CN111344417A - 作为帕金森病生物标记物的miRNA及利用其的诊断试剂盒 - Google Patents
作为帕金森病生物标记物的miRNA及利用其的诊断试剂盒 Download PDFInfo
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Abstract
本发明涉及提供用于诊断帕金森病的信息的方法。并且,本发明涉及用于预防、改善或治疗帕金森病的组合物。由于本发明的miRNA的表达在帕金森病模型中特异性下调或上调,可有效地利用于帕金森病的诊断及治疗。
Description
技术领域
本发明涉及利用在帕金森病中表达下调或上调的至少一种miRNA的诊断帕金森病的方法及利用上述miRNA的用于预防或治疗帕金森病的组合物。
背景技术
帕金森病为中枢神经系统退行性疾病的一种,其始于中脑的黑质致密部(Substantia nigra pars compacta)的神经元退化,具有大脑体积的减少、α-突触核蛋白(α-synuclein,αSyn)的凝聚等的病态生理症状,该疾病伴随步态障碍、手颤、僵直的行动等症状。
韩国的帕金森病患者在2010年为6万1565名,年均增长8.7%,2014年增加至8万5888名,在2014年的患者中,60岁以上患者比例为95.7%,患病率与患者的年龄具有相关关系,帕金森病患者的男性和女性患者的数目分别为,男性为3万3831名,女性为5万2057名(最近5年帕金森病患者数目、诊疗费呈增加趋势,韩国青年医师,2015年)。
主要7个国家(美国、日本、法国、德国、意大利、西班牙、英国)的帕金森病患者人口在2008年约为454万名、2012年约为505万名,年均增加2.72%,在亚洲的国家中,帕金森病患者人口从2008年的198万名增加至2012年的219万名,在欧洲国家中,帕金森病患者人口从2008年的82万名增加至2012年的91万名(Product and Pipeline Analysis of theGlobal Parkinson′s Disease Therapeutic Market,F&S,2014年)。
观察世界帕金森病治疗剂产品动向,2011年的占比为47%的占比最大的多巴胺激动药预计到2021年降至42%,预期新型管线药物到2021年约占20%(R&D Trends:Parkinson′s Disease,Datamonitor,2012年)。
与帕金森病的诊断关联的各种检查包括1)正电子发射断层成像(PET)检查、2)磁共振成像(MRI)检查、3)与内科疾病有关的检查等。
脑多巴胺转运体正电子发射断层成像(dopamine transporter PET,positronemission tomography)检查为可以确定多巴胺细胞的损伤与否,若进行脑多巴胺转运体正电子发射断层成像检查,则可用于检测帕金森症状是否是由帕金森病以外的原因引起的。在药物诱发帕金森综合征、血管性帕金森综合征、阿尔茨海默病相关的帕金森症状、原发性震颤相关的帕金森症状的情况下,乍一看,通常伴随着与帕金森症状相似的震颤和运动迟缓的情况,但多巴胺神经元是正常的。
在呈现出帕金森症状的情况下,必须区分与帕金森病相似的疾病,为了将帕金森病与继发性帕金森综合征及非典型帕金森综合征等进行区分,需先进行大脑磁共振成像(MRI)检查。在患者患有帕金森病的情况下,MRI检查结果正常,相反,帕金森病以外的其他疾病的MRI检查结果具有特征性。
内科疾病情况下,在帕金森病诊断中进行与内科疾病有关的检查(血液检查、尿液检查、心电图、胸部X射线检查)是为了确认通常引起全身羸弱而被误认为帕金森症状的其他内科疾病的发作和进展。
但是,上述帕金森病诊断方法具有费用高、诊断步骤复杂的问题。专注于帕金森病的治疗及诊断的最佳企业之一的丹麦灵北(Lundbeck)公司,在其主页提及了研发用于快速诊断帕金森病的工具的重要性,但目前不销售商用化的试剂或试剂盒。并且,患有帕金森病的患者数目逐年迅速增加,帕金森病治疗剂市场也与此成比例的快速增长,但是,目前帕金森病诊断市场的发展甚微。因此,急需开发用于将帕金森病与相似疾病区分来迅速且经济地诊断帕金森病的技术及在诊断帕金森病的同时用于治疗帕金森病的技术。
