CN102811739A - T细胞活化抑制剂、含有其的药物组合物以及抑制t细胞活化的物质的筛选方法 - Google Patents
T细胞活化抑制剂、含有其的药物组合物以及抑制t细胞活化的物质的筛选方法 Download PDFInfo
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Abstract
本发明提供了T细胞活化抑制剂,其以抗RGM中和抗体等的RGM抑制物质作为有效成分。所述T细胞活化抑制剂作为多发性硬化症等的自身免疫病和/或其它的由于T细胞活化所引起的疾病的预防或治疗用药物组合物是有用的。另外,通过将受试物质与RGM接触,选择使RGM的活性水平降低的受试物质,能够筛选抑制T细胞活化的物质。
Description
技术领域
本发明涉及T细胞活化抑制剂、含有其的药物组合物以及抑制T细胞活化的物质的筛选方法,具体而言,本发明涉及以RGM抑制物质作为有效成分的T细胞活化抑制剂、含有该T细胞活化抑制剂的用于预防或治疗T细胞活化所引起的疾病的药物组合物、以及抑制T细胞活化的物质的筛选方法,所述筛选方法包括使受试物质与RGM接触的步骤。
背景技术
RGM(排斥性导向分子(repulsive guidance molecule))最初是被鉴定为视觉系统的轴突导向分子的膜蛋白质(参照非专利文献1)。已知RGM家族包含称为RGMa、RGMb以及RGMc的3类成员,至少RGMa和RGMb通过相同的信号传导机制发挥作用(参照非专利文献2)。后续的研究明确了RGM在非洲爪蟾和鸡胚中具有轴突导向和层形成(laminaformation)的功能、以及在小鼠胚中具有调节头部神经管的闭锁等功能(参照非专利文献3)。除了在发育阶段的功能外,RGM还在成年人和大鼠的中枢神经系统损伤后再表达,在大鼠中抑制RGM提高了脊髓损伤后的轴突生长、促进了功能恢复(参照非专利文献4),因此认为RGM是中枢神经系统损伤后抑制轴突再生的物质。另外,专利文献1公开了含有以抗RGM中和抗体为有效成分的轴突再生促进剂。
多发性硬化症(Multiple Sclerosis)是由于覆盖脑和脊髓的神经纤维的髓鞘质(髓鞘)发炎引起脱髓,神经的信息不能很好地传导导致出现视觉障害、运动障害、感觉低下、平衡障害等多种症状的疾病,其原因尚未清楚地阐明,且在现代医学中是不能完全治愈的慢性病。虽然多发性硬化症作为一种自身免疫病而认识,但是尚未详细阐明其发病机理。例如,非专利文献5报导了CD4+T细胞免疫地攻击脑以及脊髓的白质中的髓鞘质以及少突胶质细胞。
现有技术文献
专利文献
专利文献1:专利第3981148号公报
非专利文献
非专利文献1:Stahl,B.,Muller,B.,von Boxberg,Y.,Cox,E.C.&Bonhoeffer,F.Biochemical characterization of a putative axonal guidancemolecule of the chick visual system.Neuron 5,735-743(1990)
非专利文献2:Liu,X.,Hashimoto,M.,Horii,H.,Yamaguchi,A.,Naito,K.和Yamashita,T.,Repulsive guidance molecule b inhibits neuritegrowth and is increased after spinal cord injury.Biochem.Biophys.Res.Commun.382,795-800(2009)
非专利文献3:Yamashita,T.,Mueller,B.K.&Hata,K.,Neogenin andrepulsive guidance molecule signaling in the central nervous system.Curr.Opin.Neurobiol.17,29-34(2007)
非专利文献4:Hata,K.等人,RGMa inhibition promotes axonalgrowth and recovery after spinal cord injury.J.Cell Biol.173,47-58(2006)
非专利文献5:Trapp,B.D.,Ransohoff,R.M.,Fisher,E.&Rudick,R.Neurodegeneration in multiple sclerosis:relationship to neurologicaldisability.Neuroscientist 5,48-57(1999)
发明概述
发明欲解决的课题
本发明的目的是,发现RGM的新功能,提供新的T细胞活化抑制剂、提供用于预防或治疗T细胞活化所引起的疾病的药物组合物、抑制T细胞活化的物质的筛选方法。
用于解决课题的手段
本发明为了解决上述课题,包含以下各发明。
[1]T细胞活化抑制剂,其以RGM抑制物质作为有效成分。
[2]上述[1]所述的T细胞活化抑制剂,其中RGM抑制物质是抗RGM中和抗体。
[3]上述[1]所述的T细胞活化抑制剂,其中RGM抑制物质是RGMsiRNA。
[4]T细胞活化所引起的疾病的预防或治疗用药物组合物,其特征在于,含有上述[1]~[3]中任一项所述的T细胞活化抑制剂。
[5]上述[4]所述的药物组合物,其中T细胞活化所引起的疾病是自身免疫病。
[6]上述[5]所述的药物组合物,其中自身免疫病是多发性硬化症。
[7]抑制T细胞活化的物质的筛选方法,其特征在于包括使受试物质与RGM接触的步骤、测定所述RGM的活性水平的步骤、将所述活性水平与没有接触受试物质的RGM的活性水平进行比较的步骤、和选择使RGM的活性水平降低的受试物质的步骤。
[8]抑制T细胞活化的方法,其特征在于,包括使RGM抑制物质与RGM表达细胞接触的步骤。
[9]上述[8]所述的抑制T细胞活化的方法,其中RGM抑制物质是抗RGM中和抗体。
[10]上述[8]所述的抑制T细胞活化的方法,其中RGM抑制物质是RGM siRNA。
