CN101965356A - 以特异方式与磷脂酰丝氨酸结合的多肽及其用途 - Google Patents
以特异方式与磷脂酰丝氨酸结合的多肽及其用途 Download PDFInfo
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- CN101965356A CN101965356A CN2009801063128A CN200980106312A CN101965356A CN 101965356 A CN101965356 A CN 101965356A CN 2009801063128 A CN2009801063128 A CN 2009801063128A CN 200980106312 A CN200980106312 A CN 200980106312A CN 101965356 A CN101965356 A CN 101965356A
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Abstract
本发明涉及一种以特异方式与磷脂酰丝氨酸结合的多肽及其用途,具体涉及一种具有序列号码为1的氨基酸序列并以特异方式与磷脂酰丝氨酸结合的多肽、包含该多肽作为有效成分的用于检测磷脂酰丝氨酸的组成物、利用该组成物的磷脂酰丝氨酸检测方法、包含该组成物作为有效成分的用于检测凋亡细胞的组成物、包含该组成物作为有效成分的用于传达药物的组成物、用于防治肿瘤疾病的组成物以及用于患肿瘤部位成像的组成物等。本发明中具有序列号码为1的氨基酸序列的多肽以特异方式与磷脂酰丝氨酸结合。因此,本发明的多肽可以广泛应用于磷脂酰丝氨酸的检测,更进一步,可以广泛应用于将磷脂酰丝氨酸异位表达在细胞表面的凋亡细胞及肿瘤细胞的检测或者成像等。
Description
技术领域
本发明涉及一种以特异方式与磷脂酰丝氨酸结合的多肽及其用途,具体涉及一种具有序列号码为1的氨基酸序列并以特异方式与磷脂酰丝氨酸结合的多肽、其检测磷脂酰丝氨酸的用途、利用其检测磷脂酰丝氨酸的方法、其检测凋亡细胞的用途、其传达药物用途、其治疗肿瘤病症用途以及患肿瘤部位成像用途等。
背景技术
磷脂酰丝氨酸(phosphatidylserine,PS)是巨噬细胞识别凋亡细胞而去除的重要的标志物(Schlegel,R.A.et al.,Cell Death and Differentiation,2001,8:551-563;Lauber,K.et al.,Mol Cell 2004,14:277-287 Henson,P.M.et al.,Curr Biol 2001,11:R795-805Grimsley,C.et al.,Trends Cell Biol.2003,13:648-656)。在正常情况下,磷脂酰丝氨酸存在于细胞膜内部,或者细胞接到凋亡信号,或者红血球老化时,会显露到细胞膜的外部(Fadeel,B.et al.,Cell Mol Life Sci,2003,60:2575-2585)。对此,由巨噬细胞通过细胞表面的接受体进行识别而吞噬(Fadok,V.A.et al.,J immunol 1992,148:2207-2216;Fadok,V.A.et al.,Nature 2000,405:85-90;Park,S.Y.et.al.,Cell Death and__Differentiation,in press)。
除了凋亡细胞之外,磷脂酰丝氨酸也同样在各种疾患状态下显露到细胞膜的外部(Zwaal,R.F.A.et al.,Cell.Mol.Life Sci 2005,62:971-988),如斯科特综合症(Scottsyndrome)、抗磷脂综合症(antiphospholipid syndrome)、镰状细胞性贫血(sickle cellanemia)、地中海型贫血(thalassemia)、口形红细胞增多症(stomatocytosis)、尿毒症(uremia)、肾结石病(kidney stone disease)、糖尿病(diabetes)、血糖过高症(hyperglycemia)、病毒及微生物感染症、疟疾(malaria)、子痫前症(pre-eclampsia)、高胆红素血症(hyperbilirubinemia)及瘤变(neoplasia)等。特别是,很多肿瘤细胞向细胞膜的外部异位表达磷脂酰丝氨酸(Utsugi,T.et al.,Cancer Res.1991,15:3062-3066;Rao,L.etal.,Thromb Res.1992,67:517-531;Sigimura,M.et al.,Fibrinolysis.1994,5:365-373;Ran,S.et al.,Cancer Res.2002,62:6132-6140;Woehlecke,H.et al.,Biochem J.2003,376:489-495),这种现象在未分化的细胞瘤(undifferentiatedtumorigenic cell)里更明显。肿瘤组织也同样伸出向细胞膜的外部显露磷脂酰丝氨酸的小血管(Ran,S.et al.,Cancer Res.2002,62:6132-6140;Zwaal,R.F.A.et al.,Blood.1997,89:1121-1132)。
而且,在各种细胞内生理过程中的非凋亡细胞也可以观察到磷脂酰丝氨酸。,如血小板的活化过程、肌肉细胞的融合过程、合体滋养细胞(syncytiotrophoblast)的形成、肥大细胞(mast cells)的免疫球蛋白依赖性刺激及T细胞的移动等(Fadeel,B.et al.,CellDeathDiffer 2006,13:360-2 Ran,S.et al.,Int J Radiat Oncol Biol Phys 2002,54:1479-84 Schlegel,R.A.etal.,Cell Death Differ 2001,8:551-63.)。其中,特别是,阻止在活化血小板的过程中异位表达的磷脂酰丝氨酸时,通过抑制由于动脉硬化可能会形成的血栓形成(thrombosi s)过程而表达其治疗效果(Cederholm,A.and Frosteg,J.,AnnN Y Acad Sci.2007,1108:96-103Cederholm,A.and Frosteg,J.,Drug News Perspect.200720(5):321-6)。因此,由于上述磷脂酰丝氨酸起到的作用,特别是,在包括肿瘤性及炎症性疾病在内的各种情况下,磷脂酰丝氨酸正在成为诊断、治疗及治疗追踪的目标物质。
另外,据有关资料的报告内容,抑制磷脂酰丝氨酸的识别可以增强被检测淋巴瘤(irradiated lymphoma)的细胞免疫性(immunogenicity)(Bondanza,A.et al.,J Exp Med2004、200:1157-65)。因此,可以与磷脂酰丝氨酸有效结合的蛋白质阻碍凋亡细胞识别磷脂酰丝氨酸和去除免疫抑制性,可以用于增强以凋亡细胞为基础的疫苗(apoptotic cell-basedvaccines)的效果。
鉴于此,本发明人探索和研究可以将磷脂酰丝氨酸作为目标物的新蛋白质或者其片段的结果发现,具有序列号码为1的氨基酸序列的多肽可以以特异方式与磷脂酰丝氨酸结合,并完成了本发明。
发明技术问题的公开
为了解决以上问题,本发明提供一种以特异方式与磷脂酰丝氨酸结合的多肽及其用途。
技术方案