如上所述的背景技术的说明仅用于增进与本发明的背景有关的理解,不应理解为属于本技术领域的普通技术人员所知的现有技术。
发明内容
技术问题
本发明人努力并深入地研发了用于将帕金森病与相似疾病区分来迅速且经济地诊断帕金森病的技术及在诊断帕金森病的同时用于治疗帕金森病的技术。结果发现,帕金森病模型中的特定miRNA的表达水平特异性上调或下调,并确认可以利用其来有效地诊断及治疗帕金森病,基于该发现完成了本发明。
因此,本发明的一个目的在于,提供一种提供用于诊断帕金森病的信息的方法。
本发明的再一个目的在于,提供用于帕金森病的诊断或预后分析的试剂盒。
本发明的另一个目的在于,提供用于筛选诱发帕金森病的物质的方法。
本发明的还有一个目的在于,提供用于筛选帕金森病治疗剂的方法。
本发明的又一个目的在于,提供用于预防、改善或治疗帕金森病的组合物。
本发明的又一个目的在于,提供用于治疗帕金森病的方法。
本发明的其他目的及优点可通过下述发明的详细说明、权利要求及附图变得更加明确。
解决问题的手段
本发明的一个方面提供一种提供用于诊断帕金森病的信息的方法,包括:
(a)将选自从对象获取的样品中miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324及miR-4726-5p中的一种或多于一种的miRNA的表达水平与正常样品中所选miRNA的表达水平进行比较;以及
(b)在上述对象的样品中所选miRNA的表达水平比正常样品中所选miRNA的表达水平减少的情况下,诊断为帕金森病。
根据本发明的优选实施方案,所述方法还包括:
(a)将选自从对象获取的样品中miR-1226-5p、miR-4767及miR-3064-5p中的一种或多于一种的miRNA的表达水平与正常样品中所选miRNA的表达水平进行比较;以及
(b)在上述对象的样品中所选miRNA的表达水平比正常样品中所选miRNA的表达水平增加的情况下,诊断为帕金森病。
本发明的另一个方面提供一种提供用于诊断帕金森病的信息的方法,包括:
(a)将选自从对象获取的样品中miR-1226-5p、miR-4767及miR-3064-5p中的一种或多于一种的miRNA的表达水平与正常样品中所选miRNA的表达水平进行比较;以及
(b)在上述对象的样品中所选miRNA的表达水平比正常样品中所选miRNA的表达水平增加的情况下,诊断为帕金森病。
在本说明书中,除非另外说明,否则术语“miRNA”用于表示15个至25个碱基的非编码RNA,其作为发夹状结构的RNA前体来转录,并被具有RNA酶III切割活性的dsRNA切割酶切割来导入至称为RISC的蛋白质复合物,涉及参与mRNA的翻译抑制。并且,在本说明书中使用的miRNA不仅包括以特定核苷酸序列(或序列)示出的miRNA,还包括上述miRNA的前体(pre-miRNA、pri-miRNA)、生物功能与之等同的miRNA,如同源物(即,同系物或直系同源物)、基因多态性等的变体及其衍生物。这些前体、同源物、变体或衍生物可通过miRBase release 20(http://www.mirbase.org/)具体确定,在严格条件下,其实例包括具有与序列1至序列9的miRNA的互补序列杂交的序列的miRNA。并且,在本说明书中使用的miRNA也可以为miR基因的基因产物,这种基因产物包括成熟miRNA(例如,参与如上所述的mRNA的翻译抑制的15个至25个碱基或19个至25个碱基的非编码RNA)或miRNA前体(例如,如上所述的pre-miRNA或pri-miRNA)。
在本说明书中,术语“核酸”是指包含RNA、DNA及RNA/DNA(嵌合体)的核酸。上述DNA包括cDNA、基因组DNA及合成DNA的任一种。上述RNA包括总RNA、mRNA、rRNA、miRNA、siRNA、snoRNA、snRNA、非编码RNA及合成RNA的任一种。在本说明书中,术语“合成DNA”及“合成RNA”是指根据规定的碱基序列(天然序列或非天然序列中的任一种),如通过使用自动核酸合成仪人工制备的DNA及RNA。在本说明书中,术语“非天然序列”为以广义使用的,包括与天然序列不同的,例如包括一种或多于一种的核苷酸的取代、缺失、插入和/或添加的序列(即,变体序列)、包括一种或多于一种的修饰核苷酸的序列(即,修饰序列)等。并且,在本说明书中,多核苷酸与核酸互换地使用。