[11]预防或治疗自身免疫病的方法,其特征在于包括将有效量的上述[1]~[3]中任一项所述的T细胞活化抑制剂施与哺乳动物。
[12]上述[1]~[3]中任一项所述的T细胞活化抑制剂的用途,用于制备自身免疫病的预防或治疗用药物组合物。
[13]上述[1]~[3]中任一项所述的T细胞活化抑制剂,用于预防或治疗自身免疫病。
发明的效果
通过本发明,能够提供新的T细胞活化抑制剂。所述T细胞活化抑制剂用于预防或治疗T细胞活化所引起的疾病,例如自身免疫病。另外,通过本发明的筛选方法得到的抑制T细胞活化的物质成为T细胞活化所引起的疾病的预防药或治疗药的有效成分的候选。
附图简述
图1是显示通过实时定量PCR测定用LPS刺激的骨髓来源的树状细胞(BMDCs)中RGMa mRNA表达的结果的图。
图2是显示通过蛋白质印迹检测用LPS刺激的BMDCs中RGMa的表达结果的图。
图3是显示通过流式细胞术分析CD4+T细胞以及CD11b+巨噬细胞与各种浓度的人RGMa-Fc的结合的结果图。
图4(A)是显示通过蛋白质印迹检测CD4+T细胞以及CD11b+巨噬细胞中总Rap1以及活性型Rap1的结果图。
图4(B)是显示将图4(A)的蛋白质印迹条带用Scion image定量,算出相对Rap1活化水平的结果图。
图5(A)是显示在有或没有RGMa刺激下,测定脾细胞向纤连蛋白的粘附率的结果图。
图5(B)是显示在有或没有RGMa刺激下,测定CD4+T细胞向ICAM1的粘附率的结果图。
图6是显示在施与抗RGMa中和抗体的多发性硬化症模型小鼠和施与对照抗体的多发性硬化症模型小鼠中,通过评分实验性自身免疫性脑脊髓炎(EAE)的临床症状而数值化、观察随时间变化的结果图。
图7(A)是显示在每组中EAE的临床症状发生率的图;(B)是显示在每组中EAE发病日的平均值的图;(C)是显示每组中各小鼠的EAE评分最大值的平均值的图;(D)是显示每组中各小鼠的EAE评分累积值的平均值的图。
图8(a)以及(b)是施与对照抗体的多发性硬化症模型小鼠的颈髓的HE染色组织图像;(c)以及(d)是施与抗RGMa中和抗体的多发性硬化症模型小鼠的颈髓的HE染色组织图像。
图9是显示在施与对照抗体的多发性硬化症模型小鼠的骨髓组织标本以及施与抗RGMa中和抗体的多发性硬化症模型小鼠的骨髓组织标本中,基于炎症指数而将炎症程度数值化的结果的图。
图10是显示自施与对照抗体的多发性硬化症模型小鼠以及施与抗RGMa中和抗体的多发性硬化症模型小鼠制备的脾细胞,研究其抗原特异的T细胞活化以及非特异的T细胞活化的结果的图,(a)是显示细胞增殖的结果的图,(b)是显示IL-2的分泌量的结果的图,(c)是显示IFN-γ的分泌量的结果的图,(d)是显示IL-17的分泌量的结果的图。
图11是显示在施与抗RGMa中和抗体的再发性多发性硬化症模型小鼠和施与对照抗体的多发性硬化症模型小鼠中,通过评分EAE的临床症状而数值化、观察随时间变化的结果图。
图12是显示对敲低了RGMa基因的BMDCs或没有敲低RGMa基因的BMDCs进行MOG刺激后,将这些BMDCs移植至接受体小鼠,在接受体小鼠中通过评分EAE的临床症状而数值化、观察随时间变化的结果图。
图13是显示对自施与了抗RGMa中和抗体或对照抗体、以及MOG的供体小鼠制备的CD4+T细胞,进行MOG刺激后,将这些CD4+T细胞移植至接受体小鼠,在接受体小鼠中通过评分EAE的临床症状而数值化、观察随时间变化的结果图。
用于实施发明的实施方案
本发明者们首次发现骨髓来源的树状细胞(BMDCs)表达RGM;CD4+T细胞以及CD11b+巨噬细胞表达RGM受体;RGM通过与CD4+T细胞或CD11b+巨噬细胞的RGMa受体结合而活化Rap1,其结果是发现CD4+T细胞或CD11b+巨噬细胞的细胞粘附活性增强。另外,本发明者们明确了,在使用多发性硬化症模型小鼠的实验中,施与抗RGM中和抗体减轻了髓鞘少突胶质细胞糖蛋白(MOG)诱导的多发性硬化症模型小鼠(实验性自身免疫性脑脊髓炎(EAE)小鼠)的临床症状以及组织病变这两者、自施与抗RGM中和抗体的多发性硬化症模型小鼠采取的脾细胞显著减轻了抗原特异的以及非特异的T细胞活化。进一步地,发现了抗RGM中和抗体的施与在髓鞘蛋白脂质蛋白(PLP)诱导的再发性多发性硬化症模型小鼠中抑制EAE临床症状的出现;对敲低了RGMa的BMDCs进行MOG刺激并移植至小鼠的情形,在接受移植的小鼠中抑制EAE临床症状的出现;在自施与了抗RGM中和抗体以及MOG的小鼠制备的CD4+T细胞给予MOG刺激并移植至小鼠的情形,在接受移植的小鼠中抑制EAE临床症状的出现(参照实施例)。
〔T细胞活化抑制剂〕
本发明提供了以RGM抑制物质作为有效成分的T细胞活化抑制剂。作为本发明的T细胞活化抑制剂的有效成分的RGM抑制物质可以是任一抑制RGM的活性(RGM活性)的物质和抑制RGM表达的物质。
作为抑制RGM活性的物质,可以列举例如抑制RGM活性的低分子化合物、抗RGM中和抗体等。作为RGM活性的指标,可以列举例如RGM向RGM受体的结合活性(参照实施例1的(1-2))、Rap1活化的诱导活性(参照实施例1的(1-3))、T细胞对ICAM的粘附增强活性(参照实施例1的(1-4))等,使用这些指标评价时抑制(减弱)RGM活性的物质称为RGM抑制物质。作为抑制RGM活性的低分子化合物,可以列举例如作为Rho激酶抑制剂而已知的Y27632(M.Uehata等人,Calciumsensitization of smooth muscle mediated by a Rho-associated proteinkinase in hypertension.Nature 389,990(1997))。
抗RGM中和抗体可以是结合RGM并抑制其活性的抗体,例如,可以列举结合RGM,使RGM不能结合RGM受体的抗RGM抗体等。能够使用RGM或其片段作为免疫原,以公知的方法制备抗RGM中和抗体。能够使用上述RGM活性的指标确认所获得的抗体是中和抗体。作为RGM,可以列举例如包含序列编号2所示的氨基酸序列的人RGMa、包含序列编号4所示的氨基酸序列的大鼠RGMa等,但不限于此。能够合适地使用多种生物来源的RGM作为免疫原。这些氨基酸序列能够容易地获自公知的数据库(Protein Data Bank等)。