为了实现本发明的上述目的,本发明提供一种具有序列号码为1的氨基酸序列并以特异方式与磷脂酰丝氨酸(phosphatidylserine)结合的多肽。
为了实现本发明的另一目的,本发明还提供一种对于所述多肽实施加密的多聚核苷酸。
为了实现本发明的又另一目的,本发明还提供一种包含所述多肽作为有效成分的用于检测磷脂酰丝氨酸的组成物。
为了实现本发明的又另一目的,本发明还提供一种检测磷脂酰丝氨酸的所述多肽的用途。
为了实现本发明的又另一目的,本发明还提供一种将所述多肽与饲料混合并去除未结合或者以特异方式结合的多肽之后确认所述多肽和饲料是否结合以及位置的磷脂酰丝氨酸的检测方法。
为了实现本发明的又另一目的,本发明还提供一种包含所述多肽作为有效成分的用于检测凋亡细胞(apoptotic cell)的组成物。
为了实现本发明的又另一目的,本发明还提供一种检测凋亡细胞(apoptotic cell)的所述多肽的用途。
为了实现本发明的又另一目的,本发明还提供一种包含所述多肽作为有效成分的用于传达药物的组成物。
为了实现本发明的又另一目的,本发明还提供一种传达药物的所述多肽的用途。
为了实现本发明的又另一目的,本发明还提供一种包含所述多肽以及与其结合的抗肿瘤性药物制剂作为有效成分的用于防治肿瘤病症的药物性组成物。
为了实现本发明的又另一目的,本发明还提供一种制造肿瘤病症治疗制剂的所述多肽以及与其结合的抗肿瘤制剂的用途。
为了实现本发明的又另一目的,本发明还提供一种包含所述多肽作为有效成分的用于患肿瘤部位成像的组成物。
为了实现本发明的又另一目的,本发明还提供一种成像患肿瘤部位的所述多肽的用途。
为了实现本发明的又另一目的,本发明还提供一种包含所述多肽作为有效成分的用于诊断斯科特综合症等病症的诊断用组成物。
为了实现本发明的又另一目的,本发明还提供一种诊断斯科特综合症等病症的所述多肽的用途。
以下详细说明本发明。
本发明根据具有序列号码为1的氨基酸的多肽以特异方式与显露在细胞表面的磷脂酰丝氨酸结合的特性,基于具有序列号吗为1的氨基酸的新序列多肽及其用途提供包含所述多肽的用于检测磷脂酰丝氨酸(phosphatidylserine)的组成物等。
本发明中多肽是指以特异方式与磷脂酰丝氨酸结合并具有序列号码为1的氨基酸序列的多肽。本发明中肽片段是指包含所有种类的肽、多肽、蛋白质、肽模仿物、化合物及生物制剂且具有可以以特异方式与异位表达在肿瘤细胞等细胞的表面的磷脂酰丝氨酸结合的活性的物质。本发明中多肽可以来自天然,也可以利用公知肽合成方法通过合成制造而成。
而且,本发明进一步提供一种对于多肽实施加密的具有碱基序列的多聚核苷酸。
另外,本发明提供一种对于本发明中多肽实施加密的具有碱基序列的载体及基因转殖成所述载体的基因转殖体。
本发明中载体包括质粒载体、噬菌粒载体、噬菌体载体及病毒载体等,但并不局限于此。本发明中载体可以是通常的选殖载体或者异位表达载体,该表达载体除了包含促进剂、操作符、起始密码、终止密码、多聚腺苷酸信号及增强剂(促进基因)等已位表达调整序列之外,还包含以膜定位或者分泌为目的的信号序列或者前导序列,并根据目的制造成多种形态。另外,所述载体包含选择内含载体的宿主细胞的选择标记。如果是可以复制的载体,会包含复制起始点。
可以利用本发明所属领域技术人员公知基因转殖技术基因转殖成所述载体。优选地,利用基因枪法(microprojectile bombardment)、电穿孔法(electroporation)、磷酸钙(CaPO4)沉淀、氯化钙(CaCl2)衬垫、PEG-介导融合法(PEG-mediated fusion)、微量注射法(microinjection)及脂质体介导法(liposome-mediated method),所述基因转殖体可以是大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、链霉菌(Streptomyces)、假单胞菌(Pseudomonas)、变形杆菌(Proteus mirabilis)、葡萄球菌(Staphylococcus)及根癌土壤杆菌(Agrobacterium tumefaciens),但并不局限于此。
本发明人为了核实以特异方式与磷脂酰丝氨酸结合的所述多肽的功能进行各种试验的结果发现,本发明的肽以特异方式识别老化的细胞以及异位表达在凋亡细胞的表面的磷脂酰丝氨酸,从而介导所述细胞的粘附和吞噬。而且,本发明的肽以特异方式与肿瘤细胞结合,从而在活体内(in vivo)或者在活体外(ex vivo)确认及成像肿瘤细胞。由此本发明人理解到,本发明的肽可以利用为检测磷脂酰丝氨酸的组成物,更进一步讲,本发明的肽可以利用为识别肿瘤组织内磷脂酰丝氨酸的用于诊断或者治疗追踪的制剂或者特异的治疗肿瘤制剂以及防治肿瘤病症的药物性组成物等。
更具体地讲,本发明一实施例利用常用M13噬菌体库筛选了以特异方式与磷脂酰丝氨酸结合的噬菌体。其结果,通过共4轮筛选终于筛选出了以特异方式与磷脂酰丝氨酸结合的噬菌体。分析其序列的结果发现,筛选的噬菌体主要是具有CLSYYPSYC的氨基酸共同序列的肽。
而且,还核实了本发明另一实施例筛选的噬菌体与磷脂酰丝氨酸结合时显示出的特异性。其结果,对比群噬菌体几乎不会与磷脂酰胆碱或者磷脂酰丝氨酸。与此相反,被筛选的噬菌体以特异方式与磷脂酰丝氨酸结合。另外,被筛选的噬菌体以特异方式与磷脂酰丝氨酸脂质体,且由于膜联蛋白V的参与阻碍与磷脂酰丝氨酸之间的结合。
另外,本发明又另一实施例核实了被筛选的噬菌体克隆体或者本发明中肽是否与凋亡细胞结合。核实结果发现,被筛选的噬菌体克隆体和本发明中肽与显露在凋亡细胞的磷脂酰丝氨酸之间的结合状态良好。
本发明又另一实施例核实了本发明中肽是否可以引导到被移植的肿瘤部位以及是否可以将其成像。核实结果发现,在本发明中肽进行处理的群中,本发明中肽确实被引导到肿瘤组织,且可以通过标志核实其过程。
总而言之,本发明中多肽以特异方式与磷脂酰丝氨酸结合,并在或体内识别和吞噬凋亡细胞和肿瘤细胞。
上述内容中核苷酸及蛋白质作业可以参考以下文献(Maniatis et al.,MolecularCloning:A Laboratory Manual,Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y.(1982);Sambrook et al.,Molecular Cloning:A Laboratory Manual,2d Ed.,ColdSpring Harbor Laboratory Press(1989);Deutscher,M.,Guide to Protein PurificationMethods Enzymology,vol.182.Academic Press.Inc.,San Diego,CA(1990))。
因此,本发明提供一种包含序列号码为1的氨基酸序列的本发明中多肽作为有效成分的用于检测磷脂酰丝氨酸(phosphatidylserine)的组成物。
而且,本发明还提供一种检测磷脂酰丝氨酸的具有序列号码为1的氨基酸序列的本发明中多肽的用途。
而且,由于本发明的多肽以特异方式与磷脂酰丝氨酸结合,本发明提供一种包括如下步骤磷脂酰丝氨酸检测方法,其步骤如下:
(a)将本发明中多肽与饲料混合的步骤;
(b)去除未结合或者以非特异方式结合的所述多肽的步骤;
(c)核实所述多肽是否结合以及位置的步骤。