在本说明书中使用的“对象”理解为包括哺乳动物,例如人、黑猩猩的灵长类,狗、猫等的宠物,牛、马、羊、山羊等的家畜,小鼠、大鼠等的啮齿类等。在本说明书中使用的“正常组”也与“对象”具有相同的含义,表示检测到未患有帕金森病的对象。
只要本发明所包含的样品自然地或人为地从对象分离,包括对象的帕金森病相关遗传信息,并无限制。优选地,样品是从粪便、细胞、血液、血浆、血清、毛发或尿液等分离的,更优选地,样品是从身体分离的血液、血浆、血清等。
本发明的提供用于诊断帕金森病的信息的方法可使用公知的各种试验方法,例如可利用杂交法、免疫分析方法或基因扩增方法来实施,但并不限定于此。
上述杂交法通过利用探针来确认miRNA的存在。
在本说明书中使用的术语“探针”为自然或修饰的单体或连接的线性低聚物,探针旨在包括脱氧核糖核苷酸及核糖核苷酸,可与靶核苷酸序列特异性杂交,是自然存在或人为合成的。优选地,本发明的探针为单链,为寡脱氧核糖核苷酸。
在本发明的诊断方法可以以杂交方式中的微阵列方式实施的情况下,将如上所述的探针用作杂交阵列元件,固定于基质上。优选的基质为刚性或半刚性的载体,其实例包括膜、过滤器、芯片、载玻片、晶片、纤维、磁珠或非磁珠、凝胶、管、板、聚合物、微粒和毛细管。上述杂交阵列元件排列在如上所述的基质上并固定。如上所述的固定化通过化学结合或共价键和的方法,如紫外线(UV)来实施。例如,上述杂交阵列元件可与玻璃表面结合,因此需要包含环氧化合物或醛基的修饰,并且,可在聚赖氨酸涂敷表面通过UV结合。或者,上述杂交阵列元件可通过连接物(例:乙二醇低聚物及二胺)与基质结合。
另一方面,适用于本发明的微阵列的样品DNA可选地被标记(labeling),并与微阵列上的阵列元件杂交。杂交能够在各种方式下进行。并且,可根据标记物质以多种方式实施杂交程度的检测及分析。
探针的标记可提供能够检测杂交是否发生的信号,并且标记的探针可与寡核苷酸连接。适合的标记包括具有如荧光基团(例如,荧光素(fluorescein)、藻红蛋白(phycoerythrin)、罗丹明、丽丝胺(lissamine)、Cy3和Cy5(法玛西亚(Pharmacia)公司))、末端脱氧核苷酰转移酶(TdT,terminal deoxynucleotidyl transferase)、发色团、化学发光团、磁性粒子、放射性同位素(P32或S35)、质量标记、电子致密粒子、酶(碱性磷酸酶或辣根过氧化物酶)、辅因子、与酶有关的底物、重金属(例如,金)、具有特异性结合伴侣的半抗原,例如抗体、链霉亲和素、生物素、地高辛和螯合基团,但并不限定于此。标记可通过本领域通常实施的各种方法,如切口平移方法(nick translation)、随机引物方法(MultiprimeDNA labelling systems booklet,“Amersham”(1989))及5’末端磷酸化方法(Maxam&Gilbert,Methods in Enzymology,65:499(1986))实施。标记提供可通过荧光、辐射能、发色检测、重量检测、X射线衍射或吸收、磁、酶活性、质量分析、结合亲和力、杂交、高频、或纳米晶体检测的信号。
在利用探针的情况下,使探针与cDNA分子杂交。在本发明中,可通过最佳化程序以一连串的过程确定适合的杂交条件。为了在研究室确立所要使用的方案,普通技术人员通过一连串的过程实施这种程序。例如,温度、成分的浓度、杂交及清洗时间、缓冲液成分及它们的pH及离子强度等的条件根据探针的长度及GC含量、靶核苷酸序列等的各种因素变化。用于杂交的具体条件可在Joseph Sambrook,et al.,Molecular Cloning,A LaboratoryManual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.(2001);及M.L.M.Anderson,Nucleic Acid Hybridization,Springer-Verlag New York Inc.N.Y.(1999)中确认。