抗RGM中和抗体可以是多克隆抗体,也可以是单克隆抗体。另外,其可以是完全的抗体分子,也可以是能够与抗原特异性结合的抗体片段(例如,Fab、F(ab’)2、Fab’、Fv、scFv等)。多克隆抗体例如能够如下制备并获得。即,将抗原(RGM或它们的片段)溶解在PBS中,根据需要将适量混合了常规佐剂(例如弗氏完全佐剂)的物质作为免疫原免疫哺乳动物(小鼠、大鼠、兔、山羊、马等)。对免疫方法没有特别的限定,但是例如优选皮下注射或腹腔内注射1次或以适当的间隔注射多次的方法。接下来,根据常规方法,能够通过从经免疫的动物采集血液,分离血清,纯化多克隆抗体级分而取得。单克隆抗体是将上述经免疫的哺乳动物得到的免疫细胞(例如脾细胞)与骨髓瘤细胞融合得到杂交瘤,能够通过自该杂交瘤的培养物采集抗体而获得。另外,从杂交瘤中克隆抗体基因,整合入适当的载体,将所述载体导入宿主细胞,使用基因重组技术能够产生重组型的单克隆抗体。进一步地,也能够使用噬菌体展示法来制备。
抗RGM中和抗体优选是人型嵌合抗体或人源化抗体。人型嵌合抗体是由人以外的动物来源的抗体的重链可变区以及轻链可变区、和人抗体的重链恒定区以及轻链恒定区组成的抗体。人源化抗体是指将人以外的动物来源的抗体的CDR(互补决定区:complementarity determining region)移植至人抗体的CDR的抗体,也称为CDR移植抗体、重构抗体等。将人源化抗体的FR(构架区:framework region)选择为使CDR形成良好的抗原结合部位的FR。为了使人源化抗体的CDR形成合适的抗原结合部位,根据需要也可以替换抗体可变区中FR的氨基酸序列。人抗体的恒定区的氨基酸序列能够从公知的数据库(Protein Data Bank等)获得。
作为抑制RGM表达的物质,可以列举例如RGM基因的siRNA(短干扰RNA)、shRNA(短发夹RNA)、反义寡核苷酸等。作为RGM基因,可以列举例如包含序列编号1所示碱基序列的人RGMa基因、包含序列编号3所示碱基序列的大鼠RGMa基因等,但不限于此。能够容易地从公知数据库(GenBank等)获得多种生物来源的RGM基因的碱基序列。siRNA是能够抑制目标基因(本发明中是RGM基因)表达的短的双链RNA。只要作为siRNA发挥功能,对其碱基序列和/或长度(碱基长)无特别限定,但优选地是不到约30个碱基、更优选地是约19~27个碱基、进一步优选地是约21~25个碱基。shRNA是指通过在单链RNA中包含部分回文状的碱基序列,形成分子内的双链结构,在3′末端具有突出部的短的发夹结构的约20个碱基对以上的分子。这样的shRNA在导入细胞内后,在细胞内分解成约20个碱基(代表性地为例如21个碱基、22个碱基、23个碱基)的长度、与siRNA同样地能够抑制目标基因(本发明中是RGM基因)的表达。只要是能够抑制RGM基因的表达,siRNA以及shRNA可以是任何形式。siRNA或shRNA能够人工地化学合成。另外,例如使用T7RNA聚合酶以及T7启动子,能够自模板DNA体外合成反义以及有义的RNA。反义寡核苷酸可以是与RGM基因的DNA序列中连续5个至100个碱基序列互补的或杂交的核苷酸、是DNA或RNA均可。另外,只要不阻碍功能,也可以是经修饰的。能够通过常规方法合成反义寡核苷酸,例如,通过市售的DNA合成装置能够容易地合成。
因此,RGM抑制物质通过与RGM表达细胞(表达RGM的细胞或具有表达RGM能力的细胞)接触,能够抑制T细胞活化。因此,本发明也包含抑制T细胞活化的方法,其特征在于包括使RGM抑制物质与RGM表达细胞接触的步骤。
〔药物组合物〕
本发明提供了对T细胞活化所引起的疾病的预防或治疗用药物组合物,其包含上述本发明的T细胞活化抑制剂。本发明的药物组合物换言之是以RGM抑制物质作为有效成分的、用于预防或治疗T细胞活化所引起的疾病的药物组合物。
作为T细胞活化所引起的疾病,可以列举自身免疫病、脑血管病等。作为自身免疫病,可以列举例如细胞性自身免疫病、风湿症、多发性硬化症、神经自身免疫病(急性热病性多神经炎、神经贝切特病等)、恶性贫血、I型(胰岛素依赖型)糖尿病、系统性红斑狼疮(SLE)、炎症性肠疾病(IBD)、斯耶格伦综合征、遗传过敏性皮炎、古德帕斯彻综合征、格雷夫斯病、自身免疫性溶血性贫血、自身免疫性血小板减少性紫癜病、肾小球肾炎、重症肌无力症、桥本病等。作为脑血管病,有脑梗塞、脑出血、蛛网膜下出血等。其中,本发明的药物组合物优选地用于预防或治疗自身免疫病,特别优选地用于预防或治疗多发性硬化症。
本发明的药物组合物能够将作为有效成分的RGM抑制物质与药学上容许的载体或添加剂适宜地混合而制剂化。具体而言,能够制剂为锭剂、包衣锭剂、丸剂、散剂、颗粒剂、胶囊剂、液体剂、悬浮剂、乳剂等经口剂;注射剂、输液、栓剂、软膏、贴剂等非经口剂。关于载体或添加剂的混合比例,基于医药品领域通常采用的范围合适地设定即可。对于能够用来混合的载体或添加剂,没有特别的限制,可以列举例如水、生理盐水、其它水性溶剂、水性或油性基剂等各种载体;赋形剂、结合剂、pH调节剂、崩解剂、吸收促进剂、润滑剂、着色剂、矫味剂、香料等各种添加剂。
