特异性识别肺癌细胞的小分子肽筛选及应用研究
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摘要
研究背景
肺癌目前在我国城市恶性肿瘤的发病中已居于首位,且其发病率仍呈明显的上升趋势,每年增长率高达百分之二十六点九,预计到2025年中国将成为世界第一肺癌大国。肺癌患者的预后差,平均存活时间仅为一年,一方面是因为尚未建立如前列腺癌、结肠癌等肿瘤的早期、特异性的筛选或诊断方法,患者在明确诊断后病程往往已发展至中、晚期,部分已发生转移;另一方面,缺乏理想的治疗手段,临床观察仅约20%的非小细胞肺癌患者对抗癌药物有一定疗效,且副作用大,对骨髓造血等重要脏器的功能有严重的抑制作用。建立肺癌早期、特异性的诊断和治疗方法已成为临床肿瘤学亟需解决的重点问题之一。
应用杂交瘤技术产生的识别细胞表面特异性抗原或受体的单克隆抗体已广泛用于肿瘤的临床诊断,对乳腺癌、结肠癌和前列腺癌等肿瘤的单克隆抗体已常规用于相应肿瘤的临床筛选和诊断工作;已开展了包括肺癌、乳腺癌及淋巴瘤等分子靶向治疗,并取得了较好的疗效,如抗EGFR(Gefitinib)抗体用于治疗肺癌,抗Her2/neu抗体(Herceptin)治疗乳腺癌,抗CD20单克隆抗体(Rituxan)治疗B细胞淋巴瘤等。但单克隆抗体用于肿瘤临床治疗尚存在着以下几个问题,从而影响了其临床治疗效果,(1)、单克隆抗体属分子量较大的蛋白质,难有效地穿过靶细胞达到最佳治疗目的:(2)、抗体在网状内皮系统的非特异性聚集可能导致其抗肿瘤的效价减低:(3)、抗体的鼠源性或嵌合体存在着潜在的免疫原性;(4)、制作成本较高。近年来研究发现小分子肽可弥补单克隆抗体的某些不足,其优越性表现在(1)、分子量小,比单克隆抗体更有效地穿透瘤体,如十肽分子量仅约1000 Da;(2)、小分子肽与毒素或放射性物质结合容易且稳定性较单克隆抗体高;(3)、用非天然氨基酸合成的小分子肽链能防止蛋白酶水解,增加肽的稳定性;(4)、小分子肽很少被网状内皮系统所吞噬;(5)免疫原性低:(6)小分子肽可大量合成,制作成本明显低于抗体。可见,小分子肽在肿瘤的临床诊断和治疗方面有着更为明显的优势。
目前小分子肽在肿瘤诊断和治疗方面的研究呈现出良好和广阔的应用前景,主要表现在以下三个方面:(1)利用与上皮癌细胞相结合的小分子肽俘获分离循环血中的转移癌细胞;(2)用顺磁性原子、荧光素、同位素等标记小分子肽制备分子靶向探针,如标记小分子肽RGD能显著提高MRI成像的敏感性:
(3)用放射性核素标记的小分子肽可作为肿瘤靶向放射性治疗剂,如~(111)In和~(99m)Tc标记含有RGD的小分子肽对肿瘤生长有明显的抑制作用。
目前用于小分子肽筛选的常用方法有二种:一为`一个珠子一条肽链`的组合化学肽库,二为噬菌体展示肽库法。`一个珠子一条肽链`的组合化学肽库技术是1991年由Lam教授首先提出并建立的。其原理是随机把天然和非天然氨基酸合成于树脂珠子上,根据实验需要可合成4、6、8个氨基酸等不同长度和构象(环状或线状)的肽链,每个珠子上仅存在一种肽链,一次实验即可对数万种肽链进行筛选,筛选效率很高。除外组合化学肽库技术,噬菌体展示肽库技术也是目前小分子肽筛选的主要方法,而由于前者在肽链合成过程中可随机加入非天然氨基酸,因此筛选得到的肽链不易被蛋白酶水解,结构较后者稳定,是小分子肽筛选的较理想方法。
国外学者应用组合化学肽库技术已成功筛选得到多种与细胞粘附相关的小分子肽。Pennington等(1996年)用前列腺癌细胞株(DU-H)在150万个肽链中筛选出两种序列分别为LNIVS-VNGRHX(RU-1)和DNRIRLQKXX(RX-1)与细胞粘附有关的肽链。DeRoock等(2001年)筛选得到的两个6个氨基酸短肽,kmviywkag(RZ-3)和kikmviswkg(HYD-1)能支持前列腺癌细胞粘附,并抑制癌细胞与细胞外基质蛋白粘附。Mikawa(2004年)等发现小分子肽NIeDI/V/Nle能特异性与肺泡细胞癌细胞粘附。Aina(2005年)等筛选得到的小分子cDGXGXXc能特异性粘附卵巢癌细胞。
基于国外应用组合化学肽库技术筛选小分子肽的成功报道,本研究拟利用`一个珠子一条肽链`的组合化学肽库技术,分别用非小细胞肺癌细胞A549和小细胞肺癌细胞DMS53筛选6个氨基酸的小分子肽库,以期获得与非小细胞肺癌细胞(A549)和小细胞肺癌细胞(DMS53)特异性结合的小分子肽,并将获得的肺癌特异性小分子肽合成至树脂珠子上,探讨其对提高临床肺癌胸水脱落细胞的阳性诊断率,另外,本研究还将利用生物素和荧光素标记小分子肽,评价小分子肽作为非小肺癌细胞特异性标记物的临床意义。本文共包括以下三个部分。
第一部分:特异性识别非小细胞肺癌A549细胞的小分子肽筛选
目的:应用“一个珠子一个化合物”的组合化学肽库,以期筛选得到特异性识别非小细胞肺癌细胞(A549)的小分子肽。
方法:用非小细胞肺癌细胞A549筛选“一个珠子一个化合物”的6个氨基酸的组合化学肽库,对获得的小分子肽作进一步分析(1)通过三个实验验证小分子肽与非小细胞肺癌结合的特异性(A)用A549、Calu-1等12种非小细胞肺癌、小细胞肺癌及其它瘤细胞系,观察小分子肽与A549等细胞的结合情况;(B)用相应游离小分子肽阻断细胞与小分子肽珠子结合,观察其对细胞与珠子结合的阻断率;(C)将游离小分子肽包被在玻片上,观察比较A549、Calu-1等12种细胞与玻片上小分子肽的粘附情况;(2)将小分子肽中的氨基酸分别用丙氨酸替代或删除小分子肽中每一种氨基酸,观察细胞与不同小分子肽珠子的粘附结合情况,分析肽链的长度及特定氨基酸对小分子肽的粘附活性的影响,确定小分子肽的结构稳定性;(3)应用RAGE、流式细胞仪及免疫荧光染色技术分析非小细胞肺癌细胞表面与细胞粘附关系密切的整合素的表达情况,然后利用抗体阻断实验确定A549细胞与小分子肽的粘附结合位点。实验还采用免疫荧光染色技术观察小分子肽和A549细胞的结合过程中,整合素α3与局部粘着斑激酶(focaladhesion kinase,FAK)、paxillin及vinculin三种重要的细胞粘附相关的细胞信号转导分子的关系。
结果:(1)初次筛选共得到29个与A549阳性结合的珠子,经氨基酸序列分析后发现含有-NGXG-肽链结构的序列共有10个;(2)选择cNGQGEQc作进一步的细胞特异性研究,发现cNGQGEQc与非小细胞肺癌A549、Calu-1及H178的粘附特异性明显高于其他细胞系;(3)对cNGQGEQc的结构分析显示-NGXG-及六肽长度对小分子肽与A549细胞的粘附非常重要(4) RAGE、流式细胞仪及免疫荧光染色技术显示A549,Calu-1等非小细胞肺癌细胞表面存在整合素α3的阳性表达,小细胞肺癌为阴性表达;(5)用抗整合素的抗体(α1-6,v/β1-5)阻断小分子肽与A549细胞表面的相应受体结合,结果显示α3与β亚单位的任何组合均对cNGQGEQc与A549细胞的粘附有明显的阻断作用;(5)在小分子肽和A549细胞的结合过程中整合素α3与FAK、paxillin及vinculin间存在密切的共表达关系。