在杂交反应之后,检测通过杂交反应产生的杂交信号。杂交信号,可根据如与探针结合的标记的种类以各种方法实施。例如,在探针被酶标记的情况下,可通过使上述酶的底物与杂交反应产物进行反应来确认杂交与否。可使用的酶/底物的组合为过氧化物酶(例如,辣根过氧化物酶)和氯萘酚、氨基乙基咔唑、二氨基联苯胺、D-荧光素、光泽精(双N-甲基吖啶硝酸盐)、苄氧基试卤灵、鲁米诺、Amplex Red试剂(10-乙酰-3,7-二羟基吩嗪)、HYR(对苯二胺-HCl和邻苯二酚)、四甲基联苯胺(TMB)、2,2-连氮基-二(3-乙基-苯并噻唑啉磺酸酯)(ABTS)、邻苯二胺(OPD)及萘酚/焦宁;碱性磷酸酶和磷酸溴氯吲哚酯(BCIP)、氮蓝四唑(NBT)、萘酚-AS-B1-磷酸酯及ECF底物;葡萄糖氧化酶和t-氮蓝四唑(t-NBT)及吩嗪硫酸二甲酯(m-PMS)等。在探针被金粒子标记的情况下,可利用硝酸银以银染色方法检测。
生物样品与正常样品相比与上述miRNA的序列有关的杂交信号上调或下调时,将对象诊断为患有帕金森病。
本发明的提供用于诊断帕金森病的信息的方法可通过免疫分析方法实施。上述免疫分析方法包括辐射能免疫分析、辐射能免疫沉淀、免疫沉淀、酶联免疫吸附试验(ELISA)、捕捉ELISA、抑制或竞争分析、三明治免疫分析、流式细胞分析(flow cytometry)、免疫荧光染色及免疫亲和纯化,按并不限定于此。上述免疫分析方法记载于Enzyme Immunoassay,E.T.Maggio,ed.,CRC Press,Boca Raton,Florida,1980;Gaastra,W.,Enzyme-linkedimmunosorbentassay(ELISA),inMethodsinMolecularBiology,Vol.1,Walker,J.M.ed.,Humana Press,NJ,1984;及Ed Harlow and David Lane,UsingAntibodies:A LaboratoryManual,Cold Spring Harbor Laboratory Press,1999,上述文献通过引用并入本说明书中。
本发明的提供用于诊断帕金森病的信息的方法可通过基因扩增方法实施。利用基因扩增方法的miRNA的检测可利用本领域公知的各种方法来实施,在此情况下,可利用miRNA的引物或探针。
在利用引物的情况下,通过实施基因扩增反应来研究上述miRNA的基因的表达水平。由于本发明的诊断方法是基于对上述基因的表达水平的分析,因此,在分析样品(例如,细胞)中用上述标记物研究mRNA量来确定上述标记物的基因的表达水平。因此,本发明原则上将生物样品内的mRNA用作模板,利用与mRNA或cDNA结合的引物来实施基因扩增反应。
首先,为了获取mRNA,从试样分离总RNA。可根据本领域公知的常规方法分离总RNA(参照:Sambrook,J.et al.,MolecularCloning.A Laboratory Manual,3rd ed.ColdSpring Harbor Press(2001);Tesniere,C.et al.,PlantMol.Biol.Rep.,9:242(1991);Ausubel,F.M.et al.,CurrentProtocolsinMolecularBiology,John Willey&Sons(1987);及Chomczynski,P.et al.,Anal.Biochem.162:156(1987))。例如,可利用Trizol容易地分离细胞内的总RNA。