当RGM抑制物质是抗RGM中和抗体的情形,优选地将与药学上容许的载体一同制剂的注射剂或输液通过非经口施与途径施与,例如静脉内、肌内、皮内、腹腔内、皮下或局部施与。包含抗RGM中和抗体的注射剂或输液能够作为溶液、悬液或乳液使用。作为其溶剂,能够使用例如注射用蒸馏水、生理盐水、葡萄糖溶液以及等张液(例如,氯化钠、氯化钾、甘油、甘露醇、山梨醇、硼酸、硼砂、丙二醇等溶液)。进一步地,该注射剂或输液可以包含稳定剂、助溶剂、悬浮化剂、乳化剂、无痛化剂、缓冲剂、保存剂、防腐剂、pH调节剂等。作为稳定剂,可以使用例如白蛋白、球蛋白、明胶、甘露醇、葡萄糖、葡聚糖、乙二醇、丙二醇、抗坏血酸、亚硫酸氢钠、硫代硫酸钠、EDTA钠、柠檬酸钠、二丁羟基甲苯等。作为助溶剂,可以使用例如醇(例如,乙醇等)、多元醇(例如,丙二醇、聚乙二醇等)、非离子型表面活性剂(例如,失水山梨醇聚氧乙烯(20)醚油酸酯(注册商标)、HCO-50等)等。作为悬浮化剂,可以使用例如单硬脂酸甘油酯、单硬脂酸铝、甲基纤维素、羧甲基纤维素、羟甲基纤维素、十二烷基硫酸钠等。作为乳化剂,可以使用例如阿拉伯胶、藻酸钠、西黄蓍胶等。作为无痛化剂,可以使用例如苯甲醇、氯丁醇、山梨醇等。作为缓冲剂,可以使用例如磷酸缓冲液、乙酸缓冲液、硼酸缓冲液、碳酸缓冲液、柠檬酸缓冲液、三羟甲基氨基甲烷缓冲液等。作为保存剂,可以使用例如对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯、对羟基苯甲酸丁酯、氯丁醇、苯甲醇、氯化苯甲烃铵、脱氢乙酸钠、乙二胺四乙酸钠、硼酸、硼砂等。作为防腐剂,可以使用例如氯化苯甲烃铵、对羟基苯甲酸、氯丁醇等。pH调节剂,可以使用例如盐酸、氢氧化钠、磷酸、乙酸等。
当RGM抑制物质是核酸(siRNA、shRNA、反义寡核苷酸等)的情形,能够以非病毒载体或病毒载体的形式施用。在非病毒载体形式的情形,能够利用使用脂质体导入核酸分子的方法(脂质体法、HVJ-脂质体法、阳离子脂质体法、脂质转染法、脂质转染胺试剂(1ipofectamine)法等)、微注射法、用基因枪(Gene Gun)将运载体(金属粒子)与核酸分子一同转移入细胞的方法等。将siRNA或shRNA使用病毒载体施与生物的情形时,能够利用重组腺病毒、逆转录病毒等病毒载体。通过使细胞或组织感染重组病毒,能够将基因导入细胞或组织内,其中所述重组病毒是向已无毒化的逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒、痘苗病毒、痘病毒、脊髓灰质炎病毒、辛德比斯病毒、仙台病毒、SV40等DNA病毒或RNA病毒中导入表达siRNA或shRNA的DNA。
通过将由此所获得的制剂以有效量施与例如人或其它哺乳动物(例如,大鼠、小鼠、兔、绵羊、猪、牛、猫、狗、猴等)能够预防或治疗T细胞活化所引起的疾病。对于施与量,通过考虑目的、疾病的严重程度、患者的年龄、体重、性别、过往病史、有效成分的种类等而合适地设定。例如,当有效成分是抗RGM中和抗体的情形,在以具有约65~70kg体重的通常人为对象的情形时,优选地每日施用0.02mg~4000mg左右,更优选地每日施用0.1mg~200mg左右。每日的总施与量可以是单一施与量,也可以是分开的施与量。
〔筛选方法〕
对于本发明的筛选方法,只要所述方法包括使受试物质与RGM接触的步骤、测定与受试物质接触过的RGM的活性水平的步骤、将该测定的活性水平与没有接触受试物质的RGM的活性水平比较的步骤、选择使RGM的活性水平降低的受试物质的步骤即可。通过本发明的筛选方法,能够简便且有效地筛选到抑制T细胞活化的物质。
能够通过例如向RGM溶液中添加受试物质使其溶解或悬浮而实施受试物质与RGM的接触。对于RGM,能够使用市售的重组RGM或根据常规方法自制的重组RGM。对于接触时间、接触温度没有特别的限定,进行适宜选择即可。另外,优选地设定不与受试物质接触的对照组。RGM的活性水平能够以RGM与RGM受体的结合活性(参照实施例1的(1-2))、诱导Rap1活化的活性(参照实施例1的(1-3))、T细胞向ICAM的粘附增强活性(参照实施例1的(1-4))等为指标而测定。
通过将所测定的活性水平与没有接触受试物质的RGM的活性水平比较,能够确定受试物质是否是抑制T细胞活化的物质。与没有接触受试物质的RGM的活性水平比较,如果接触了受试物质的RGM的活性水平降低,则确定受试物质是抑制T细胞活化的物质,可以选择该物质。优选地,接触了受试物质的RGM的活性水平为50%以下,更优选地,为25%以下时,则确定是抑制T细胞活化的物质。通过本发明的筛选方法获得的抑制T细胞活化的物质作为自身免疫病等由T细胞活化所引起的疾病的预防药或治疗药的有效成分候选物质是有用的。特别地,作为多发性硬化症的预防药或治疗药的有效成分候选物质是极有用的。
实施例
以下通过实施例详细说明本发明,但是本发明不受限于这些实施例。
〔实施例1:免疫细胞中RGMa以及RGMa受体的表达〕
(1-1)骨髓来源的树状细胞(BMDCs)中RGMa的表达
按照Lutz等人的方法(Lutz,M.B.,等人,J.Immunol.Methods 223,77-92(1999)),自C57BL/6小鼠(8~10周龄)采取骨髓细胞,通过在含有GM-CSF(20ng/mL,Sigma-Aldrich)的培养基中培养获得BMDCs。实验中,使用培养开始后6天的BMDCs。
通过实时定量PCR测定BMDCs中RGMa mRNA的表达。即,向培养开始后6天的BMDCs添加0、0.01、0.1或1μg/mL LPS(Sigma-Aldrich),培养24小时,收集活化的BMDCs。使用RNeasy Kit(QUIAGEN)自BMDCs提取总RNA,使用逆转录酶(GE Healthcare)获得cDNA。以得到的cDNA为模板,按照制造商的方案进行TaqMan实时PCR(ABI Prism7500 Seq uence Detection System),定量分析RGMa的mRNA表达。自Applied Biosystems购买特异的引物和探针。
此外,通过蛋白质印迹检测BMDCs中RGMa的表达。即,将添加了LPS 24小时后的BMDCs(2×106个)裂解于2×样本缓冲液(250mMTris-HCl、4%SDS、20%甘油、0.02%溴酚兰、以及10%β-巯基乙醇),煮沸5分钟后,将等量的各样本在还原条件下进行15%SDS-PAGE。泳动后将蛋白质转移至PVDF膜(Immobilon-P,Millipore)。用包含5%脱脂乳以及0.05%吐温20的PBS封闭后,与抗RGMa抗体(本发明者们制备的抗体,参照非专利文献3)反应,使用HRP标记的二次抗体(CellSignaling Technology)以及ECL化学发光系统(GE Healthcare)检测。使用Scion image进行蛋白质条带的定量。