结论:(1)筛选得到的小分子六肽cNGQGEQc能特异性与非小细胞肺癌细胞A549结合,其与A549细胞结合的配体是细胞表面的整合素α3;(2)整合素α3可作为非小细胞肺癌的分子标记物和靶向治疗的靶点,本研究发现的小分子肽可望为肺癌的分子诊断和靶向治疗提供一种理想的载体。
第二部分:特异性识别小细胞肺癌细胞DMS53的小分子肽筛选
目的:应用“一个珠子一个化合物”的组合化学肽库技术,以期筛选得到特异性识别小细胞肺癌细胞(DMS53)的小分子肽。
方法:用小细胞肺癌细胞DMS53筛选“一个珠子一个化合物”的6个氨基酸的组合化学肽库,对获得的小分子肽作进一步分析(1)通过三个实验验证小分子肽与小细胞肺癌结合的特异性(A)用DMS53等小细胞肺癌细胞系,观察小分子肽与DMS53等细胞的结合情况;(B)用相应游离小分子肽阻断细胞与小分子肽珠子结合,观察其对细胞与珠子结合的阻断率;(C)将游离小分子肽包被在玻片上,观察比较DMS53等细胞与玻片上小分子肽的粘附情况;(2)将小分子肽中的氨基酸分别用丙氨酸替代或删除小分子肽中每一种氨基酸,观察细胞与不同小分子肽珠子的粘附结合情况,分析肽链的长度及特定氨基酸对小分子肽的粘附活性的影响,确定小分子肽的结构稳定性;(3)应用RAGE、流式细胞仪及免疫荧光染色技术分析DMS53细胞表面与细胞粘附关系密切的整合素的表达情况,然后利用抗体阻断实验确定DMS53细胞与小分子肽的粘附结合位点。
结果:(1)初次筛选共得到32个与DMS53阳性结合的珠子,经氨基酸序列分析后发现含有cNGRXXXc或cXNGRXXc肽链结构的序列共有10个;再次合成三种有代表性的小分子肽,发现cFNGRQQc与DMS53的结合率明显高于其他小分子肽;(2)选择cFNGRQQc作进一步的细胞特异性研究,发现cFNGRQQc与DMS53的粘附特异性明显高于其他细胞系;(3)对c-FNGRQQ-c的结构分析显示-NGR-及六肽长度对小分子肽与DMS53细胞的粘附非常重要;(4) RAGE、流式细胞仪及免疫荧光染色技术发现DMS53细胞存在α5和β1表达;(5)使用不同浓度的阻断抗体包括抗整合素α和β亚单位,E-cadherin,NCAM及ICAM抗体,结果显示上述抗体均未能明显地阻断
结论:筛选得到的六肽分子cFNGRQQc是一种能特异性识别小细胞肺癌细胞DMS53并与之结合的小分子肽,但c-FNGRQQ-c与DMS53的结合位点尚不清楚。同时也提示我们c-FNGRQQ-c有别于已发现的其他小分子肽,可能不是通过细胞表面整合素受体与DMS53结合,与粘附分子E-cadherin,NCAM及ICAM也不相关。小分子肽与DMS53细胞表面的结合位点有待于进一步证实。
第三部分:肺癌细胞特异性小分子肽的应用研究
目的:利用前期研究获得的肺癌特异性小分子肽cNGQGEQc,探讨其对提高临床肺癌胸水脱落细胞的阳性诊断率及作为非小肺癌细胞的特异性标记物的临床意义。
方法:(1)将小分子肽cNGQGEQc合成于树脂珠子上,将A549细胞混合于正常人血中,检验小分子肽珠子分离A549细胞的特异性,然后对取自64例包括肺癌、间皮瘤、胃癌、结肠癌、乳腺癌、肺炎和胸膜结核等患者的胸水与小分子肽珠子共培养,分离粘附在树脂珠子上的细胞,显微镜下观察细胞形态。(2)用生物素或荧光素(FITC)标记小分子肽cNGQGEQc,免疫染色A549等细胞株及肺癌组织中的肺癌细胞,评价cNGQGEQc作为肺癌细胞分子标记物的应用价值。
结果:(1)常规细胞学方法仅发现1例阳性病例,而经小分子肽树脂珠子分离胸水中细胞,再结合常规细胞学方法检出5例阳性病例,阳性率提高了10.81%;(2) cNGQGEQc能特异性标记A549和Calu-1非小细胞肺癌细胞,而与小细胞肺癌DMS53等其它细胞无反应:全部18例肺腺癌和大部分鳞癌(7/9)细胞能与小分子肽结合,而与小细胞肺癌细胞呈阴性反应。
结论:(1)小分子肽珠子结合常规细胞学方法可提高胸水中肺癌细胞的阳性检出率;(2)小分子肽cNGQGEQc小分子肽cNOQGEQc能特异性标记非小细胞肺癌细胞,有望作为非小细胞肺癌细胞新的分子标记物。
Background
Lung cancer is the leading malignant tumor with 26.9%increasing incidence per year in our country especially in city.China will be the biggest country with lung cancer patients in the world in 2025.Lung cancer is associated with high mortality and morbidity because most cases present as middle-advanced stage with partly metastasis after diagnosis.The long-term outlook for patients with advanced lung cancer is poor with median survival and is typically less than one year.Only about 20%of non-small lung cancer(NSCLC) patients response to anticancer drugs and have to give up chemotherapy with severe side-effect such as hematopoiesis inhibition.Moreover,there has been no effective way for screening or detection of early lung cancer up to now,in contrast to what have been established for prostate cancers and colon cancer.Obviously,new strategies are needed for early detection and treatment for lung cancer.