为了本发明中多肽的核实是否结合、检测及定量变容易,本发明中多肽可以以标志的状态提供。、即,可以涂在可以检测的标志上(如,共价贴附或者交联)提供。所述可检测标志可以是显色酶(如,过氧化酶、碱性磷酸盐)、放射形同位素(如,125I、32P、35S)、发色团(chromophore)、发光物质或者荧光物质(如,FITC、RITC、荧光蛋白质(GFP(Green FluorescentProtein)和EGFP(Enhanced Green Fluorescent Protein)、RFP(Red Fluorescent Protein)和DsRed(Discosoma sp.redfluorescent protein)和CFP(Cyan Fluorescent Protein)、CCFP(Cyan GreenFluorescent Protein)、YFP(Yellow Fluorescent Protein)、Cy3、Cy5及Cy7.5)、超顺磁性纳米粒子(super paramagnetic particles)或者超小型顺磁性纳米粒子(ultrasuper paramagnetic particles)。
依据标志的检测方法是本发明所属领域公知技术,例如,可以采用如下方法实施。采用可检测标志利用荧光物质时,可以利用免疫荧光染色法。例如,使以荧光物质标记的本发明中多肽与试剂产生反应并去除未结合或者特异的结合物之后,采用荧光显微镜观察由于肽产生的荧光。另外,将可检测标志用于酶时,依据通过酶反应产生的基质的显色反应检测吸光度。如果是放射性物质,可以通过检测放射线的放射量达到检测目的。
而且,本发明的多肽可以使磷脂酰丝氨酸以特异方式与显露在细胞表面的细胞结合。鉴于磷脂酰丝氨酸在凋亡细胞(apoptotic cell)显露到细胞膜,本发明提供一种包含本发明中多肽作为有效成分的用于检测凋亡细胞(apoptotic cell)的组成物。而且,本发明还提供一种检测凋亡细胞的本发明中多肽的用途。上述内容中,本发明并不限定凋亡细胞的检测方法,例如,可以采用检测所述磷脂酰丝氨酸时的方法。如上所述,为了使本发明中多肽的核实是否结合、检测及定量变得更容易,本发明的多肽可以以标志的状态提供,其检测结果可以依据基于检测标志公开的成像方法实施成像。
另外,由于本发明中多肽可以使磷脂酰丝氨酸以特异方式与显露在细胞表面的细胞结合,可以用作可以将药物选择性地传达给所述细胞的智能型传达药物剂。鉴于此,本发明提供一种包含本发明中多肽作为有效成分的用于传达药物的组成物。
如上所述,越来越多的磷脂酰丝氨酸异位表达在细胞膜外部的情况出现在黑色素瘤及大肠癌细胞(Utsugi,T.et al.,Cancer Res.1991,15:3062-3066)、卵巢癌细胞(Rao,L.etal.,Thromb Res.1992,67:517-531)、胃癌及肝癌细胞(Sugimura,M.et al.,Blood Coagul.Fibrinolysis.1994,5:365-373;Woehlecke,H.et al.,Biochem J.2003,376:489-495)、癌组织的内皮细胞(Ran,S.et al.,Cancer Res.2002,62:6132-6140)等各种肿瘤细胞中,这种情况在未分化的细胞瘤(undifferentiated tumorigenic cell)里更明显。
肿瘤组织也同样伸出向细胞膜的外部显露磷脂酰丝氨酸的小血管(Ran,S.etal.,Cancer Res.2002,62:6132-6140;Zwaal,R.F.A.et al.,Blood.1997,89:1121-1132)。
因此,所述用于传达药物的组成物有可能对于肿瘤病症起到特异作用。肿瘤病症是指由于恶性肿瘤出现病理性症状的疾病,如,大肠癌、肺癌、胃癌、食道癌、胰腺癌、胆囊癌、肾癌、膀胱癌、前列腺癌、睾丸癌、子宫颈癌、子宫内膜癌、绒毛癌、卵巢癌、乳房癌、甲状腺癌、脑癌、头颈部癌、恶性黑色素瘤、皮肤癌、肝癌、白血病(leukemia)、淋巴瘤(lymphoma)、多发性骨髓瘤(multiple myeloma)、慢性骨髓性白血病(chronic myelogenous leukemia)、神经母细胞瘤(neuroblastoma)及再生障碍性贫血,但是,并不局限于此。
如果将包含于本发明中用于传达药物的组成物的本发明多肽和现有抗肿瘤性药物制剂联系起来治疗病症,就可以通过本发明的多肽使所述制剂选择性地只传达到肿瘤细胞之中。如此,不仅可以提高药效,还可以显著减少对于正常组织产生的副作用。
本发明不特别限定可以与本发明多肽联系起来的抗肿瘤性药物制剂,只要是用于治疗肿瘤的现有药物均可,如紫杉醇、阿霉素、长春新碱、柔红霉素(daunorubicin)、长春花碱(vinblastine)、放线菌素-D(actinomycin-D)、多烯紫衫醇(docetaxel)、依托泊苷(etoposide)、替尼泊苷(teniposide)、比生群(bisantrene)、高三尖杉酯碱(homoharringtonine)、格里维克(Gleevec;STI-571)、顺铂(cisplain)、5-氟尿嘧啶(5-fluouracil)、亚德里亚霉素(adriamycin)、甲氨蝶呤(methotrexate)、白消安(busulfan)、苯丁酸氮芥(chlorambucil)、环磷酰胺(cyclophosphamide)、美法仑(melphalan)、氮芥(ni trogen mustard)及亚硝基尿素(ni trosourea)等。所述制剂和本发明中多肽的联系是可以通过本发明所属领域公知技术方法实施,如共价贴附、交联等。为此,本发明的肽根据需要在不丢失其活性的范围内可以对其实施化学改性(modification)。包含于本发明组成物的本发明肽的量根据结合的抗肿瘤药物的种类和量不同。
另外,本发明以本发明中多肽及与其结合的抗肿瘤性药物制剂为有效成分提供一种用于防治肿瘤病症的药物性组成物。而且,本发明还提供一种制造肿瘤病症治疗制剂的本发明中多肽以及与其结合的抗肿瘤制剂的用途。
此时,所述药物性组成物中的抗肿瘤性药物制剂、结合方法及肿瘤病症相关说明与上述内容相同。
另外,本发明中药物性组成物可以以所述多肽的纯粹形态提供或者与药物学方面允许的载体一起配制成妥当的形态来提供。’药物学方面允许’是指生理学方面允许,且投药给人类时,通常不会引起胃肠障碍、眩晕等过敏性反应或者与其类似的反应的非毒性组成物。所述载体包含左右种类的溶剂、方差矩阵、水中油或者油中水乳剂、水性组成物、脂质体、微型珠及微粒体。
而且,本发明的药物性组成物可以根据给药途径与妥当的载体一起配制。所述本发明中药物性组成物的给药途径可以采用口腔给药方式也可以采用非口腔给药方式,但是,其给药途径并不限于此。非口腔给药方式包括各种途径,如,透皮、鼻腔、腹腔、肌肉、皮下或者静脉等。
通过口腔给药本发明的药物性组成物时,本发明的药物性组成物可以根据本发明所属领域公知技术方法,与适合口腔给药的载体一起配制成粉末、颗粒、片剂、丸剂、糖丸、胶囊、药液、凝胶制剂、糖浆制剂、悬浮液、薄片等形态。适合用作载体的有内含乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、木糖醇、赤藓糖醇及麦芽糖醇等的糖类和内含玉米淀粉、小麦淀粉、大米淀粉及土豆淀粉等的淀粉类、内含纤维素、甲基纤维素、羧甲基纤维素钠及羟丙基甲基纤维素等的纤维素类以及凝胶、聚乙烯吡咯烷酮等填充物。而且,根据情况,还可以添加交联结合聚乙烯吡咯烷酮、琼脂、海藻酸或者海藻酸钠等作为崩解剂。进一步,所述药物性组成物还可以包含抗凝结剂、润滑剂、湿润剂、香辛料、乳化剂及防腐剂等。