实时PCR的结果示于图1,蛋白质印迹的结果示于图2。由图1可见,LPS的刺激增强了BMDCs中RGMa mRNA的表达。另外,由图2可见,存在抗RGMa抗体结合的35kDa以及50kDa的条带,通过LPS的刺激,细胞内的量增加。50kDa的条带相当于RGMa的全长,35kDa是RGMa的C末端片段,推定是RGMa的成熟体(Mueller,B.K.,Yamashita,T.,Schaffar,G.& Mueller,R.Philos.Trans.R.Soc.Lond.B Biol.Sci.361,1513-1529(2006))。这些结果表明在活化的BMDCs中诱导了RGMa的表达。
(1-2)CD4+T细胞以及CD11b+巨噬细胞中RGMa受体的表达
自C57BL/6小鼠(8~10周龄)摘出脾脏,快速置于RPMI1640培养基(Invitrogen)中,轻轻挤压,用ACK裂解缓冲液(Lonza Walkersville)进行除去红细胞的处理,获得细胞悬液。用RPMI1640洗3次后,将细胞悬液通过70μm的细胞滤过器,制备脾细胞的单细胞悬液。向该悬液添加各种浓度的人RGMa的C末端片段(下文中称为“RGMa-Fc”),孵育30分钟。洗净后,组合使用PE标记的抗CD11b抗体或APC标记的抗CD4抗体、和FITC标记的抗人RGMa-Fc抗体(BD Biosciences)进行免疫染色,通过流式细胞术分析测定细胞与人RGMa-Fc的结合。在流式细胞术分析中使用了FACSCalibur(BD Biosciences)以及CellQuest软件(BDBiosciences)。
结果示于图3。由图3可见,来自脾脏的CD4+T细胞以及CD11b+巨噬细胞均浓度依赖地与人RGMa-Fc结合。由该结果可见,这些细胞表达RGMa受体。
(1-3)研究CD4+T细胞以及CD11b+巨噬细胞中的Rap1活化
接下来,研究了通过结合CD4+T细胞以及CD11b+巨噬细胞的RGMa而介导的可能的信号传导。已知在T淋巴细胞中,TCR连接作用引起暂时活化Rap1以及在T细胞的与抗原呈递细胞的接触面蓄积Rap1-GTP,这调节由LFA-1(整联蛋白):ICAM-1(整联蛋白配体)介导的信号(Katagiri,K.等人,Mol.Cell Biol.20,1956-1969(2000);Katagiri,K.,Hattori,M.,Minato,N.& Kinashi,T.Mol.Cell Biol 22,1001-1015(2002))。本文评价了CD4+T细胞以及CD11b+巨噬细胞的Rap1活化中RGMa的参与。
自上述(1-2)制备的脾细胞的单细胞悬液分别使用抗CD4磁珠以及抗CD11b磁珠(Miltenyi Biotec),通过阳性分选分离CD4+T细胞以及CD11b+巨噬细胞,测定活性型Rap1。在活性型Rap1的测定中,使用Rap1活化测定法试剂盒(Upstate Biotech);使用分离CD4+T细胞以及CD11b+巨噬细胞之前的脾细胞作为对照。即,向各细胞悬液中添加2μg/mL的重组小鼠RGMa(R&D Systems),处理5分钟后,用Mg2+裂解缓冲液(包含25mM HEPES(pH 7.5),150mM NaCl,1%Igepal CA-630,10mMMgCl2,1mM EDTA,2%甘油,2mM原钒酸钠,1mM乙磺酰氯,以及蛋白酶抑制剂混合物(Roche Diagnostics))裂解。将细胞裂解液用于总Rap1的测定。结合有RalGDS的琼脂糖与细胞裂解液于4℃反应30分钟,沉淀结合至Ral结合结构域的活性型Rap1。将珠用Mg2+裂解缓冲液洗净,再悬浮于2×样本缓冲液,进行蛋白质印迹。在蛋白质印迹中使用了试剂盒所附带的抗Rap1抗体,以上述(1-1)同样的步骤实施。使用Scion image进行条带的定量,算出相对的Rap1活化水平。
蛋白质印迹结果示于图4(A),显示相对的Rap1活性的图示于图4(B)中。在图4(B)中,相对活性值的图以3~4次独立实验的平均值以及标准误差表示,“*”表示Student t检验中P<0.05。由图4(A)可见,小鼠RGMa处理使任一细胞均增加了活性型Rap1(图中是下拉的(Pulldown)Rap1)。而且,如图4(B)所示,通过RGMa刺激5分钟,脾细胞、CD4+T细胞以及CD11b+巨噬细胞的Rap1均显著地被活化。该结果教导了,树状细胞表达的RGMa通过CD4+T细胞以及CD11b+巨噬细胞表面的RGMa受体而可以活化细胞内的Rap1。
(1-4)淋巴细胞结合测定法
通过整联蛋白介导的粘附在T细胞运输以及活化中发挥关键的作用。进一步地,TCR诱导的粘附需要Rap1的活化(Katagiri,K.等人,Mol.CellBiol.20,1956-1969(2000);Reedquist,K.A.等人,J.Cell Biol.148,1151-1158(2000);Suga,K.等人,FEBS Lett.489,249-253(2001))。因此,为了确认通过RGMa诱导的活性型Rap1是否参与粘附活性,进行了淋巴细胞结合测定法。
根据现有技术文献(Sebzda,E.,Bracke,M.,Tugal,T.,Hogg,N.&Cantrell,D.A.,Nat.Immunol.3,251-258(2002);Duchniewicz,M.等人,Mol.Cell Biol.26,643-653(2006))所述的方法进行细胞粘附的评价。即,在96孔平底板(NUNC,MaxiSorp)中添加2μg/mL重组小鼠ICAM-1/Fc(R&D Systems)或4μg/mL纤连蛋白(Sigma-Aldrich),4℃静置一夜,进行预包被。用PBS洗净板,为了防止非特异的结合,使用包含2%BSA的RPMI1640(Invitrogen)于37℃、1小时进行封闭。将ICAM-1/Fc包被的板用于CD4+T细胞的评价;将纤连蛋白包被的板用于脾细胞评价。