Monoclone antibodies specific for antigen or receptor on cell surface generated by hybridoma technique,have been widely applied in the clinical diagnosis of breast cancer,colon cancer and prostate cancer.Moreover,molecular-targeting therapy has become a reality in treatment of lung cancer,breast cancer and lymphoma.For example,inhibitors of epidermal growth factor receptor,gefitinib and erlotinib,have recently been used to treat NSCLC refractory to traditional chemotherapy.Herceptin and Rituxan have been used for breast cancer and B cell leukemia.However,clinical application of monoclone antibodies in cancer treatment still remains several practical problems which affect the clinic therapeutic efficacy as follow:(1) Monoclone antibody may not permeate the target tumor mass effectively because of large molecule protein;(2) The non-specific aggregation of antibody in reticuloendothelial system may lead to a reduction of its anticancer potency;(3) Antimouse antibody or the chimera of antibody have potential immunogenicity.Recent studies have showed that small molecule peptide can retrieve a few shortage of antibody.Small molecule peptide has more advantage in the following aspects:(1) Effective to penetrate the tumor mass with less small molecule than a antibody,such as ten peptide have a molecular weight of approximately 1000 Da;(2) Easier and more constant to conjugated with toxin and radioactive agent than a antibody;(3) More stable and effective to protect amino acide from proteolysis by protein enzyme than a antibody; (4) Less to be phagocytosed by reticuloendothelial system;(5) Less likely to provoke an immunoresponse than proteins;(6) Much less cost in synthesis than antibody.We believe that small molecule peptide has more promising approach in cancer diagnosis and treatment.
Small molecule peptide has basically been used to apply in cancer diagnosis and treatment in three aspects:(1) Small molecule peptide can capture metastatic cancer cell from circulation;(2) Small molecule peptide labelled with magnetic atom or fluorescein or radiation isotope will be molecule probes,for example,small molecule peptide RGD labelled with isotope can remarkably promote the sensitivity of MRI;(3) Small molecule peptide labelled with radiation isotope will be targeting radiative therapeutic agent for cance patients,for example,peptide containing RGD sequence labelled with ~(111)In or ~(99m)Tc can dramatically inhibit tumor growth.
Now there are two general screening methods for small molecule peptides:one is "one-bead one-peptide" combinatorial chemistry peptide library,another is phage-displaying peptide library."one-bead one-peptide" combinatorial chemistry peptide library was firstly introduced by Pro.Lam in 1991.The library is synthesized using the natural and unnatural amino acide on resin beads at random.According to the experiments,libraries with one peptide on one bead can be synthesized with 4 or 6 or 8 peptides length and loop or liner conformation.The efficiency of screening is very high because millions of peptides can be screened at one time.Phage-displaying peptide library is one of the main methods for peptides screening at present as well. However,"one-bead one-peptide" combinatorial chemistry peptide library can be added into unnatural amino acide randomly during the synthesis process and therefore peptides are generally resistant to proteolysis and more stable than the latter.So "one-bead one-peptide" combinatorial library is regard as more ideal method for small molecule peptide screening.
Many scholars have successfully found some small molecule peptides bind specifically to cancer cells using "one-bead one-peptide" combinatorial libraries. Pennington et al(1996) identified two peptides,RU-1(LNIVSVNGRHX) and RX-1 (DNRIRLQAKXX) by scanning for prostate cancer cell among 1.5 million peptide beads.DeRoock et al(2001) found two peptides with six amino acid,RZ-3 (kmviywkag) and HYD-1(kikmviswkg).These two peptides support tumor cell adhesion and can inhibit cancer cell adhesion to extracellular matrix protein.Mikawa et al(2004) identified a small molecular peptide NleDI/V/NIe can specifically adhere to bronchioloalveolar carcinoma cells.Aina et al(2005) reported a novel small molecule peptide cDGXGXXc by screening a cyclic random 8-mer library can specifically adhere to ovarian cancer cells.
In this study,we intended to identify small molecule peptides specific for NSCLC A549 and SCLC DMS53 cells by screening for "one-bead one-peptide" combinatorial chemistry library.And applications of the small molecule peptide is also investigated in promoting cytopathological positive diagnosis rate of pleural fluid with lung cancer by synthesis the peptide on beads and is evaluated to be as specific molecular marker of NSCLC cells.The full text consists of the three following sections:
Section one:A novel specific small molecule peptide for non-small cell lung cancer cell A549
Aim:To screen small molecule peptide specific binding to non-small cell lung cancer cell(A549) using the "one-bead one-peptide" combinatorial library. Materials and Methods:A "one-bead one-peptide" combinatorial library with six amino acids was used to screen for specific binding peptides to non-small cell lung cancer cell(A549).Beads with cell adhesion and growth(positive beads) were isolated,stripped,and microsequenced.The novel consensus peptides will be re-synthesized and further tested as follow:(1) Cell-type specificity was analysized by three independent experiments(A) A panel of cell lines including non-small-cell lung cancer A549,Calu-1 and other cancer cells were used to study their specificity to novel peptides;(B) Free peptides were used to block the binding between cancer cells and peptide beads;(C) Adhesion to a slide coated with free peptides was tested by twelve different cancer cells including A549 and Calu-1.(2) The strategies of site-directed deletion and alanine scanning were employed to determine the structure either length or specific amino acid for cell adhesion.(3) Expression profling of integrins for cell adhesion was investigated using restriction analysis of gene expression(RAGE),flow cytometry and immunofluorescence staining technique. Antibody blocking assay was then used to confirm the binding site between A549 cells and small molecule peptides.Immunofluorescence technique was also used to observe the relationship between integrin a3 and focal adhesion kinase(FAK) and paxillin and vinculin which are all important cell adhesion associated signal transduction molecules.