另外,通过非口腔途径给药时,本发明的药物性组成物可以根据本发明所属领域公知技术方法,适合采用非口腔给药方式的载体一起配制成注射剂、透皮制剂及鼻腔吸入制剂形态。所述注射剂必须要彻底杀菌,保证不受到病毒和真菌等微生物的污染。适合于注射剂的载体可以是水、乙醇、多元醇(例如,甘油、丙二醇及液状聚乙二醇等)、其混合物及/或者内含植物油的溶剂或者方差矩阵。进一步优选地,适合的载体有内含平衡盐溶液、林格液、三乙醇胺的PBS(phosphate buffered saline)或者用于注射的灭菌水、10%乙醇、40%丙二醇及5%葡萄糖等等渗溶液等。为了保护所述注射剂免收微生物的污染,还可以进一步包含灭菌剂、氯丁醇、苯酚、山梨酸、消毒液等各种抗菌剂及抗真菌剂。而且,所述注射剂在大部分情况下还可以进一步包含糖或者氯化钠等等渗剂。
透皮剂包含软膏剂、乳膏剂、涂剂、凝胶剂、外用药水、糊剂、擦剂、气孔剂等形态。所述“透皮给药”是指将药物性组成物局部投放到皮肤里,使内含于药物性组成物的有效量活性成分传达到皮肤内部的给药方式,其配制方式可以采用制药化学行业通常公知的处方索引文献(Remington’s Pharmaceutical Science,15th Edition,1975,Mack PublishingCompany,Easton,Pennsylvania)里。
吸入制剂中,本发明使用的化合物可以使用妥当的推进剂,例如,使用二氯氟甲烷、三氯氟乙烷、四氟二氯乙烷、二氧化碳或者其它妥当的气体,以气雾剂喷雾形态从加压包或者雾化器顺利传达。使用加压气雾剂时,可以通过提供传达已计量好的投入量的管路决定给药单位。例如,用于吸入器或者吹入器的软胶囊剂和卡局式剂型可以内含化合物及乳糖或者淀粉等妥当的粉末状粉末混合物。
要想了解除此之外的药理上允许采用的载体,可以参考以下文献中记载的内容(Remington’s Pharmaceutical Sciences,19th ed.,Mack Publishing Company,Easton,PA,1995)。
而且,本发明中药物性组成物进一步包含一种以上缓冲剂(如生理盐水或者PBS)、碳水化物(如葡萄糖、甘露糖、蔗糖或者葡萄聚糖)、稳定剂(亚硫酸氢钠、亚硫酸钠或者抗坏血酸)、抗氧化剂、抑菌剂、螯合萃取剂(如EDTA或者谷胱甘肽)、辅助剂(如氢氧化铝)、悬浮剂、增稠剂及/或抗菌防腐剂(氯化苯甲烃胺、甲基-或丙基-灭菌剂及氯丁醇)。
而且,本发明中药物性组成物可以采用本领域公知的方法制备,从而保证将其投入到哺乳动物之后迅速、持续或者推迟放出活性成分。
可以通过包括口腔、透皮、皮下、静脉或者肌肉在内的各种途径投入通过上述方法制备而成的药物性组成物。所述’有效量’是指投入给患者时可以追踪其诊断或者治疗效果的化合物或者提取物的量。本发明中药物性组成物的的投入量可以根据投入途径、投入对象、对象病症及其重症程度、年龄、性别、体重、体质差异和患病状态妥当选择。优选地,包含本发明多肽的药物性组成物可以根据患病程度改变有效成分的含量,可通常情况下,以成人为准时,可以以一次10μg至10mg有效容量每日反复投入数次。
而且,由于本发明中多肽以特异方式与磷脂酰丝氨酸结合,可以有效使用于肿瘤病症患病部位的成像。鉴于此,本发明提供一种包含所述多肽作为有效成分的用于肿瘤病症部位成像的组成物。而且,本发明还提供一种成像肿瘤病症部位的本发明中多肽的用途。为了易于确认是否结合、检测及定量,可以以标记的状态提供所述多肽,其方法可遵照上述内容里记载的方法。
另外,除了上述肿瘤病症之外的各种患病状况下,磷脂酰丝氨酸也显露到细胞膜外部(Zwaal,R.F.A.et al.,Cell.Mol.Life Sci.2005,62:971-988),例如,斯科特综合症(Scott syndrome)、抗磷脂综合症(antiphospholipid syndrome)、镰状细胞性贫血(sicklecell anemia)、地中海型贫血(thalathemia)、口形红细胞增多症(stomatocytosis)、尿毒症(uremia)、肾结石病(kidney stone disease)、糖尿病(diabetes)、血糖过高症(hyperglycemia)、病毒及微生物感染症、疟疾(malaria)、子痫前症(pre-eclampsia)、高胆红素血症(hyperbilirubinemia)及瘤变(neoplasia)等。
鉴于此,本发明提供一种包含本发明中多肽作为有效成分且由斯科特综合症(Scottsyndrome)、抗磷脂综合症(antiphospholipid syndrome)、镰状细胞性贫血(sickle cellanemia)、地中海型贫血(thalathemia)、口形红细胞增多症(stomatocytosis)、尿毒症(uremia)、肾结石病(kidney stone disease)、糖尿病(diabetes)、血糖过高症(hyperglycemia)、病毒及微生物感染症、疟疾(malaria)、子痫前症(pre-eclampsia)、高胆红素血症(hyperbilirubinemia)及瘤变(neoplasia)组成的群中选择且用于诊断病症的组成物。
而且,本发明还提供一种由斯科特综合症(Scott syndrome)、抗磷脂综合症(antiphospholipid syndrome)、镰状细胞性贫血(sickle cell anemia)、地中海型贫血(thalathemia)、口形红细胞增多症(stomatocytosis)、尿毒症(uremia)、肾结石病(kidneystone disease)、糖尿病(diabetes)、血糖过高症(hyperglycemia)、病毒及微生物感染症、疟疾(malaria)、子痫前症(pre-eclampsia)、高胆红素血症(hyperbilirubinemia)及瘤变(neoplasia)组成的群中选择且用于诊断病症的本发明中多肽的用途。
有益效果
如上所述,本发明中具有序列号码为1的氨基酸序列的多肽以特异方式与磷脂酰丝氨酸结合。因此,本发明中多肽可以广泛应用于检测磷脂酰丝氨酸乃至检测将磷脂酰丝氨酸异位表达在细胞表面的凋亡细胞及肿瘤细胞或者成像等。
附图简要说明
图1图示了在噬菌体库筛选对于磷脂酰丝氨酸特别的噬菌体时其筛选过程(A)、确认上包磷脂酰丝氨酸和磷脂酰胆碱的细胞培养板(B)以及各个筛选步骤中噬菌体滴定量(C)和筛选结果(D)(PC:磷脂酰胆碱、PS:磷脂酰丝氨酸)。
图2核实了被筛选噬菌体和对比群噬菌体库的磷脂酰丝氨酸(噬菌体滴定量(A)、噬菌体ELISA(B))和磷脂酰丝氨酸脂质体(C)的结合特异性以及处理膜联蛋白V时抑制结合(D)(PhageLib.:噬菌体库(对比群)、CLSYYPSYC:本发明中肽的序列)。
图3核实了本发明中多肽与凋亡细胞结合时特异性(A、B、C)以及附着在凋亡细胞的噬菌体对于磷脂酰丝氨酸的特异性(D)(Normal:正常细胞菌、Apoptotic:凋亡细胞群、Conpeptide:对比群肽)。
图4中采用采用FACS(A)、免疫组织化学法(B)和共聚焦显微镜(C)核实了本发明中多肽与凋亡细胞结合时特异性。
图5图示了本发明中多肽对于肿瘤病症部位的活体内(in vivo)引导和成像(A)以及活体外(ex vivo)引导和成像(B)的结果。