将新鲜制备的脾细胞或CD4+T细胞用2.5μM BCECF-AM(2’7’-二-(2-羧乙基)-5-(和-6)-羧基荧光素,乙酰氧基甲酯,Calbiochem)于37℃标记30分钟,随后用包含0.5%BSA的RPMI1640洗净。将5×105个细胞加至预包被的板,在含或不含2μg/mL重组RGMa、含有0.5%BSA的RPMI1640中于37℃孵育1小时。将没有粘附的细胞用温的含有0.5%BSA的RPMI1640洗净3次而除去。使用Spectra MAX(Molecular Devices),在激发波长485nm和荧光波长538nm处定量粘附。用相对于1孔中播种的总细胞数的百分率表示粘附率。
脾细胞的结果示于图5(A),CD4+T细胞的结果示于图5(B)。粘附率的图用3次独立实验的平均值以及标准误差表示,“*”以及“**”表示在Student t检验中分别为P<0.05以及P<0.01。由图5(A)可见,与没有受RGMa刺激的脾细胞相比较,受RGMa刺激的脾细胞对纤连蛋白的粘附率显著提高。同样地,由图5(B)可见,与没有受RGMa刺激的CD4+T细胞相比较,受RGMa刺激的CD4+T细胞对ICAM-1的粘附率显著提高。由这些结果可见,RGMa通过活化Rap1,增强了脾细胞以及CD4+T细胞的粘附活性。
〔实施例2:在多发性硬化症模型小鼠中研究抗RGMa中和抗体的效果〕
(2-1)多发性硬化症模型小鼠的制备
作为多发性硬化症的临床以及病理学特征的研究模型而广泛接受的模型动物,使用以髓鞘少突胶质细胞糖蛋白(myelin oligodendrocyteglycoprotein,下文中称为“MOG”)诱导的实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,下文中称为“EAE”)小鼠。具体而言,使用包含100μg(MOG)35-55肽(MEVGWYRSPFSRVVHLYRNGK(序列编号5),Greiner Bio-one)的100μL PBS以及包含500μg结核死菌(H37Ra,Difco)的100μL弗氏完全佐剂的乳浊液200μL胁腹皮下施与C57BL/6小鼠(8~10周龄)而诱导EAE。进一步地,MOG施与后以及MOG施与48小时后,静脉内施与200ng百日咳毒素(List Biological Laboratories)。
(2-2)通过向多发性硬化症模型小鼠施与抗RGMa中和抗体观察临床症状
MOG施与后第7天以及第10天日腹腔内施与本发明者们制备的抗RGMa中和抗体(参照非专利文献3)或对照抗体(兔IgG,Sigma-Aldrich)各400μg。抗RGMa中和抗体施与组由9只、对照抗体施与组由11只EAE小鼠组成。自MOG施与日(0天)至21天进行观察,根据以下标准评价EAE的临床症状。
0:无异常
0.5:丧失尾的紧张
1:丧失尾的反射性
2:丧失尾的反射性、正向反射低下、一肢的知觉异常
3:一肢的知觉异常以及麻痹
3.5:两后肢的麻痹
4:前肢以及后肢的麻痹
5:濒死、死亡
观察结束后将小鼠安乐死,摘出脊髓,用低聚甲醛固定、制备石蜡包埋切片,供组织学评价。将切片用苏木精-伊红(HE)染色、评价炎症。每只小鼠从颈髓至胸髓观察20-30个切片,按照以下标准双盲地进行半定量的炎症组织学评价(炎症指数)。对抗RGMa中和抗体施与组中的5只、对照抗体施与组中的6只进行评价。
0:没有炎症
1:仅血管周围以及髓膜的细胞浸润
2:骨髓实质的轻度的细胞浸润
3:骨髓实质的中等程度的细胞浸润
4:骨髓实质的重度的细胞浸润
图6示出了观察期间EAE评分的变化。用平均值以及标准误差来表示EAE评分。由图6可见,与对照抗体施与组相比较,第14天以后抗RGMa中和抗体施与组的EAE评分是显著降低的值。虽然在图中没有显示,但是Student t检验的结果显示第14天P<0.05、第15天以后P<0.01,两组间有统计上的显著性差异。
图7(A)显示了在每组中EAE的临床症状发生率、图7(B)显示了在每组中EAE发病日的平均值、图7(C)显示了各组的各小鼠的评分最大值的平均值、图7(D)显示了各组的各小鼠的评分累积值的平均值。图中“n.s.”表示在Student t检验中没有显著性差异、“*”以及“**”表示在Student t检验中分别是P<0.05以及P<0.01。由图7(A)可见,对照抗体施与组全部(100%)表现出EAE的临床症状;但是在抗RGMa中和抗体施与组中有44%的小鼠没有表现出EAE的临床症状。另外,由图7(B)可见,两组间的发病日没有差异,施与抗RGMa中和抗体不推迟EAE的发病。另一方面,由图7(C)以及(D)可见,评分最大值的平均值以及评分累积值的平均值均是抗RGMa中和抗体施与组显示显著低于对照抗体施与组的值。这些结果表明,抗RGMa中和抗体的施与对抑制出现EAE的症状以及抑制其进展是有效的。
图8示出了颈髓的HE染色组织图像。(a)以及(b)是对照抗体施与组的HE染色组织图像;(c)以及(d)是抗RGMa中和抗体施与组的HE染色组织图像,比例尺表示250μm。由图8(a)以及(b)可见,其显示了对照抗体施与组的骨髓中广泛存在炎症性单核细胞、大范围的炎症性病变。另一方面,由(c)以及(d)可见,其显示了抗RGMa中和抗体施与组的骨髓中炎症性单核细胞显著减少、炎症病变减轻。
图9显示了炎症指数的评价结果。图中“*”表示在Student t检验中P<0.05。由图9可见,与对照抗体施与组相比较,其显示了抗RGMa中和抗体施与组的炎症指数表现显著的低数值。自以上的使用多发性硬化症模型小鼠的实验结果,明确了抗RGMa中和抗体的施与减轻了EAE的临床症状以及组织学的病变这两者。
〔实施例3:研究施与抗RGMa中和抗体的多发性硬化症模型小鼠的脾细胞中的T细胞活化〕