Results:(1) Twenty-nine positive beads binding to A549 cell were totally obtained after primary screening.Consensus peptide sequence of-NGXG-was identified by amino acid sequencing in ten beads.(2) Peptide cNGQGEQc was re-synthesized on beads and further studied for its cell specificity.Peptide cNGQGEQc was showed to be specific for cell attachment to non-small cell lung cancer cells including A549, Calu-1 and H178,but not to other cancer cell lines.(3) Both motif of-NGXG-and the length of peptide are very important for A549 adhesion.(4) Integrin a3 was presented on non-small cell lung cancer cells A549 and Calu-1 but not on small lung cancer cell by RAGE,flow cytometry and immunofluorescence staining technique. (5) In an blocking assay with anti-integrin antibodies(α1-6,v/β1-5),cell adhesion of A549 to peptide beads was obviously inhibited by integfinα3 combining with anyβsubunits.(6) There is intensive coexpression relationship between integrinα3 and FAK and paxillin and vinculin during the binding process between small molecule peptide and A549.
Conclusion:The results suggested that(1) small molecule peptide cNGQGEQc can bind specifically to non-small cell lung cancer cell A549 via integrinα3 on cell surface;(2) Integrinα3 represents a potential molecular marker and target for molecular therapy in non-small cell lung cancer.The novel small molecule peptide obtained in this study will be used as an ideal carder for molecular diagnosis and targeting therapy of lung cancer.
Section two:A novel specific small molecule peptide for small cell lung cancer cell DMS-53
Aim:To screen small molecule peptide specific binding to small cell lung cancer cell (DMS53) using the "one-bead one-peptide" combinatorial chemistry technology. Materials and Methods:A "one-bead one-peptide" combinatorial library with six amino acids was used to screen for small molecule peptide specific binding to small cell lung cancer cell(DMS53).Beads with cell adhesion and growth(positive beads) were isolated,stripped,and microsequenced.The novel consensus peptides will be re-synthesized and further tested as follow:(1) Cell-type specificity was analysized by three independent experiments(A) A panel of cell lines including small cell lung cancer cell DMS53 and other cancer cells were used to study their specificity to novel peptides;(B) Free peptides were used to block the binding between cancer cells and peptide beads;(C) Adhesion to a slide coated with free peptides was tested by different cancer cells including small cell lung cancer cell(DMS53).(2) The strategies of site-directed deletion and alanine scanning were employed to determine the structure either length or specific amino acid for cell adhesion.(3) Expression profling of integrins for cell adhesion was investigated using restriction analysis of gene expression(RAGE),flow cytometry and immunofluorescence staining technique.Antibody blocking assay was then used to confirm the binding site between DMS53 cells and small molecule peptide.
Results:(1) Thirty two positive beads binding to DMS53 were totally obtained after primary screening.Consensus peptide sequences of cXNGRXXc and cNGRXXXc were identified by amino acid sequencing in ten beads.Three representative peptides were re-synthesized on beads.Secondary screening showed cell adhesion percentage of cFNGRQQc was higher than the other two peptides.(2) cFNGRQQc was further studied for cell specificity,alanine scanning and site-directed deletion.The results showed that cFNGRQQc is specific for promoting cell adhesion to DMS53 but not other human cancer cell lines.(3) Both motif of-NGR-and the length of peptide are important for DMS53 attachment.(4) Integrinα5 andβ1 were presented on DMS53 by RAGE,flow cytometry and immunofluorescence staining technique.(5) In an antibody blocking assay,cell adhesion of DMS53 to peptide beads was not inhibited by antibodies including integrin,E-cadherin,NCAM and ICAM.
Conclusion:The results suggested that(1) small molecule peptide cFNGRQQc can bind specifically to small cell lung cancer cell DMS53,but the binding site between the peptide and DMS53 is still unknown;(2) The binding site between the peptide cFNGRQQc and DMS53 is different from other peptide via integrin and may be not associated with adhesion molecules E-cadherin,NCAM and ICAM.The binding site on DMS53 surface for cFNGRQQc peptide need to be proven in the future.
Section three:Application of specific small molecule peptide in lung cancer
Aim:To investigate the clinic significance of the specific small molecule peptide obtained in provious study for lung cancer in promoting cytopathological positive diagnosis rate of pleural fluid with lung cancer by synthesis the peptide on beads and is evaluated to be as specific molecular marker of NSCLC cells.
Materials and Methods.(1) Small molecule peptide cNGQGEQc synthesized on resin beads was tested the the specificity in capturing A549 cells mixed with normal human blood.Small molecule peptide beads were then co-cultured with pleural fluid obtained from sixty-four cases including lung cancer,mesothelioma,gastric carcinoma,colon cancer,breast cancer,pneumonia and tuberculosis of pleura and so on.Capturing of cancer cells on the beads was accomplished using method as described above.Cells captured on beads were subsequently stripped off the beads and cell smears were prepared on slides for microscopic examination.(2) The small molecule peptide labelled with biotin or FITC was used to stain A549 cell as well as other cancer cells.The specificity of small molecule peptide binding to lung cancer cells was also tested in lung cancer tissues.
Results:(1) Only one positive case was found.by routine cytology method,while five positive cases(10.81%) were present by routine cytologic method combining with capturing cancer cell with peptide beads.(2) Non-small lung cancer cells A549 and Calu-1 but not DMS53,were specifically recognised by peptide "cNGQGEQc".All the eighteen samples of adenocarcinoma and most of squemous cancinoma(7/9) of lung were immunostained with peptide but not in small cell lung cancer.
Conclusion:(1) Small molecule peptide bead technology combining with routin cytology method can be used for isolating and enriching the scanty number of cancer cells in pleural effusion.(2) Small molecule peptide "cNGQGEQc" can specifically label the non-samll lung cancer and will be the novel helpful biological marker for NSCLC cells.