图6通过冷冻切片组织的肿瘤血管(A)和凋亡细胞(B)的免疫组织染色核实了本发明中多肽对于肿瘤病症部位的活体内(in vivo)引导。
实施本发明的最佳方式
以下详细说明本发明实施例。
但是,以下实施例只用于说明本发明而非限制本发明。
<实施例1>
细胞培养及细胞培养板的制作
<1-1>细胞培养
本发明使用的H460及H157人类肺癌细胞株和U937白血病细胞株是在包含添加抗生素(青霉素及链霉素)的10%胎牛血清(FBS,Fetal bovine serum)的RMPI 1640培养基进行培养,并每隔3日至4日进行了传代培养。
<1-2>制作上包磷脂酰胆碱或者磷脂酰丝氨酸的细胞培养板
为了制作出上包磷脂酰胆碱或者磷脂酰丝氨酸的细胞培养板(或者细胞),首先,用乙醇溶化磷脂酰胆碱或者磷脂酰丝氨酸之后(3/ml,100),放入96-细胞免疫吸附1B微滴板(Immulon1Bmicrotiter plates,Thermo,Milford,MA,USA)并在空气中干燥6个小时。为了防止非特异性结合,加入包含10mg/ml BSA(bovine serum albumin)的TBS(Tris-HCl 50mM,pH 7.4,NaCl150mM)并在室温环境下堵塞了(blocking)了1个小时。通过以下磨、膜联蛋白V结合测定(annexin V binding test)评估上包通过上述方法制造的磷脂酰胆碱或者磷脂酰丝氨酸的细胞培养板,并选用上包状态好的细胞培养板。
为了膜联蛋白V的结合,采用包含10mg/ml BSA的TBS缓冲剂溶化附着组氨酸的重组膜联蛋白V蛋白质(his-tagged recombinant annexin Vprotein(20/ml))之后,放入上包磷脂酰胆碱或者磷脂酰丝氨酸的ELISA细胞培养板之中。然后,在室温状态下放置1个小时。对其采用TBS-T缓冲剂淘洗3次,使结合的膜联蛋白V蛋白质与上述结合HRP的老鼠抗-组氨酸IgG抗体(Santa Cruz Biotechnology)反应之后采用TBM基质引起反应而进行检测。
其结果,如图1B(上包磷脂酰丝氨酸之后进行测试的结果),本发明使用的细胞培养板与膜联蛋白V的结合状态良好,由此可知,所述细胞培养板的上包状态也良好。上包的细胞培养板用于以下测试当中。
<实施例2>
筛查与磷脂酰丝氨酸的结合的噬菌体
<2-1>筛查与磷脂酰丝氨酸的结合的噬菌体
采用显露溶入pIII的7-mer随机环肽库(random cyclic peptides)的M13噬菌体库(NewEngland Biolabs,Ipswich,MA)筛查通过以下方法与磷脂酰丝氨酸结合的肽。
在包含10mg/ml BSA的TBS缓冲剂(100)里具备2×1011噬斑形成单位(plaque-formingunits,pfu)的所述M13噬菌体库(phage library,10)放入上包磷脂酰胆碱的细胞之中,在室温状态下缓慢搅拌(gentle shaking)并放置1个小时(subtraction step)。然后,将去除(subtracted)附着于磷脂酰胆碱的噬菌体的噬菌体库放入上包磷脂酰丝氨酸的细胞之中,在室温状态下缓慢搅拌并放置1个小时。为了去除没有结合或者结合力弱的噬菌体,对其采用TBS-T(TBS containing 0.05%Tween-20)缓冲剂强力淘洗(washing)10次以上。在室温状态下,采用包含1mg/ml BSA的100的0.2M glycine-HCl(pH 2.2)缓冲剂洗提(elution)10分钟与上包磷脂酰丝氨酸的细胞结合的噬菌体。立即采用1M Tris-HCl(pH 9.1)中和被洗提的噬菌体,并按照制造公司的操作指南,扩增到20ml的ER2738细菌(New England Biolabs公司)(参考图1A)。
扩增之后,采用聚乙二醇/NaCl溶液沉淀培养液上清液中噬菌体之后,检测滴定量(titer)。检测滴定量时,使用包含IPTG和X-gal的LB细胞培养板,并在37℃温度环境下培养一夜之后,计算蓝色菌落的数量。
对于利用检测到的滴定量首次筛选的噬菌体,按照2×1011pfu标准进行进一步的筛选(2次至4次),其筛选过程与上述内容相同。
<2-2>核实对于磷脂酰丝氨酸特异的噬菌体克隆体
为了核实上述实施例<2-1>中对于磷脂酰丝氨酸特异的噬菌体是否得到浓缩(enrichment),在上述实施例<2-1>中各个环节(1次至4次)进行筛选时,连上包磷脂酰胆碱的细胞也包括在其中。而且,相互对比了上包磷脂酰胆碱和磷脂酰丝氨酸的细胞的噬菌体滴定量。
其结果,如图1C所示,对于磷脂酰丝氨酸特异的肽从第二次环节开始得到了浓缩。
<2-3>筛选对于磷脂酰丝氨酸特异的噬菌体
如上述实施例<2-1>,总共通过4个筛选环节筛选出对于磷脂酰丝氨酸特异的噬菌体,并从被筛选的噬菌体克隆体中分离出单链DNA。从至少30个以上噬菌体克隆体分离出DNA,并分析(sequencing)了其碱基序列。将通过碱基序列分析结果得到的氨基酸序列排列成CLUSTAL W算法来确认共同的肽序列(consensus peptide sequence)。为了确认与所述肽的序列之间的同源性强的蛋白质,实施高级局域序列对位排列算法搜索(advanced BLAST search(EMBL/GenBank/DDBJ))功能。
其结果,如图1D所示,主要筛选出了具备CLSYYPSYC的氨基酸共同序列的肽。经核实,与具有所述CLSYYPSYC的氨基酸序列的肽之间的同源性强的人类蛋白质如下表1。
表1
Example of human proteins containing identical amino acid sequences to peptide
<实施例3>
核实被筛选噬菌体对于磷脂酰丝氨酸的结合特异性
<3-1>通过检测噬菌体滴定量核实结合特异性
通过以下噬菌体结合测试(phage binding tests)测定被筛选噬菌体克隆体对于磷脂酰丝氨酸的结合特异性(binding specificity)。
将被筛选噬菌体克隆体(1×109pfu)和被扩增M13噬菌体库(对比群,1×109pfu)放入100的TBS缓冲剂里添加到上包通过所述实施例<1-2>所示方法制造的磷脂酰胆碱或者磷脂酰丝氨酸的细胞之中。然后,缓慢摇晃并在室温状态下放置(incubate)1个小时。对其采用TBS-T(TBS containing 0.05%Tween-20)缓冲剂强力淘洗10次,采用包含1mg/ml BSA的100的0.2Mglycine-HCl(pH 2.2)缓冲剂在室温状态下洗提10分钟与细胞结合的噬菌体,立即采用1M Tris-HCl(pH 9.1)中和被洗提噬菌体之后,分别检测各个被洗提噬菌体的滴定量。
其结果,如图2A所示,对比群M13噬菌体库几乎不与上包磷脂酰胆碱或者磷脂酰丝氨酸的细胞。与此相反,本发明筛选的噬菌体克隆体强烈地与磷脂酰丝氨酸结合。尽管部分被筛选噬菌体克隆体与磷脂酰胆碱结合,可是,比起与磷脂酰丝氨酸之间的结合,结合比率达不到大约5%。由此可知,其以特异方式与磷脂酰丝氨酸结合。
<3-2>通过噬菌体ELISA核实结合特异性
通过以下噬菌体ELISA检测被筛选噬菌体克隆体对于磷脂酰丝氨酸的结合特异性(binding specificity)。
简而言之,如所述实施例<1-2>所示,用磷脂酰胆碱或者磷脂酰丝氨酸上包免疫吸附1B微滴板(Immulon 1B microtiter plates)。然后,在室温状态下,采用包含10mg/ml BSA的TBS培养基堵塞(blocking)1个小时。