自结束了实施例2的临床症状观察的多发性硬化症模型小鼠(EAE小鼠)在MOG施与后第21天摘出脾脏,分离脾细胞,以5×105个/孔播种在96孔板中,培养3天,分析细胞增殖。对于培养基,使用包含谷氨酰胺、丙酮酸钠、青霉素、链霉素、2-ME、以及10%热灭活FBS的RPMI1640培养基。为了再刺激CD4+T细胞,当培养开始时添加20μg/mL的MOG肽或5μg/mL的抗CD3单克隆抗体(2C11,BD Biosciences)。按照Kubo等人的方法(Kubo,T.,等人,J.Immunol.173,7249-7258(2004)),评价培养结束前18小时的[3H]胸苷摄入。
为了测定IFN-γ、IL-2、IL-4以及IL-17的生成量,将上述脾细胞以2×106个/孔播种在24孔板,在20μg/mL的MOG肽或5μg/mL的抗CD3单克隆抗体存在下或不存在下进行培养。将培养开始后24小时的培养上清用于IL-2的测定;将培养开始后72小时的培养上清用于其他细胞因子的测定。使用ELISA试剂盒(Invitrogen)按照说明书所述进行细胞因子的测定。
图10中显示了结果。(a)是细胞增殖的测定结果、(b)是IL-2的测定结果、(c)是IFN-γ的测定结果、(d)是IL-17的测定结果。各图用平均值以及标准误差(对照抗体施与组n=6、抗RGMa中和抗体施与组n=5)表示,“*”以及“**”分别表示在Student t检验中P<0.05以及P<0.01。由图10(a)可见,与自对照抗体施与组的小鼠获得的脾细胞的细胞增殖相比较,在MOG肽进行抗原特异的刺激以及抗CD3单克隆抗体进行非特异的刺激的任一情形中均显示出自抗RGMa中和抗体施与组的小鼠获得的脾细胞的细胞增殖显著地低。另外,由图10(b)、(c)、(d)可见,与自对照抗体施与组的小鼠获得的脾细胞的各细胞因子分泌量相比较,在MOG肽进行抗原特异的刺激以及抗CD3单克隆抗体进行非特异的刺激的任一情形中均显示出自抗RGMa中和抗体施与组的小鼠获得的脾细胞的IL-2、IFN-γ以及IL-17的分泌量显著地低。另一方面,虽然没有给出数据,但是抗RGMa中和抗体的施与不影响IL-4的分泌量。这些结果表明,向EAE小鼠施与抗RGMa中和抗体显著减弱了EAE小鼠中抗原特异的以及非特异的T细胞活化。
〔实施例4:在再发性多发性硬化症模型小鼠中研究抗RGMa中和抗体的效果〕
(4-1)再发性多发性硬化症模型小鼠的制备
代替MOG,将髓鞘蛋白脂质蛋白(myelin proteolipid protein,以下称为“PLP”)施与SJL/J小鼠,制备再发性EAE模型小鼠。具体而言,再发性EAE是通过将包含100μg(PLP)139-151肽(HSLGKWLGHPDKF(序列编号6),Greiner Bio-one)的100μL PBS和包含500μg结核死菌(H37Ra,Difco)的100μL弗氏完全佐剂的乳浊液200μL胁腹皮下施与SJL/J小鼠(8~10周龄)而诱导的。进一步地,PLP施与后以及PLP施与48小时后,静脉内施与200ng百日咳毒素(List Biological Laboratories)。
(4-2)通过向再发性多发性硬化症模型小鼠施与抗RGMa中和抗体观察临床症状
PLP施与后第25天以及第28天,腹腔内施与各400μg的本发明者们制备的抗RGMa中和抗体(参照非专利文献3)或对照抗体(兔IgG,Sigma-Aldrich)。抗RGMa中和抗体施与组由9只、对照抗体施与组由11只的EAE小鼠组成。自PLP施与(第0天)至第45天进行观察,根据上述实施例2所述的标准评价EAE的临床症状。
图11示出了观察期间EAE评分的变化。用平均值以及标准误差表示EAE评分。图中“*”表示在Student t检验中P<0.05。由图11可见,与对照抗体施与组的EAE评分相比较,抗体施与后的抗RGMa中和抗体施与组的EAE评分是显著的低值。该结果表明,通过在最初麻痹表现期的后期施与抗RGMa中和抗体,能够预防EAE的再发。
〔实施例5:在MOG刺激的BMDCs移植小鼠中研究RGMa基因敲低对EAE临床症状的效果〕
(5-1)RGMa基因敲低的BMDCs的制备
购买和使用以下所示碱基序列的小鼠RGMa siRNA(Stealth RNAi(商品名),Invitrogen)。另外,使用非靶向dsRNA(Invitrogen)作为对照siRNA。
有义链:5’-AAAGAGGCCGCAGUGAGUGUAGUUG-3’(序列编号7)
反义链:5’-CAACUACACUCACUGCGGCCUCUUU-3’(序列编号8)
使用培养开始后7天的BMDCs(参照实施例1)。在包含500pmol小鼠RGMa siRNA或对照siRNA的100μL核酸导入用溶液中悬浮1×106个BMDCs。将样本移至小杯中,使用Nucleofector(注册商标)装置(Lonza社),按照制造者所附的说明书,将siRNA导入细胞。在siRNA导入后,使用添加了谷氨酰胺、丙酮酸钠、青霉素、链霉素、2-ME、10%热灭活FBS的RPMI1640培养基培养细胞。导入了小鼠RGMa siRNA的BMDCs中的RGMa的表达显著降低。
(5-2)MOG刺激以及向小鼠的移植
向导入了小鼠RGMa siRNA或对照siRNA的BMDCs添加100μg/mL的(MOG)35-55肽,培养4~6小时,从而给予抗原刺激。将刺激后的6×105个活细胞静脉内施与接受体的小鼠(C57BL/6)。接下来,皮下施与包含500μg结核死菌(H37Ra,Difco)的弗氏完全佐剂200μL。进一步地,在48小时后静脉内施与200ng百日咳毒素(List Biological Laboratories)。
(5-3)EAE临床症状的观察
对RGMa siRNA组(5只)以及对照siRNA组(5只),自细胞移植(第0天)至第21天进行观察,按照上述实施例2所述的标准评价EAE的临床症状。
