,Shimaoka M,Carman CV,et al.Immunology dimerization and the effectiveness of ICAM-1 in mediating LFA-1-dependent adhesion.Natl Acad Sci USA,2001,98(12):6830-6835.
6.Harlan JM,Winn RK.Leukocyte-endothelial interactions:clinical trials of anti-adhesion therapy.Crit Care Med,2002,30(2):214-219.
7.Smith GP,Petrenko VA.Phage display.Chem Rev,1997,97(6):391-410.
8.Smith GP.Filamentous fusion phage:novel expression vector that display cloned antigens on the viron surface.Science,1985,228(16):1315-1317.
9.武婕,陈惠鹏.噬菌体肽库研究进展.军事医学科学院院刊,2000,01(17):91-95.
10.Geysen HM,Barteling SJ,Meloen RH,et al.Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein.Proc Natl Acad Sci USA,1985,82(1):178-182.
11.Ruoslahti E,Pierschbacher MD.Arg-Gly-Asp:a versatile cell recognition signal.Cell,1986,44(4):517-518.
12.Ringe D.What makes a binding site a binding site? Curr Opin Struct Biol,1995,5(6):825-829.
13.Pasqualini R,Ruoslahti E.Organ targeting in vivo using phage display peptide libraries.Nature,1996,380(6572):364-366.
14.田媛,胡宝成.体内筛选技术在靶向治疗中的新进展.中国生物工程杂志,2004,24(11):117-120.
15.阚文宏,闫文生,姜勇等.P38 MAPK在内毒素休克小鼠肺组织诱导型一氧化氮合酶表达中的作用.中华老年多器官疾病杂,2002,1(1):41-47.
16.Kuzu I,Bicknell R,Fletcher CD,et al.Expression of adhesion molecules on the endothelium of normal tissue vessels and vascular tumors.Lab Invest,1993,69(12):322-328.
17.Dale JC,Elizabeth BG,Benson RE,et al.Phage display for target-based antibacterial drug discovery.Drug Discovery Today,2001,6(7):721-727.
18.Lu D,Jimenez X,Zhang H,et al.Selection of high affinity human neutralizing antibodies to VEGFR2 from a large antibody phage display library for antiangiogenesis therapy. Int J Cancer, 2002,97(3): 393-399.
19. Pinilla C, Rubio-Godoy V, Dutoit V, et al. Combinatorial peptide libraries as an alternative approach to the identification of ligands for tumor-reactive cytolytic T lymphocytes. Cancer Res, 2001, 61(13): 5153-5160.
20. Keresztessy Z, Csosz E, Harsfalvi J, et al. Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase. Protein Sci, 2006, 15(11):2466-2480.
21. Nixon AE. Phage display as a tool for protease ligand discovery. Curr Pharm Biotechnol,2002,3(1): 1-12.
22. Fredericks ZL, Forte C, Capuano IV, et al. Identification of potent human anti-IL-1RI antagonist antibodies. Protein Eng Des Sel, 2004, 17(1): 95-106.
23. Venkatesh N, Zaltsman Y, Somjen D, et al. A synthetic peptide with estrogen-like activity derived from a phage-display peptide library. Peptides, 2002,23(3): 573-580.
24. Lohse PA, Wright MC. In vitro protein display in drug discovery. Curr Opin Drug Discov Devel,2001,4(2): 198-204.
25. Arap W, Pasqualini R, Ruoslahti E, et al. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse mode. Science, 1998, 279(5349): 377-380.
26. Essler M, Ruoslahti E. Molecular specialization of breast vasculature: a breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature. Proc Natl Acad Sci USA, 2002, 99(4): 2252-2257.
27. Liu C, Bhattacharjee G, Boisvert W, et al. In vivo interrogation of the molecular display of atherosclerotic lesion surfaces. Am J Pathol, 2003, 163(5): 1859-1871.
28. Hong HY, Lee HY, Kwak WJ, et al. Phage display selection of peptides that home to atherosclerotic plaques: IL-4 receptor as a candidate target in atherosclerosis. J Cell Mol Med,2007, 12(10):180-186.
29. Arap W, Kolonin MG, Trepel M, et al. Steps toward mapping the human vasculture by phage display. Nat Med, 2002, 8(2): 121-127.
30. Ladner RC. Constrained peptides as binding entities. Trends Biotechnol, 1995, 13 (10):426-430.
31. Trepel M, Arap W, Pasqualini R. In vivo phage display and vascular heterogeneity:implications for targeted medicine. Curr Opin Chem Biol, 2002,6(3): 399-404.
32. Mizutani Y, Kihara A, Iqarashi Y, et al. Mammalian Lass6 and its related family members regulate synthesis of specific ceramides. Biochem J. 2005,390(1): 263-271.
33. Li W, Godzik A. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics, 2006, 22(13): 1658-1659.
34. Higgins D, Thompson T, Gibson JD, et al. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res, 1994,22(22): 4673-4680.
35. Altschul SF, Gish W, Miller EW, et al. Basic local alignment search tool. J Mol Biol, 1990,215(3): 403-410.
36. Henrik N, Jacob E, Sren B, et al. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering, 1997,10(1): 1-6.
37. Krogh A, Larsson B, Heijne B, et al. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mo Biol, 2001, 305(3): 567-80.
38. Samuel AL, Steven W, Sophien K, et al. An analysis of the Candida albicans genome database for soluble secreted proteins using computer-based prediction algorithms. Yeast,2003,20(7): 595-610.