肺癌目前在我国城市恶性肿瘤的发病中已居于首位,且其发病率仍呈明显的上升趋势,每年增长率高达百分之二十六点九,预计到2025年中国将成为世界第一肺癌大国。肺癌患者的预后差,平均存活时间仅为一年,一方面是因为尚未建立如前列腺癌、结肠癌等肿瘤的早期、特异性的筛选或诊断方法,患者在明确诊断后病程往往已发展至中、晚期,部分已发生转移;另一方面,缺乏理想的治疗手段,临床观察仅约20%的非小细胞肺癌患者对抗癌药物有一定疗效,且副作用大,对骨髓造血等重要脏器的功能有严重的抑制作用。建立肺癌早期、特异性的诊断和治疗方法已成为临床肿瘤学亟需解决的重点问题之一。
应用杂交瘤技术产生的识别细胞表面特异性抗原或受体的单克隆抗体已广泛用于肿瘤的临床诊断,对乳腺癌、结肠癌和前列腺癌等肿瘤的单克隆抗体已常规用于相应肿瘤的临床筛选和诊断工作;已开展了包括肺癌、乳腺癌及淋巴瘤等分子靶向治疗,并取得了较好的疗效,如抗EGFR(Gefitinib)抗体用于治疗肺癌,抗Her2/neu抗体(Herceptin)治疗乳腺癌,抗CD20单克隆抗体(Rituxan)治疗B细胞淋巴瘤等。但单克隆抗体用于肿瘤临床治疗尚存在着以下几个问题,从而影响了其临床治疗效果,(1)、单克隆抗体属分子量较大的蛋白质,难有效地穿过靶细胞达到最佳治疗目的:(2)、抗体在网状内皮系统的非特异性聚集可能导致其抗肿瘤的效价减低:(3)、抗体的鼠源性或嵌合体存在着潜在的免疫原性;(4)、制作成本较高。近年来研究发现小分子肽可弥补单克隆抗体的某些不足,其优越性表现在(1)、分子量小,比单克隆抗体更有效地穿透瘤体,如十肽分子量仅约1000 Da;(2)、小分子肽与毒素或放射性物质结合容易且稳定性较单克隆抗体高;(3)、用非天然氨基酸合成的小分子肽链能防止蛋白酶水解,增加肽的稳定性;(4)、小分子肽很少被网状内皮系统所吞噬;(5)免疫原性低:(6)小分子肽可大量合成,制作成本明显低于抗体。可见,小分子肽在肿瘤的临床诊断和治疗方面有着更为明显的优势。
目前小分子肽在肿瘤诊断和治疗方面的研究呈现出良好和广阔的应用前景,主要表现在以下三个方面:(1)利用与上皮癌细胞相结合的小分子肽俘获分离循环血中的转移癌细胞;(2)用顺磁性原子、荧光素、同位素等标记小分子肽制备分子靶向探针,如标记小分子肽RGD能显著提高MRI成像的敏感性:
(3)用放射性核素标记的小分子肽可作为肿瘤靶向放射性治疗剂,如~(111)In和~(99m)Tc标记含有RGD的小分子肽对肿瘤生长有明显的抑制作用。
目前用于小分子肽筛选的常用方法有二种:一为`一个珠子一条肽链`的组合化学肽库,二为噬菌体展示肽库法。`一个珠子一条肽链`的组合化学肽库技术是1991年由Lam教授首先提出并建立的。其原理是随机把天然和非天然氨基酸合成于树脂珠子上,根据实验需要可合成4、6、8个氨基酸等不同长度和构象(环状或线状)的肽链,每个珠子上仅存在一种肽链,一次实验即可对数万种肽链进行筛选,筛选效率很高。除外组合化学肽库技术,噬菌体展示肽库技术也是目前小分子肽筛选的主要方法,而由于前者在肽链合成过程中可随机加入非天然氨基酸,因此筛选得到的肽链不易被蛋白酶水解,结构较后者稳定,是小分子肽筛选的较理想方法。
国外学者应用组合化学肽库技术已成功筛选得到多种与细胞粘附相关的小分子肽。Pennington等(1996年)用前列腺癌细胞株(DU-H)在150万个肽链中筛选出两种序列分别为LNIVS-VNGRHX(RU-1)和DNRIRLQKXX(RX-1)与细胞粘附有关的肽链。DeRoock等(2001年)筛选得到的两个6个氨基酸短肽,kmviywkag(RZ-3)和kikmviswkg(HYD-1)能支持前列腺癌细胞粘附,并抑制癌细胞与细胞外基质蛋白粘附。Mikawa(2004年)等发现小分子肽NIeDI/V/Nle能特异性与肺泡细胞癌细胞粘附。Aina(2005年)等筛选得到的小分子cDGXGXXc能特异性粘附卵巢癌细胞。
基于国外应用组合化学肽库技术筛选小分子肽的成功报道,本研究拟利用`一个珠子一条肽链`的组合化学肽库技术,分别用非小细胞肺癌细胞A549和小细胞肺癌细胞DMS53筛选6个氨基酸的小分子肽库,以期获得与非小细胞肺癌细胞(A549)和小细胞肺癌细胞(DMS53)特异性结合的小分子肽,并将获得的肺癌特异性小分子肽合成至树脂珠子上,探讨其对提高临床肺癌胸水脱落细胞的阳性诊断率,另外,本研究还将利用生物素和荧光素标记小分子肽,评价小分子肽作为非小肺癌细胞特异性标记物的临床意义。本文共包括以下三个部分。
第一部分:特异性识别非小细胞肺癌A549细胞的小分子肽筛选
目的:应用“一个珠子一个化合物”的组合化学肽库,以期筛选得到特异性识别非小细胞肺癌细胞(A549)的小分子肽。
方法:用非小细胞肺癌细胞A549筛选“一个珠子一个化合物”的6个氨基酸的组合化学肽库,对获得的小分子肽作进一步分析(1)通过三个实验验证小分子肽与非小细胞肺癌结合的特异性(A)用A549、Calu-1等12种非小细胞肺癌、小细胞肺癌及其它瘤细胞系,观察小分子肽与A549等细胞的结合情况;(B)用相应游离小分子肽阻断细胞与小分子肽珠子结合,观察其对细胞与珠子结合的阻断率;(C)将游离小分子肽包被在玻片上,观察比较A549、Calu-1等12种细胞与玻片上小分子肽的粘附情况;(2)将小分子肽中的氨基酸分别用丙氨酸替代或删除小分子肽中每一种氨基酸,观察细胞与不同小分子肽珠子的粘附结合情况,分析肽链的长度及特定氨基酸对小分子肽的粘附活性的影响,确定小分子肽的结构稳定性;(3)应用RAGE、流式细胞仪及免疫荧光染色技术分析非小细胞肺癌细胞表面与细胞粘附关系密切的整合素的表达情况,然后利用抗体阻断实验确定A549细胞与小分子肽的粘附结合位点。实验还采用免疫荧光染色技术观察小分子肽和A549细胞的结合过程中,整合素α3与局部粘着斑激酶(focaladhesion kinase,FAK)、paxillin及vinculin三种重要的细胞粘附相关的细胞信号转导分子的关系。
结果:(1)初次筛选共得到29个与A549阳性结合的珠子,经氨基酸序列分析后发现含有-NGXG-肽链结构的序列共有10个;(2)选择cNGQGEQc作进一步的细胞特异性研究,发现cNGQGEQc与非小细胞肺癌A549、Calu-1及H178的粘附特异性明显高于其他细胞系;(3)对cNGQGEQc的结构分析显示-NGXG-及六肽长度对小分子肽与A549细胞的粘附非常重要(4) RAGE、流式细胞仪及免疫荧光染色技术显示A549,Calu-1等非小细胞肺癌细胞表面存在整合素α3的阳性表达,小细胞肺癌为阴性表达;(5)用抗整合素的抗体(α1-6,v/β1-5)阻断小分子肽与A549细胞表面的相应受体结合,结果显示α3与β亚单位的任何组合均对cNGQGEQc与A549细胞的粘附有明显的阻断作用;(5)在小分子肽和A549细胞的结合过程中整合素α3与FAK、paxillin及vinculin间存在密切的共表达关系。