观察期间的EAE评分的变化示于图12。EAE评分用平均值以及标准误差表示。图中“*”以及“**”分别表示在Student t检验中P<0.05以及P<0.01。由图12可见,与对照siRNA组的EAE评分相比较,RGMa siRNA组的EAE评分是显著的低值。由该结果可见,RGMa siRNA有效地预防或治疗多发性硬化症。
〔实施例6:在受MOG刺激的CD4+T细胞移植小鼠中研究抗RGMa中和抗体对EAE临床症状的效果〕
(6-1)供体小鼠的CD4+T细胞的制备
向供体小鼠(C57BL/6)腹腔内施与本发明者们制备的抗RGMa中和抗体(参照非专利文献3)或对照抗体(兔IgG,Sigma-Aldrich)各400μg(第-2天)。2天后(第0天),第二次施与抗RGMa中和抗体或对照抗体,同时皮下施与包含100μg(MOG)35-55肽的100μL PBS和包含500μg结核死菌(H37Ra,Difco)的100μL弗氏完全佐剂的乳浊液200μL。进一步地,在其5天后(第5天),第三次施与抗RGMa中和抗体或对照抗体。自MOG施与的第10天,将小鼠安乐死,取出脾脏以及引流区淋巴结,分离细胞,制备细胞悬液。为了再刺激CD4+T细胞,用包含40μg/mL的(MOG)35-55肽的培养基培养细胞(1×106个/mL)3天。培养结束后收集细胞,使用CD4+T细胞分离试剂盒(Miltenyi Biotec)分离CD4+T细胞。
(6-2)向接受体小鼠的移植以及EAE临床症状的观察
预先用亚致死量(500Gy)的放射线照射接受体小鼠(C57BL/6)。将6×105个CD4+T细胞(活细胞)静脉内施与接受体小鼠。对于移植来自施与了抗RGMa中和抗体的供体小鼠的CD4+T细胞的小鼠(抗RGMa中和抗体组,7只)以及移植来自施与了对照抗体的供体小鼠的CD4+T细胞的小鼠(对照抗体组,7只),自细胞移植(第0天)至第21天进行观察,按照上述实施例2所述的标准评价EAE的临床症状。
观察期间的EAE评分的变化示于图13。EAE评分用平均值以及标准误差表示。图中“*”以及“**”分别表示在Student t检验中P<0.05以及P<0.01。由图13可见,与对照抗体组的EAE评分相比较,抗RGMa中和抗体组的EAE评分是显著的低值。由该结果可见,由抗RGMa中和抗体所致的对多发性硬化症的预防或治疗效果是基于抗RGMa中和抗体抑制抗原特异的T细胞活化和/或自身反应性T细胞的分化。
以上结果解明了,RGMa参与了通过单核细胞对脊髓的浸润以及T细胞活化等增大免疫应答引起的EAE的发病,其中所述T细胞活化是T细胞的增殖和/或EAE相关细胞因子IL-2、IL-17以及IFN-γ的产生等。进一步地,显示了抗RGMa中和抗体和RGMa siRNA抑制T细胞的活化,对于预防或治疗多发性硬化症是有效的。
另外,本发明不限于上述各实施方案以及实施例,而是能在权利要求所示的范围进行多种变更,将不同实施方案中分别公开的各技术的手段进行适宜组合得到的实施方案也包含在本发明的技术范围中。另外,本说明书中所述的全部学术文献以及专利文献均在本说明书中援引作为参考。
产业上的利用可能性
本发明在医药品产业具有极高的利用价值。
Claims (7)
1.T细胞活化抑制剂,其以RGM抑制物质作为有效成分。
2.权利要求1所述的T细胞活化抑制剂,其中RGM抑制物质是抗RGM中和抗体。
3.权利要求1所述的T细胞活化抑制剂,其中RGM抑制物质是RGMsiRNA。
4.T细胞活化所引起的疾病的预防或治疗用药物组合物,其特征在于,含有权利要求1至3中任一项所述的T细胞活化抑制剂。
5.权利要求4所述的药物组合物,其中T细胞活化所引起的疾病是自身免疫病。
6.权利要求5所述的药物组合物,其中自身免疫病是多发性硬化症。
7.抑制T细胞活化的物质的筛选方法,其特征在于包括使受试物质与RGM接触的步骤、测定所述RGM的活性水平的步骤、将所述活性水平与没有接触受试物质的RGM的活性水平进行比较的步骤、和选择使RGM的活性水平降低的受试物质的步骤。
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TWI732757B (zh) * | 2015-04-28 | 2021-07-11 | 日商田邊三菱製藥股份有限公司 | RGMa結合蛋白質及其使用 |
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US8962803B2 (en) | 2008-02-29 | 2015-02-24 | AbbVie Deutschland GmbH & Co. KG | Antibodies against the RGM A protein and uses thereof |
ES2562832T3 (es) | 2009-12-08 | 2016-03-08 | Abbvie Deutschland Gmbh & Co Kg | Anticuerpos monoclonales contra la proteína RGM para su uso en el tratamiento de la degeneración de la capa de fibra nerviosa de la retina |
BR112014018592B1 (pt) | 2012-01-27 | 2022-03-15 | Abbvie Inc. | Anticorpo monoclonal isolado que se liga à molécula de orientação repulsiva a (rgma) |
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KR20210034055A (ko) | 2018-07-19 | 2021-03-29 | 고쿠리츠다이가쿠호우진 도쿄다이가쿠 | Htlv-1 관련 척수증(ham) 치료 또는 예방제, 및 ham의 치료 방법 |
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