接着,从分离的mRNA合成cDNA,并扩增上述cDNA。本发明的总RNA从人的样品分离,因此,在mRNA的末端具有多聚A尾,可通过使用利用这种序列特性的寡dT引物及逆转录酶来容易合成cDNA(参照:PNAS USA,85:8998(1988);Libert F,et al.,Science,244:569(1989);及Sambrook,J.et al.,Molecular Cloning.A LaboratoryManual,3rd ed.ColdSpring Harbor Press(2001))。接着,可通过基因扩增反应对合成的cDNA进行扩增。在本发明中利用的引物在模板的一个位点杂交或退火来形成双链结构。适合形成这种双链结构的核酸杂交的条件记载于Joseph Sambrook等人,MolecularCloning,A LaboratoryManual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.(2001)及Haymes,B.D.,等人,Nucleic Acid Hybridization,A PracticalApproach,IRL Press,Washington,D.C.(1985)。
各种DNA聚合酶可利用于本发明的扩增,包括大肠杆菌(E.coli)DNA聚合酶I的“克列诺”片段、热稳定性DNA聚合酶及噬菌体T7DNA聚合酶。具体地,聚合酶为可从各种细菌种获取的热稳定性DNA聚合酶,细菌种包括Thermus aquaticus(Taq)、Thermusthermophilus(Tth)、Thermusfiliformis、Thermisflavus、Thermococcus literalis及Pyrococcusfuriosus(Pfu)。优选地,当实施聚合反应时,向反应容器提供过量的每种反应所需的成分。扩增反应所需的过量为扩增反应实质上不被成分的浓度限制的量。期望以可实现所期望的扩增程度的量向反应混合物提供如Mg2+的辅因子、dATP、dCTP、dGTP及dTTP。在扩增反应中利用的所有酶可在相同的反应条件下呈活性状态。其实,使用缓冲液使得接近所有酶达到最佳反应条件。因此,可用单一反应物实施本发明的扩增过程而不需要改变条件,如其他反应物的添加。
在本发明中,在允许靶核苷酸序列与引物之间特异性结合的严格条件下实施退火或杂交。用于退火的严格条件具有序列依赖性,根据周围环境变化而不同。
本说明书的术语“扩增反应”为扩增核酸分子的反应。本领域已报道了各种扩增反应,其实例包括聚合酶链式反应(以下,称为PCR)(美国专利第4683195号、第4683202号及第4800159号)、逆转录-聚合酶链式反应(以下,称为RT-PCR)(Sambrook等人,MolecularCloning.A LaboratoryManual,3rd ed.Cold Spring Harbor Press(2001))、Miller,H.I.(WO 89/06700)及Davey,C.等人(EP 329822)的方法、连接酶链反应(ligase chainreaction;LCR)(17,18)、缺口-LCR(WO 90/01069)、修复链反应(repair chain reaction;EP 439182)、转录介导扩增(transcription-mediated amplification;TMA)(19)(WO 88/10315)、自我持续序列复制(self sustained sequence replication)(20)(WO 90/06995)、靶多核苷酸碱基序列的选择性扩增(selective amplification of targetpolynucleotide sequences)(美国专利第6410276号)、一致序列引物聚合酶链反应(consensus sequence primed polymerase chain reaction;CP-PCR)(美国专利第4437975号)及环介导等温扩增反应(loop-mediated isothermal amplification;LAMP),但并不限定于此。可使用的其他扩增方法记载于美国专利第5242794号、第5494810号、第4988617号及美国专利申请序列第09/854317号。根据美国专利第4683195号、第4683202号及第4800159号公开的PCR(polymerase chain reaction)实施本发明的基因扩增方法。