结论:(1)筛选得到的小分子六肽cNGQGEQc能特异性与非小细胞肺癌细胞A549结合,其与A549细胞结合的配体是细胞表面的整合素α3;(2)整合素α3可作为非小细胞肺癌的分子标记物和靶向治疗的靶点,本研究发现的小分子肽可望为肺癌的分子诊断和靶向治疗提供一种理想的载体。
第二部分:特异性识别小细胞肺癌细胞DMS53的小分子肽筛选
目的:应用“一个珠子一个化合物”的组合化学肽库技术,以期筛选得到特异性识别小细胞肺癌细胞(DMS53)的小分子肽。
方法:用小细胞肺癌细胞DMS53筛选“一个珠子一个化合物”的6个氨基酸的组合化学肽库,对获得的小分子肽作进一步分析(1)通过三个实验验证小分子肽与小细胞肺癌结合的特异性(A)用DMS53等小细胞肺癌细胞系,观察小分子肽与DMS53等细胞的结合情况;(B)用相应游离小分子肽阻断细胞与小分子肽珠子结合,观察其对细胞与珠子结合的阻断率;(C)将游离小分子肽包被在玻片上,观察比较DMS53等细胞与玻片上小分子肽的粘附情况;(2)将小分子肽中的氨基酸分别用丙氨酸替代或删除小分子肽中每一种氨基酸,观察细胞与不同小分子肽珠子的粘附结合情况,分析肽链的长度及特定氨基酸对小分子肽的粘附活性的影响,确定小分子肽的结构稳定性;(3)应用RAGE、流式细胞仪及免疫荧光染色技术分析DMS53细胞表面与细胞粘附关系密切的整合素的表达情况,然后利用抗体阻断实验确定DMS53细胞与小分子肽的粘附结合位点。
结果:(1)初次筛选共得到32个与DMS53阳性结合的珠子,经氨基酸序列分析后发现含有cNGRXXXc或cXNGRXXc肽链结构的序列共有10个;再次合成三种有代表性的小分子肽,发现cFNGRQQc与DMS53的结合率明显高于其他小分子肽;(2)选择cFNGRQQc作进一步的细胞特异性研究,发现cFNGRQQc与DMS53的粘附特异性明显高于其他细胞系;(3)对c-FNGRQQ-c的结构分析显示-NGR-及六肽长度对小分子肽与DMS53细胞的粘附非常重要;(4) RAGE、流式细胞仪及免疫荧光染色技术发现DMS53细胞存在α5和β1表达;(5)使用不同浓度的阻断抗体包括抗整合素α和β亚单位,E-cadherin,NCAM及ICAM抗体,结果显示上述抗体均未能明显地阻断
结论:筛选得到的六肽分子cFNGRQQc是一种能特异性识别小细胞肺癌细胞DMS53并与之结合的小分子肽,但c-FNGRQQ-c与DMS53的结合位点尚不清楚。同时也提示我们c-FNGRQQ-c有别于已发现的其他小分子肽,可能不是通过细胞表面整合素受体与DMS53结合,与粘附分子E-cadherin,NCAM及ICAM也不相关。小分子肽与DMS53细胞表面的结合位点有待于进一步证实。
第三部分:肺癌细胞特异性小分子肽的应用研究
目的:利用前期研究获得的肺癌特异性小分子肽cNGQGEQc,探讨其对提高临床肺癌胸水脱落细胞的阳性诊断率及作为非小肺癌细胞的特异性标记物的临床意义。
方法:(1)将小分子肽cNGQGEQc合成于树脂珠子上,将A549细胞混合于正常人血中,检验小分子肽珠子分离A549细胞的特异性,然后对取自64例包括肺癌、间皮瘤、胃癌、结肠癌、乳腺癌、肺炎和胸膜结核等患者的胸水与小分子肽珠子共培养,分离粘附在树脂珠子上的细胞,显微镜下观察细胞形态。(2)用生物素或荧光素(FITC)标记小分子肽cNGQGEQc,免疫染色A549等细胞株及肺癌组织中的肺癌细胞,评价cNGQGEQc作为肺癌细胞分子标记物的应用价值。
结果:(1)常规细胞学方法仅发现1例阳性病例,而经小分子肽树脂珠子分离胸水中细胞,再结合常规细胞学方法检出5例阳性病例,阳性率提高了10.81%;(2) cNGQGEQc能特异性标记A549和Calu-1非小细胞肺癌细胞,而与小细胞肺癌DMS53等其它细胞无反应:全部18例肺腺癌和大部分鳞癌(7/9)细胞能与小分子肽结合,而与小细胞肺癌细胞呈阴性反应。
结论:(1)小分子肽珠子结合常规细胞学方法可提高胸水中肺癌细胞的阳性检出率;(2)小分子肽cNGQGEQc小分子肽cNOQGEQc能特异性标记非小细胞肺癌细胞,有望作为非小细胞肺癌细胞新的分子标记物。
Background
Lung cancer is the leading malignant tumor with 26.9%increasing incidence per year in our country especially in city.China will be the biggest country with lung cancer patients in the world in 2025.Lung cancer is associated with high mortality and morbidity because most cases present as middle-advanced stage with partly metastasis after diagnosis.The long-term outlook for patients with advanced lung cancer is poor with median survival and is typically less than one year.Only about 20%of non-small lung cancer(NSCLC) patients response to anticancer drugs and have to give up chemotherapy with severe side-effect such as hematopoiesis inhibition.Moreover,there has been no effective way for screening or detection of early lung cancer up to now,in contrast to what have been established for prostate cancers and colon cancer.Obviously,new strategies are needed for early detection and treatment for lung cancer.