将被筛选噬菌体克隆体放入100TBS缓冲剂里添加到上包磷脂酰胆碱或者磷脂酰丝氨酸的细胞之中。然后,将其缓慢摇晃并在室温环境下放置(incubate)1个小时。采用被扩增M13噬菌体库(1×109pfu)作为对比群。对其采用TBS-T(TBS containing 0.05%Tween-20)缓冲剂强力淘洗6次之后,在室温环境下,使与细胞结合的噬菌体和HRP(horseradishperoxidase)-被结合抗-M13抗体(New England Biolabs)(dilution,1∶4000 in TBS-T)结合1个小时并进行检测。然后,采用HRP基质溶液(TMB,Pierce)引起显色反应。添加2N H2SO4中断显色反应并利用酶标仪(microplate reader,Bio-Rad,model 550)在450nm条件下检测吸光度。
其结果,如图2B所示,对比群M13噬菌体库几乎没有检测出磷脂酰丝氨酸,而被筛选噬菌体库以特异方式强烈地与磷脂酰丝氨酸结合。
<3-3>核实对于磷脂酰丝氨酸脂质体的结合特异性
为了了解除了磷脂酰丝氨酸之外的磷脂酰丝氨酸脂质体中本发明肽是否也具有结合特异性,按照以下方法,在上包磷脂酰丝氨酸脂质体的微型板实施噬菌体ELISA。
为了制造出磷脂酰胆碱或者磷脂酰胆碱/磷脂酰丝氨酸脂质体(50∶50Mol.%),利用公知方法(Oka,K.et al.,Proc.Natl.Acad.Sci.USA.95:9535-9540,1998;Fadok,V.A.et al.,Nature 405:85-90,2000)以如下方法制造。更具体地讲,在真空状态下干燥三氯甲烷中将磷脂酰胆碱或者磷脂酰丝氨酸和磷脂酰胆碱分别以50∶50的摩尔比混合的混合物之后在TBS缓冲剂进行水化处理(hydrate),并在冰冻(ice)状态下进行5至10分钟的超声波处理(sonication)。
对于免疫吸附1B微滴板,在4℃温度环境下,采用100的脂质体(20/ml)上包一夜。在室温状态下,采用包含10mg/ml BSA的TBS培养液堵塞(blocking)之后,将被筛选噬菌体克隆体放入100TBS缓冲剂里添加到上包的细胞之中。然后,将其缓慢摇晃并室温状态下放置(incubate)1个小时。采用被扩增M13噬菌体库(1×109pfu)作为对比群。对其采用TBS-T(TBS containing 0.05%Tween-20)缓冲剂强力淘洗6次之后,在室温状态下,使与细胞结合的噬菌体和HRP(horseradish peroxidase)-被结合抗-M13抗体(New England Biolabs)(dilution,1∶4000in TBS-T)结合1个小时并进行检测。然后,采用HRP基质溶液(TMB,Pierce)引起显色反应。添加2N H2SO4中断显色反应并利用酶标仪(microplate reader,Bio-Rad,model 550)在450nm条件下检测吸光度。
其结果,如图2C所示,对比群M13噬菌体库几乎没有检测出磷脂酰丝氨酸,而被筛选噬菌体库以特异方式强烈地与磷脂酰丝氨酸结合,这与实施例<3-2>的结果相同。
<3-4>通过处理膜联蛋白V核实与磷脂酰丝氨酸之间的抑制组合
在存在膜联蛋白V(annexin V)的状态下,通过核实是否与磷脂酰丝氨酸结合而检测被筛选噬菌体克隆体对于磷脂酰丝氨酸的特异性。
简言之,按照所述实施例<1-2>内容,用磷脂酰丝氨酸上包免疫吸附1B微滴板(Immulon 1Bmicrotiter plates),然后,在室温状态下,采用包含10mg/ml BSA的TBS培养基堵塞(blocking)1个小时。
将被筛选噬菌体克隆体放入100TBS缓冲剂里添加到上包磷脂酰丝氨酸的细胞之中,缓慢摇晃并在室温下放置(incubate)1个小时。添加膜联蛋白V的群中,在存在膜联蛋白V蛋白质(10nM)的情况下培养所述噬菌体克隆体之后,与上述方法相同,放入100TBS缓冲剂里添加到上包磷脂酰丝氨酸的细胞之中。然后,将其缓慢地摇晃并在室温下放置1个小时。对其采用TBS-T(TBS containing 0.05%Tween-20)缓冲剂强力淘洗6次之后,在室温环境下,使与细胞结合的噬菌体和HRP(horseradish peroxidase)-被结合抗-M13抗体(New England Biolabs)(dilution,1∶4000in TBS-T)结合1个小时并进行检测。然后,采用HRP基质溶液(TMB,Pierce)引起显色反应。采用2N H2SO4中断显色反应并利用酶标仪(microplate reader,Bio-Rad,model 550)在450nm条件下检测吸光度。
其结果,如图2D所示,添加膜联蛋白V时,很大程度上抑制本发明中肽与磷脂酰丝氨酸之间的结合。
<实施例4>
核实对于凋亡细胞的结合特异性
<4-1>核实噬菌体对于凋亡细胞的结合
通过噬菌体斑法(phage plaque assay)了解被筛选噬菌体克隆体是否与各种凋亡细胞(apoptotic cells)结合。对于H460细胞、H157细胞和U937细胞处理依托泊苷(etoposide,50uM,Sigma)并引导细胞凋亡(apoptosis)。上述处理过程对于U937细胞进行4个小时,对于H460细胞和H157细胞进行18个小时。
为了核实凋亡细胞表面是否显露磷脂酰丝氨酸分子,根据制造公司的指南,用膜联蛋白V(BD Biosciences公司)染色之后进行FACS分析(fluorescence activated cell sortinganalyses)。
分析噬菌体结合时,用PBS淘洗凋亡细胞之后放入包含10mg/ml BSA的DMEM培养基并在室温中预培养30分钟。预培养的细胞里放入被扩增噬菌体库(对比群)或者被筛选噬菌体克隆体1×109pfu的量缓慢摇晃且在4℃培养1个小时。对于没有被结合的噬菌体,采用包含10mg/ml的BSA和包含0.05%双-20(Tween-20)的DMEM培养基强力淘洗。对于被结合的噬菌体,在室温状态下采用包含1mg/ml BSA的1ml 0.2M甘氨酸-HCl(pH 2.2)处理10分钟而洗提。对于被洗提噬菌体,立即采用1M三羟甲基氨基甲烷-HCl(pH 9.1)进行中和之后,检测噬菌体滴定量。
其结果,如图3A至图3C所示,H460细胞(图3A)、H157细胞(图3B)及U937细胞(图3C)表面的磷脂酰丝氨酸分子显露状况良好(请参考图3A至图3C右侧板面),对比群M13噬菌体库几乎没有结合。本发明筛选的噬菌体几乎不与正常细胞结合,可与凋亡细胞之间的结合状态良好。
<4-2>核实对于凋亡细胞的结合特异性
通过核实与膜联蛋白V之间是否发生竞争性抑制来检测附着在凋亡细胞的噬菌体对于磷脂酰丝氨酸的特异性。
为此,使用将凋亡细胞(H460细胞)与噬菌体一同培养之前用膜联蛋白V(10)预培养30分钟的群和不采用膜联蛋白预培养的对比群。对于噬菌体,如所述实施例<4-1>所示内容进行结合和洗提而检测滴定量。
而且,根据被筛选噬菌体克隆体中出现的肽序列,即,CLSYYPSYC的氨基酸序列,检测处理被合成肽(50)的群以及用未处理群和对比群肽进行处理的对比群。对于噬菌体,如所述实施例<4-1>所示内容进行结合和洗提而检测滴定量。