PCR为众所周知的核酸扩增方法,并研发了其的诸多变形和应用。例如,为了增进PCR的特异性或灵敏度,对常规PCR步骤进行变形来研发了降落(touchdown)PCR、热启动(hot start)PCR、巢式(nested)PCR及增效(booster)PCR。并且,为了特定应用,研发了实时(real-time)PCR、差异显示PCR(differential display PCR:DD-PCR)、cDNA末端的迅速扩增(rapid amplification of cDNA ends:RACE)、多重PCR、反向聚合酶链式反应(inversepolymerase chain reaction:IPCR)、小载体(vectorette)PCR、热不对称交错(TAIL)-PCR(thermal asymmetric interlaced PCR)。与PCR有关的详细内容记载于McPherson,M.J.,及Moller,S.G.PCR.BIOS Scientific Publishers,Springer-Verlag New York BerlinHeidelberg,N.Y.(2000),其公开通过引用并入本说明书中。
在本发明中,用于基因扩增方法的引物为具有与上述miRNA的cDNA序列的互补序列的寡核苷酸。在本说明书中,术语“引物”为可在适当的温度、适当的缓冲液内、适当的条件(即,4种不同的核苷三磷酸及聚合反应酶)下作用为模板指导DNA合成的起始点的单链寡核苷酸。每种引物的适当的长度根据各种因子,如温度与引物的用途具有变化,通常为15个至30个核苷酸。为了短的引物分子与模板形成充分稳定的杂交复合物,通常需要较低的温度。
引物的序列无需为模板的一部分序列的完全互补序列,没有特别限制,只要在与模板杂交时起到引物固有作用的范围内具有充分的互补性就可以。因此,本发明中的引物集无需为作为模板的上述标记物的cDNA序列的完全互补序列,没有特别限制,只要在与上述序列杂交时起到引物作用的范围内具有充分的互补性就可以。优选地,本发明中利用的引物为上述标记物的cDNA序列的完全互补序列。
普通技术人员可通过参照上述miRNA的cDNA序列来容易实施这种引物的设计,例如,PRIMER 3程序可用于引物设计。
以适当的方法分析以如上所述的方式扩增的上述标记物的cDNA,来研究上述标记物的基因的表达水平。例如,对如上所述的扩增反应产物进行凝胶电泳,并观察及分析最终形成的带,从而分析研究上述标记物的基因的表达程度。
通过这种扩增反应,在生物样品的上述基因的表达比正常样品基因的表达上调或下调的情况下,将对象诊断为患有帕金森病。
在本发明中,miRNA为帕金森病中的表达下调或上调的生物分子。在本说明书中使用的术语“高表达(或超表达)”或“上调”为研究对象的生物样品中的靶核苷酸序列或蛋白质的表达水平比正常样品中的高的情况。例如,在通过本领域通常所利用的表达分析方法,例如逆RT-PCR或ELISA方法(参照:Sambrook,J.et al.,Molecular Cloning.A LaboratoryManual,3rd ed.Cold Spring Harbor Press(2001))进行表达分析的情况下,分析结果为表达上调的情况。例如,通过如上所述的诊断方法进行分析的结果,与正常样品相比,本发明的miRNA上调或下调10%以上的情况下,判断为本发明中的“高表达”或“低表达”,从而将对象诊断为患有帕金森病。
根据本发明的实施例部分,对于本发明的诊断方法所包含的上述miRNA,与健康对象相比,在帕金森病患者中经确认为上调或下调,上调或下调10%以上,优选地,上调或下调50%以上。
本发明的另一个方面提供一种用于帕金森病的诊断或预后分析的试剂盒,包含能够与选自miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324、miR-4726-5p、miR-1226-5p、miR-4767及miR-3064-5p的一种或多于一种的miRNA特异性结合的核酸。
根据本发明的优选实施方案,本发明的试剂盒还包含能够与选自hsa-miR-494-3p、hsa-miR-1244、hsa-miR-6768-5p、hsa-miR-4324、hsa-miR-4726-5p、hsa-miR-501-5p、hsa-miR-1226-5p、hsa-miR-4767及hsa-miR-3064-5p中的一种或多于一种的miRNA特异性结合的核酸。