Monoclone antibodies specific for antigen or receptor on cell surface generated by hybridoma technique,have been widely applied in the clinical diagnosis of breast cancer,colon cancer and prostate cancer.Moreover,molecular-targeting therapy has become a reality in treatment of lung cancer,breast cancer and lymphoma.For example,inhibitors of epidermal growth factor receptor,gefitinib and erlotinib,have recently been used to treat NSCLC refractory to traditional chemotherapy.Herceptin and Rituxan have been used for breast cancer and B cell leukemia.However,clinical application of monoclone antibodies in cancer treatment still remains several practical problems which affect the clinic therapeutic efficacy as follow:(1) Monoclone antibody may not permeate the target tumor mass effectively because of large molecule protein;(2) The non-specific aggregation of antibody in reticuloendothelial system may lead to a reduction of its anticancer potency;(3) Antimouse antibody or the chimera of antibody have potential immunogenicity.Recent studies have showed that small molecule peptide can retrieve a few shortage of antibody.Small molecule peptide has more advantage in the following aspects:(1) Effective to penetrate the tumor mass with less small molecule than a antibody,such as ten peptide have a molecular weight of approximately 1000 Da;(2) Easier and more constant to conjugated with toxin and radioactive agent than a antibody;(3) More stable and effective to protect amino acide from proteolysis by protein enzyme than a antibody; (4) Less to be phagocytosed by reticuloendothelial system;(5) Less likely to provoke an immunoresponse than proteins;(6) Much less cost in synthesis than antibody.We believe that small molecule peptide has more promising approach in cancer diagnosis and treatment.
Small molecule peptide has basically been used to apply in cancer diagnosis and treatment in three aspects:(1) Small molecule peptide can capture metastatic cancer cell from circulation;(2) Small molecule peptide labelled with magnetic atom or fluorescein or radiation isotope will be molecule probes,for example,small molecule peptide RGD labelled with isotope can remarkably promote the sensitivity of MRI;(3) Small molecule peptide labelled with radiation isotope will be targeting radiative therapeutic agent for cance patients,for example,peptide containing RGD sequence labelled with ~(111)In or ~(99m)Tc can dramatically inhibit tumor growth.
Now there are two general screening methods for small molecule peptides:one is "one-bead one-peptide" combinatorial chemistry peptide library,another is phage-displaying peptide library."one-bead one-peptide" combinatorial chemistry peptide library was firstly introduced by Pro.Lam in 1991.The library is synthesized using the natural and unnatural amino acide on resin beads at random.According to the experiments,libraries with one peptide on one bead can be synthesized with 4 or 6 or 8 peptides length and loop or liner conformation.The efficiency of screening is very high because millions of peptides can be screened at one time.Phage-displaying peptide library is one of the main methods for peptides screening at present as well. However,"one-bead one-peptide" combinatorial chemistry peptide library can be added into unnatural amino acide randomly during the synthesis process and therefore peptides are generally resistant to proteolysis and more stable than the latter.So "one-bead one-peptide" combinatorial library is regard as more ideal method for small molecule peptide screening.
Many scholars have successfully found some small molecule peptides bind specifically to cancer cells using "one-bead one-peptide" combinatorial libraries. Pennington et al(1996) identified two peptides,RU-1(LNIVSVNGRHX) and RX-1 (DNRIRLQAKXX) by scanning for prostate cancer cell among 1.5 million peptide beads.DeRoock et al(2001) found two peptides with six amino acid,RZ-3 (kmviywkag) and HYD-1(kikmviswkg).These two peptides support tumor cell adhesion and can inhibit cancer cell adhesion to extracellular matrix protein.Mikawa et al(2004) identified a small molecular peptide NleDI/V/NIe can specifically adhere to bronchioloalveolar carcinoma cells.Aina et al(2005) reported a novel small molecule peptide cDGXGXXc by screening a cyclic random 8-mer library can specifically adhere to ovarian cancer cells.
In this study,we intended to identify small molecule peptides specific for NSCLC A549 and SCLC DMS53 cells by screening for "one-bead one-peptide" combinatorial chemistry library.And applications of the small molecule peptide is also investigated in promoting cytopathological positive diagnosis rate of pleural fluid with lung cancer by synthesis the peptide on beads and is evaluated to be as specific molecular marker of NSCLC cells.The full text consists of the three following sections:
Section one:A novel specific small molecule peptide for non-small cell lung cancer cell A549
Aim:To screen small molecule peptide specific binding to non-small cell lung cancer cell(A549) using the "one-bead one-peptide" combinatorial library. Materials and Methods:A "one-bead one-peptide" combinatorial library with six amino acids was used to screen for specific binding peptides to non-small cell lung cancer cell(A549).Beads with cell adhesion and growth(positive beads) were isolated,stripped,and microsequenced.The novel consensus peptides will be re-synthesized and further tested as follow:(1) Cell-type specificity was analysized by three independent experiments(A) A panel of cell lines including non-small-cell lung cancer A549,Calu-1 and other cancer cells were used to study their specificity to novel peptides;(B) Free peptides were used to block the binding between cancer cells and peptide beads;(C) Adhesion to a slide coated with free peptides was tested by twelve different cancer cells including A549 and Calu-1.(2) The strategies of site-directed deletion and alanine scanning were employed to determine the structure either length or specific amino acid for cell adhesion.(3) Expression profling of integrins for cell adhesion was investigated using restriction analysis of gene expression(RAGE),flow cytometry and immunofluorescence staining technique. Antibody blocking assay was then used to confirm the binding site between A549 cells and small molecule peptides.Immunofluorescence technique was also used to observe the relationship between integrin a3 and focal adhesion kinase(FAK) and paxillin and vinculin which are all important cell adhesion associated signal transduction molecules.