其结果,如图3D所示,预处理膜联蛋白V时,会抑制噬菌体的结合而大幅减少噬菌体滴定量(左侧板面)。而且,合成肽的未处理群和对比群肽处理群之间不存在太大的差异。与此相比,处理合成的本发明中肽时,会抑制噬菌体的结合而大幅减少噬菌体滴定量(右侧板面)。
<4-3>通过FACS分析核实本发明中肽对于凋亡细胞的结合
以下,在处理标记的本发明中肽之后,通过FACS分析核实本发明中肽是否与凋亡细胞的磷脂酰丝氨酸结合。
本发明使用的肽在N-末端结合荧光素(fluorescein)且按照Fmoc方法合成,并采用HPLC(Peptron Co.)进行分离。
本发明中肽或者对比群肽(氨基酸序列:NSSVDK)用最终浓度为2.5的Hepes缓冲剂(pH7.4,Hepes 10mM NaCl2 138mM,and with/without CaCl2)进行溶解,与正常H460细胞或者凋亡H460细胞(1×105cells/ml)一起,以最终体积为0.5ml的状态下,在室温环境培养15分钟。对于所述细胞,用结合缓冲剂淘洗之后,立即实施FACS分析(FACSScan,Becton Dickinson,San Jose,CA)。
其结果,如图4A所示,比起对于正常细胞处理本发明中肽时(左侧板面)和对于凋亡细胞处理对比群肽时(右侧板面),对于凋亡细胞处理本发明中肽时,本发明中肽更积极地与凋亡细胞结合。
<4-4>利用免疫组织化学法核实本发明中肽对于凋亡细胞的结合
在小室载玻片(Nalgen Nunc Int.)培养H460细胞,且按照所述实施例<4-1>所示内容,引导细胞凋亡。用PBS淘洗凋亡细胞之后,在台氏生理盐溶液缓冲液(tyrodes buffer)里与用10μM荧光素标记的肽一起在室温环境培养30分钟。然后,在室温环境下,用膜联蛋白V荧光二抗594(annexin V Alexa fluor 594,Molecular Probes公司))培养15分钟该细胞。用PBS淘洗细胞之后,用4%聚合甲醛加固5分钟。然后,采用核染剂4‘6’-二脒基-2-苯吲哚盐酸(4’,6-diamidino-2-phenylindole,DAPI)进行复染(counterstain),处理封固液(MolecularProbes公司),并用荧光显微镜(Zeiss,Oberkochen,Germany)进行拍摄。
其结果,如图4B所示,对于正常细胞处理本发明中肽或者膜联蛋白V时不出现标记(下端板面)。如果对于凋亡细胞处理对比群肽或者膜联蛋白V,只有处理膜联蛋白V时出现标记(中端板面)。与此相比,对于凋亡细胞,处理本发明中肽或者膜联蛋白V时,两种情况都出现标记。利用电脑程序合成两张照片的结果发现,本发明中肽和膜联蛋白V在相同的部位结合(上端板面)。
而且,如图4C所示,从利用激光共聚焦显微镜(LEICA公司)拍摄的影像中可以更准确地了解到,对于凋亡细胞处理本发明中肽或者膜联蛋白V时,本发明中肽和膜联蛋白V在相同的部位结合。
<实施例5>
本发明中肽的活体内引导(in vivo homing)及成像
<5-1>本发明中肽的活体内引导及成像
所有的动物实验均遵照国立庆北大学的实验指南(guidelines)进行。为了移植肿瘤(tumor xenografts),向6周岁BALB/c雄性裸体老鼠(SLC,Inc.)的右侧肩部皮下注射悬浮在包含10%FBS的RPMI培养基的H460细胞(1×107细胞)之后,使肿瘤细胞在之后的3周间长成0.5至1cm。
将具有肿瘤细胞的老鼠分成喜树碱处理群和未处理群之后,对于喜树碱处理群,在处理肽的24小时之前,处理1次分量的喜树碱(Sigma,10mg/kg)。然后,在异氟醚麻醉状态下,向各个老鼠通过尾巴静脉注射标记荧光素的本发明肽(fluorescein-labeled CLSYYPSYC)或者对比群肽(分别50)。注射肽之后,在各个期间内,采用470nM/GFP滤波器通过光学成像系统(ART Advanced Research Technologies Inc.,Montreal,Canada)确认了引导到肿瘤的肽。注射肽之前检测使用的各个老鼠的荧光素基础数据(basel ine fluorescence)。使用eXploreOptix optiView Software处理取得的影像,以使其成为标准化(normalize)影像。注射肽2小时之后去除老鼠的肿瘤,并实施肿瘤组织的活体外(ex vivo)成像。其影像的处理方法与上述内容相同。
核实肿瘤组织活体内引导的结果,如图5A所示,从2个小时开始,处理本发明中肽的群的肿瘤组织可以强烈地检测到标记荧光素的本发明中肽的信号,可是,没有处理本发明中肽或者处理对比群肽时,可以微弱地检测到荧光素信号或者几乎没有检测到荧光素信号。另外,对于肿瘤组织实施活体外成像的结果,如图5B所示,上述已处理本发明中肽的群的肿瘤组织可以强烈地检测到荧光素信号,可是,没有处理本发明中肽或者处理对比群肽时,可以微弱地检测到荧光素信号或者几乎没有检测到荧光素信号。
<5-2>通过组织学试验核实本发明肽的活体内引导
为了实施组织学试验,麻醉上述老鼠之后开腹。通过心脏灌注PBS和4%paraformaldehyde(PFA)封固液,并去除肿瘤组织及器官。冰冻切割(Cryosections)各个组织之后,通过荧光显微镜法(fluorescence microscopy)观察本发明中肽。肿瘤血管(tumorvessels)是利用老鼠CD31的抗体(BD Pharmigen)和标记为荧光568(alexa 568)的2次抗体通过免疫组织化学法(immunohistochemistry)染色。肿瘤组织中凋亡细胞是根据制造公司(Chemicon Int.USA)的指南通过TUNEL(in vitro terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling)分析法加以核实。
其结果,如图6所示,处理本发明中肽时,从肿瘤组织中由CD31染色的部位观察到大量的本发明中肽(上端板面),但是,处理对比群肽时,几乎没有观察到本发明中肽(中端板面)。另外,对比群器官肝脏(liver)及肺部(lung)组织中没有观察到本发明中肽,可是,由于肽通过小便排到体外,从肾脏(kidney)观察到位于尿路的肽发出的荧光(下端板面)。
而且,据TUNEL分析结果,如图6B所示,从TUNEL染色部位,即,发生细胞凋亡的部位观察到大量的本发明中肽的荧光信号。
本发明的有益效果在于:
综上所述,本发明中多肽以特异方式与磷脂酰丝氨酸结合,所以,本发明中多肽可以广泛应用于磷脂酰丝氨酸的检测,进一步广泛应用于将磷脂酰丝氨酸异位表达在细胞表面的凋亡细胞及肿瘤细胞的检测或者成像等。
Claims (23)
1.具有序列号码为1的氨基酸序列且以特异方式与磷脂酰丝氨酸结合的多肽。
2.具有加密权利要求1所述多肽的碱基序列的多聚核苷酸。
3.包含权利要求2所述多聚核苷酸的载体。
4.基因转殖成权利要求3所述载体的基因转殖体。
5.包含权利要求1所述多肽作为有效成分的用于检测磷脂酰丝氨酸的组成物。
6.根据权利要求5所述的组成物,其特征在于:所述多肽采用由显色酶、放射形同位素、发色团(chromophore)、发光物质及荧光物质(fluorescer)组成的群中选择的一种做标记。
7.一种磷脂酰丝氨酸检测方法,包括以下步骤:
(a)将权利要求1所述多肽与试剂混合的步骤;
(b)去除未结合或者以非特异方式结合的所述多肽的步骤;以及
(c)核实所述多肽是否结合及位置的步骤。