除了核算外,本发明的试剂盒可包含用于检测帕金森病的本领域已知的核酸或将来可发现的核酸,本发明的试剂盒还可包含用于检测帕金森病的标记物的已知抗体。
本发明的试剂盒中所包含的上述核酸可单独包装或包装为其他容器中的任何组合。
本发明的试剂盒可包括用于从体液、细胞或组织提取核酸(例如,总RNA)的试剂盒、标记用荧光物质、核酸扩增用酶及培养基、使用说明书等。
本发明的试剂盒为用于测定帕金森病标记物的设备,其中例如核算与固相结合或附着于固相。固相材质的实例包括塑料、纸、玻璃、硅酮等,容易加工的优选的固相材质为塑料。固相的形状随机,例如可呈四边形、圆形、矩形、膜的形式等。
本发明的试剂盒可包含可分别与上述miRNA的至少一个特异性结合的核酸,优选地,为可分别与上述miRNA的至少两个特异性结合的核酸,更优选地,为可分别与上述miRNA的至少三个特异性结合的核酸。
本发明的另一个方面提供一种筛选诱发帕金森病的物质的方法,包括:
(i)用诱发帕金森病候选物质处理表达选自miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324及miR-4726-5p中的一种或多于一种的miRNA的细胞,来在经处理的细胞中确认所选miRNA的表达水平;以及
(ii)在经处理的细胞中所选miRNA的表达水平下调的情况下,判断为诱发帕金森病的物质。
根据本发明的优选实施方案,本发明的筛选方法还包括:
(i)用诱发帕金森病候选物质处理表达选自miR-1226-5p、miR-4767及miR-3064-5p中的一种或多于一种的miRNA的细胞,来在经处理的细胞中确认所选miRNA的表达水平;以及
(ii)在经处理的细胞中所选miRNA的表达水平上调的情况下,判断为诱发帕金森病的物质。
本发明的另一个方面提供一种筛选诱发帕金森病物质的方法,包括:
(i)用诱发帕金森病候选物质处理表达选自miR-1226-5p、miR-4767及miR-3064-5p中的一种或多于一种的miRNA的细胞,来在经处理的细胞中确认所选miRNA的表达水平;以及
(ii)在经处理的细胞中所选miRNA的表达水平上调的情况下,判断为诱发帕金森病的物质。
本发明的另一个方面提供一种筛选帕金森病治疗剂的方法,包括:
(i)用帕金森病治疗剂候选物质处理表达选自miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324及miR-4726-5p中的一种或多于一种的miRNA的细胞,来在经处理的细胞中确认所选miRNA的表达水平;以及
(ii)在经处理的细胞中所选miRNA的表达水平上调的情况下,判断为帕金森病治疗剂。
根据本发明的优选实施方案,本发明的筛选方法还包括:
(i)用帕金森病治疗剂候选物质处理表达选自miR-1226-5p、miR-4767及miR-3064-5p中的一种或多于一种的miRNA的细胞,来在经处理的细胞中确认所选miRNA的表达水平;以及
(ii)在经处理的细胞中所选miRNA的表达水平下调的情况下,判断为帕金森病治疗剂。
本发明的另一个方面提供一种筛选帕金森病治疗剂的方法,包括:
(i)用帕金森病治疗剂候选物质处理表达选自miR-1226-5p、miR-4767及miR-3064-5p中的一种或多于一种的miRNA的细胞,来在经处理的细胞中确认所选miRNA的表达水平;以及
(ii)在经处理的细胞中所选miRNA的表达量下调的情况下,判断为帕金森病治疗剂。
在提及本发明的筛选方法的同时使用的术语“候选物质”是指为了检查是否具有诱发、减少、防止或去除帕金森病症状的活性而在筛选过程中利用的未知物质(例如,各种天然物质、化合物文库、基因或蛋白质文库等)。
本说明书中使用的术语“帕金森病治疗剂”为以呈现对于帕金森病的药理活性而周知的或确认的药物(药剂学组合物)、健康功能食品或食疗法。例如,在本发明中,可通过使用以具有对于帕金森病的药理活性而在本领域周知的药物和功能性食品使得能够对诊断为患有帕金森病的对象灵敏的进行诊断或预测预后。如另一个实例,本发明的方法可适用于在对于帕金森病的药理活性未周知的候选物质中筛选具有对于帕金森病的药理活性的物质。