39. Feng D, Doolittle R. Progressive sequence alignment as prerequisite to correct phylogenetic trees. J Mol Evol, 1987,25(4): 351-360.
40. Klein RD, Gu Q, Goddard A, et al. Selection for genes encoding secreted proteins and receptors. Proc Natl Acad Sci USA, 1996, 93(14): 7108-7113.
41. Grabley S, Thiericke R. Bioactive agents from natural sources: trends in discovery and application. Adv Biochem Eng Biotechnol, 1999, 64(1): 101-154.
42. Sonnhammer EL, Heijne YG, Krogh A, et al. A hidden markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol, 1998, 6(2):175-182.
1. Triantafilou M, Triantafilou K. Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends Immunol, 2002, 23(6): 301-304.
2. Nachman RL, Jaffe. EA. Endothelial cell culture: beginnings of modern vascular biology. J Clin Invest, 2004, 114(8): 1037-1040.
3. Shen Q. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells.Science, 2004, 304(5675): 1338-1340.
4. Kuzu I, Bicknell R, Fletcher CD, et al. Expression of adhesion molecules on the endothelium of normal tissue vessels and vascular tumors Lab Invest, 1993, 69(12): 322
5. Tedder TF , Steeber DA, Chen A, et al. The selectins: Vascular adhesion molecules. FASEB J,1995;9(2): 816-821.
6. Essani NA, Meguire GM, Manning AM, et al. Differential induction of mRNA for ICAM - 1 and selectins in hepatocytes kupper cells and endothelia cell during endotoxemia. Biochem Biophys Res Commun, 1995, 211(1): 74-82.
7. Dustin ML, Rothlein R, Bhan AK, et al. Introduction by IL-1 and interferon tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J Immunol, 1986, 137(2): 245-249.
8. Lee JH, Sorbo LD. Intercellular adhesion molecule-1 mediates cellular cross-talk between parenchymal and immune cells after lipopolysaccharide neutralization. J Immunol, 2004,172(1): 608-616.
9. Yoko S, Yoshinori N. Effects of continuous low-dose infusion lipopolysaccharide on expression of E-selectin and intercellular adhesion molecule-1 messenger RNA and neutrophil accumulation in specific organs in dogs. Am J Veterin Res, 2005, 66(7):1259-1266.
10. Duk CJ , Shimaoka M, Carman CV, et al. Immunology dime rizat ion and the effectiveness of ICAM-1 in mediati ng LFA-1 -dependent adhesion. Natl Acad Sci USA, 2001, 98(12):6830-6835.
11. Lobb RR, Antognetti G. A direct binding assay for the vascular cell adhesion molecule-1(VACM-1) interaction with alpha 4 integrins. Cell Adhe Communi, 1995, 3(5):385-397.
12. Chesnutt BC. Induction of LFA-1-dependent neutrophil rolling on ICAM-1 by engagement of E-Selectin. Microcirculation, 2006 , 13(2): 99-109.
13. Iba T, Yagi Y, Kidoko R, et al. Increased plasma levels of soluble thrombomodulin in patients with sepsis and organ failure. Surg Today, 1995,25(7): 585-590.
14. Vallet B. Bench-to-beside review: Endothelial cell dysfunction in severe sepsis: a role in organ dysfunction. Crit Care, 2003, 7(2): 130-138.
15. Tseng Y, Yuan P. Catalytic efficiency of a thrombomodulin-functionalized membrane-mimetic film in a flow model.Biomaterials, 2006,27(13): 2768-2775.
16. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelail cell. Nature, 1988; 332(6163): 411-415.
17. Anggard E, Bottine RM, Vane IR, et al. Endothelin-derived vasoconstricting factors.lancet. 1990, 30(2): 7-20.
18. Lassala P, Molet S,Ianin A,et al. ESM-1 is a novel human endothelial cell-specific molecule expressed in lung and regulated by cytokines. J Biol Chem, 1996, 271(34): 20458-20464.
19. Tsai JC, Zhang J,Voland C, et al. Cloning and characterization of thehuman lung endothelial-cell-specific-molecule promoter. J Vase Res, 2002, 39(2): 148-159.
20. Bechard D, Scherpereel A, Hammad H, et al. Human endothelial-cell specific molecule-1 binds directly to the integrin CD11a/CD18(LFA-1) and blocks binding to intercellular adhesion molecule-1. J Immunol, 2001,167(6): 3099-106.
21. Scherpereel A. Endocan, a new endothelial marker in human sepsis. Criticl Care Med, 2006,34(2): 532-537.
22. Filep JG. Endocan or endothelial cell-specific molecule-1: a novel prognostic marker of sepsis? Crit Care Med, 2006,34(2): 574-575.
23. Harlan JM, Winn RK. Leukocyte-endothelial interactionsxlinical trials of anti-adhesion therapy. Crit Care Med, 2002,30(2): 214-219.
24. Aird WC. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood, 2003,101(10): 3763-3777.
25. Alberio L, Lammle B, Esmon CT. Protein C replacement in severe meningococcemia:rationale and clinical experience. Clin Infect Dis, 2001, 32(9): 1338-1342.
26. Scholzen TE, Kotter SC, et al. a-Melanocyte stimulating hormone prevents lipopolysaccharide-Induced vasculitis by down-regulating endothelial cell adhesion molecule expression. Endocrinology, 2003,144(1): 360-370.