8.包含权利要求1所述多肽作为有效成分的用于检测凋亡细胞(apoptotic cell)的组成物。
9.包含权利要求1所述多肽作为有效成分的用于传达药物的组成物。
10.根据权利要求9所述的组成物,其特征在于;所述组成物对于肿瘤病症具有特异性。
11.根据权利要求10所述的组成物,其特征在于:所述肿瘤病症是由大肠癌、肺癌、胃癌、食道癌、胰腺癌、胆囊癌、肾癌、膀胱癌、前列腺癌、睾丸癌、子宫颈癌、子宫内膜癌、绒毛癌、卵巢癌、乳房癌、甲状腺癌、脑癌、头颈部癌、恶性黑色素瘤、皮肤癌、肝癌、白血病(leukemia)、淋巴瘤(lymphoma)、多发性骨髓瘤(multiple myeloma)、慢性骨髓性白血病(chronic myelogenous leukemia)、神经母细胞瘤(neuroblastoma)及再生障碍性贫血组成的群中选择的一种。
12.根据权利要求9所述的组成物,其特征在于:所述多肽与由紫杉醇、阿霉素、长春新碱、柔红霉素(daunorubicin)、长春花碱(vinblastine)、放线菌素-D(actinomycin-D)、多烯紫衫醇(docetaxel)、依托泊苷(etoposide)、替尼泊苷(teniposide)、比生群(bisantrene)、高三尖杉酯碱(homoharringtonine)、格里维克(Gleevec;STI-571)、顺铂(cisplain)、5-氟尿嘧啶(5-fluouracil)、亚德里亚霉素(adriamycin)、甲氨蝶呤(methotrexate)、白消安(busulfan)、苯丁酸氮芥(chlorambucil)、环磷酰胺(cyclophosphamide)、美法仑(melphalan)、氮芥(nitrogen mustard)及亚硝基尿素(nitrosourea)组成的群中选择的抗肿瘤制剂结合。
13.包含权利要求1所述多肽以及与其结合的抗肿瘤制剂作为有效成分的用于防治肿瘤病症的药物性组成物。
14.根据权利要求13所述的组成物,其特征在于:所述抗肿瘤制剂是由紫杉醇、阿霉素、长春新碱、柔红霉素(daunorubicin)、长春花碱(vinblastine)、放线菌素-D(actinomycin-D)、多烯紫衫醇(docetaxel)、依托泊苷(etoposide)、替尼泊苷(teniposide)、比生群(bisantrene)、高三尖杉酯碱(homoharringtonine)、格里维克(Gleevec;STI-571)、顺铂(ci splain)、5-氟尿嘧啶(5-fluouracil)、亚德里亚霉素(adriamycin)、甲氨蝶呤(methotrexate)、白消安(busulfan)、苯丁酸氮芥(chlorambucil)、环磷酰胺(cyclophosphamide)、美法仑(melphalan)、氮芥(nitrogen mustard)及亚硝基尿素(nitrosourea)组成的群中选择的。
15.包含权利要求1所述多肽作为有效成分的用于成像患肿瘤部位的组成物。
16.根据权利要求15所述的组成物,其特征在于:所述多肽采用由显色酶、放射形同位素、发色团、发光物质或者荧光物质(fluorescer)、超顺磁性纳米粒子(super paramagneticparticles)及超小型顺磁性纳米粒子(ultrasuper paramagnetic particles)组成的群中选择的一种做标记。
17.包含权利要求1所述的多肽作为有效成分且由斯科特综合症(Scott syndrome)、抗磷脂综合症(antiphospholipid syndrome)、镰状细胞性贫血(sickle cell anemia)、地中海型贫血(thalassemia)、口形红细胞增多症(stomatocytosis)、尿毒症(uremia)、肾结石病(kidney stone disease)、糖尿病(diabetes)、血糖过高症(hyperglycemia)、病毒及微生物感染症、疟疾(malaria)、子痫前症(pre-eclampsia)、高胆红素血症(hyperbilirubinemia)及瘤变(neoplasia)组成的群中选择并用于诊断病症的组成物。
18.检测磷脂酰丝氨酸时权利要求1所述多肽的用途。
19.检测凋亡细胞(apoptotic cell)时权利要求1所述多肽的用途。
20.传达药物时权利要求1所述多肽的用途。
21.制造治疗肿瘤制剂时权利要求1所述多肽以及与其结合的抗肿瘤制剂的用途。
22.成像患肿瘤部位时权利要求1所述多肽的用途。
23.由斯科特综合症(Scott syndrome)、抗磷脂综合症(antiphospholipid syndrome)、镰状细胞性贫血(sickle cell anemia)、地中海型贫血(thalassemia)、口形红细胞增多症(stomatocytosis)、尿毒症(uremia)、肾结石病(kidney stone disease)、糖尿病(diabetes)、血糖过高症(hyperglycemia)、病毒及微生物感染症、疟疾(malaria)、子痫前症(pre-eclampsia)、高胆红素血症(hyperbilirubinemia)及瘤变(neoplasia)组成的群中选择的用于诊断病症的权利要求1所述多肽的用途。
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Publication number | Priority date | Publication date | Assignee | Title |
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CN106660992A (zh) * | 2014-05-22 | 2017-05-10 | 美泰康公司 | 二甲基吡啶胺衍生物及其医药用途 |
CN106660992B (zh) * | 2014-05-22 | 2021-01-08 | 美泰康公司 | 二甲基吡啶胺衍生物及其医药用途 |
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Publication number | Publication date |
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JP5055438B2 (ja) | 2012-10-24 |
KR100968839B1 (ko) | 2010-07-09 |
WO2009107971A2 (ko) | 2009-09-03 |
CN101965356B (zh) | 2014-03-26 |
US8133971B2 (en) | 2012-03-13 |
JP2011515075A (ja) | 2011-05-19 |
KR20090091589A (ko) | 2009-08-28 |
EP2280024A4 (en) | 2011-03-30 |
EP2280024B1 (en) | 2014-12-17 |
WO2009107971A3 (ko) | 2009-11-26 |
US20090214430A1 (en) | 2009-08-27 |
EP2280024A2 (en) | 2011-02-02 |
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