Results:(1) Twenty-nine positive beads binding to A549 cell were totally obtained after primary screening.Consensus peptide sequence of-NGXG-was identified by amino acid sequencing in ten beads.(2) Peptide cNGQGEQc was re-synthesized on beads and further studied for its cell specificity.Peptide cNGQGEQc was showed to be specific for cell attachment to non-small cell lung cancer cells including A549, Calu-1 and H178,but not to other cancer cell lines.(3) Both motif of-NGXG-and the length of peptide are very important for A549 adhesion.(4) Integrin a3 was presented on non-small cell lung cancer cells A549 and Calu-1 but not on small lung cancer cell by RAGE,flow cytometry and immunofluorescence staining technique. (5) In an blocking assay with anti-integrin antibodies(α1-6,v/β1-5),cell adhesion of A549 to peptide beads was obviously inhibited by integfinα3 combining with anyβsubunits.(6) There is intensive coexpression relationship between integrinα3 and FAK and paxillin and vinculin during the binding process between small molecule peptide and A549.
Conclusion:The results suggested that(1) small molecule peptide cNGQGEQc can bind specifically to non-small cell lung cancer cell A549 via integrinα3 on cell surface;(2) Integrinα3 represents a potential molecular marker and target for molecular therapy in non-small cell lung cancer.The novel small molecule peptide obtained in this study will be used as an ideal carder for molecular diagnosis and targeting therapy of lung cancer.
Section two:A novel specific small molecule peptide for small cell lung cancer cell DMS-53
Aim:To screen small molecule peptide specific binding to small cell lung cancer cell (DMS53) using the "one-bead one-peptide" combinatorial chemistry technology. Materials and Methods:A "one-bead one-peptide" combinatorial library with six amino acids was used to screen for small molecule peptide specific binding to small cell lung cancer cell(DMS53).Beads with cell adhesion and growth(positive beads) were isolated,stripped,and microsequenced.The novel consensus peptides will be re-synthesized and further tested as follow:(1) Cell-type specificity was analysized by three independent experiments(A) A panel of cell lines including small cell lung cancer cell DMS53 and other cancer cells were used to study their specificity to novel peptides;(B) Free peptides were used to block the binding between cancer cells and peptide beads;(C) Adhesion to a slide coated with free peptides was tested by different cancer cells including small cell lung cancer cell(DMS53).(2) The strategies of site-directed deletion and alanine scanning were employed to determine the structure either length or specific amino acid for cell adhesion.(3) Expression profling of integrins for cell adhesion was investigated using restriction analysis of gene expression(RAGE),flow cytometry and immunofluorescence staining technique.Antibody blocking assay was then used to confirm the binding site between DMS53 cells and small molecule peptide.
Results:(1) Thirty two positive beads binding to DMS53 were totally obtained after primary screening.Consensus peptide sequences of cXNGRXXc and cNGRXXXc were identified by amino acid sequencing in ten beads.Three representative peptides were re-synthesized on beads.Secondary screening showed cell adhesion percentage of cFNGRQQc was higher than the other two peptides.(2) cFNGRQQc was further studied for cell specificity,alanine scanning and site-directed deletion.The results showed that cFNGRQQc is specific for promoting cell adhesion to DMS53 but not other human cancer cell lines.(3) Both motif of-NGR-and the length of peptide are important for DMS53 attachment.(4) Integrinα5 andβ1 were presented on DMS53 by RAGE,flow cytometry and immunofluorescence staining technique.(5) In an antibody blocking assay,cell adhesion of DMS53 to peptide beads was not inhibited by antibodies including integrin,E-cadherin,NCAM and ICAM.
Conclusion:The results suggested that(1) small molecule peptide cFNGRQQc can bind specifically to small cell lung cancer cell DMS53,but the binding site between the peptide and DMS53 is still unknown;(2) The binding site between the peptide cFNGRQQc and DMS53 is different from other peptide via integrin and may be not associated with adhesion molecules E-cadherin,NCAM and ICAM.The binding site on DMS53 surface for cFNGRQQc peptide need to be proven in the future.
Section three:Application of specific small molecule peptide in lung cancer
Aim:To investigate the clinic significance of the specific small molecule peptide obtained in provious study for lung cancer in promoting cytopathological positive diagnosis rate of pleural fluid with lung cancer by synthesis the peptide on beads and is evaluated to be as specific molecular marker of NSCLC cells.
Materials and Methods.(1) Small molecule peptide cNGQGEQc synthesized on resin beads was tested the the specificity in capturing A549 cells mixed with normal human blood.Small molecule peptide beads were then co-cultured with pleural fluid obtained from sixty-four cases including lung cancer,mesothelioma,gastric carcinoma,colon cancer,breast cancer,pneumonia and tuberculosis of pleura and so on.Capturing of cancer cells on the beads was accomplished using method as described above.Cells captured on beads were subsequently stripped off the beads and cell smears were prepared on slides for microscopic examination.(2) The small molecule peptide labelled with biotin or FITC was used to stain A549 cell as well as other cancer cells.The specificity of small molecule peptide binding to lung cancer cells was also tested in lung cancer tissues.
Results:(1) Only one positive case was found.by routine cytology method,while five positive cases(10.81%) were present by routine cytologic method combining with capturing cancer cell with peptide beads.(2) Non-small lung cancer cells A549 and Calu-1 but not DMS53,were specifically recognised by peptide "cNGQGEQc".All the eighteen samples of adenocarcinoma and most of squemous cancinoma(7/9) of lung were immunostained with peptide but not in small cell lung cancer.
Conclusion:(1) Small molecule peptide bead technology combining with routin cytology method can be used for isolating and enriching the scanty number of cancer cells in pleural effusion.(2) Small molecule peptide "cNGQGEQc" can specifically label the non-samll lung cancer and will be the novel helpful biological marker for NSCLC cells.
引文
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