本发明的有一个方面提供一种用于预防、改善或治疗帕金森病的组合物,包含选自miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324及miR-4726-5p中的一种或多于一种的miRNA作为活性成分。
本发明的另一个方面提供一种治疗帕金森病的方法,包括向患者施用有效量的选自miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324及miR-4726-5p中的一种或多于一种的miRNA的步骤。
根据本发明的一个实施方案,本发明的组合物为药剂学组合物。
本发明的药剂学组合物可以包含的药剂学上可接受的载体,其通常是本领域已知的。适合用于本发明的药剂学组合物的载体包括乳糖、右旋糖、蔗糖、山梨糖醇、甘露醇、淀粉、阿拉伯胶、磷酸钙、海藻酸、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁及矿物油等,但并不限定于此。除上述成分之外,本发明的药剂学组合物还可包含选自润滑剂、湿润剂、甜味剂、香味剂、乳化剂、悬浮剂、防腐剂等的一种或多于一种的添加剂。适合的药剂学上可接受的载体及制剂详细记载于Remington′s Pharmaceutical Sciences(19th ed.,1995)。
本发明的药剂学组合物可口服给药或肠胃外施用,在肠胃外施用的情况下,可进行鼻腔施用、滴眼施用、静脉注射、皮下注射、肌内注射、腹腔注射、经皮给药等。
本发明的药剂学组合物的适当剂量根据如制剂化方法、施用方式、患者的年龄、体重、性别、病理状况、食物、施用时间、施用途径、排泄速度及反应灵敏性的因素不同,通常,熟练的医生可以容易地确定并处方所期望的对于治疗或预防有效的药剂学组合物的量。根据本发明的优选实施方案,本发明的药剂学组合物的每日剂量为0.001mg/kg至100mg/kg。
本发明的药剂学组合物可根据本领域所属技术人员容易实施的方法利用药剂学上可接受的载体和/或赋形剂制备为单位剂量形式或分散在多剂量容器中。此时,剂型可为油或水性介质中的溶液、混悬剂或乳剂形式,或还可为提取物、散剂、颗粒剂、片剂或胶囊形式,还可包括分散剂或稳定剂。
本发明的药剂学组合物可制备为外用皮肤制剂、气溶胶、喷雾剂、眼药水、口服制剂和注射用制剂。
根据本发明的一个实施方案,本发明的组合物为食品组合物。
在本发明的食品组合物还可以任选地包含制备食品时通常添加的一种或多于一种的成分,例如,任选的成分可以选自蛋白质、碳水化合物、脂肪、营养素、调味剂及香味剂。如上所述的碳水化合物的实例包括糖类,例如葡萄糖、果糖等的单糖,如麦芽糖、蔗糖、低聚糖等的二糖,如糊精,环糊精等的多糖以及木糖醇、山梨糖醇、赤藓糖醇等的糖醇。作为调味剂可使用天然调味剂(牛磺酸、甜菊提取液[例如,莱鲍迪甙A、甘草酸苷等])及合成调味剂(糖精、阿斯巴甜等)。
例如,在本发明的食品组合物制备为饮料的情况下,还可包含柠檬酸、高果糖玉米糖浆、糖、葡萄糖、乙酸、苹果酸、果汁、杜仲提取物、枣提取物、甘草提取物等。
发明的效果
本发明的特征及优点如下:
(i)本发明提供一种提供用于诊断帕金森病的信息的方法。
(ii)本发明提供用于预防、改善或治疗帕金森病的组合物。
(iii)本发明所用的表达的miRNA在帕金森病模型中特异性下调或上调,可有效地用于诊断及治疗帕金森病。
附图说明
图1示出在帕金森病模型细胞的凋亡步骤中表达下调的miRNA。
图2示出在帕金森病模型细胞的凋亡步骤中表达上调的miRNA。
图3为注入6-OHDA的内部对照组(internal control)的照片。在脑组织未进行任何处理的左半球的黑质保存完好(A),相反,注射的6-羟基多巴胺(6-hydroxy dopamineinjected)的右半球的黑质的神经元凋亡,并且由于组织的变形,其大小过多地收缩(B)。
图4示出在与6-OHDA一同注射494-3p的影响下的黑质的活神经元。通过H&E染色法确认相同个体内的A(正常)和B(注射)中的病变。
图5示出在与6-OHDA一同注射miR1244的影响下的黑质的活神经元。通过H&E染色法确认相同个体内的A(正常)和B(注射)中的病变。
图6示出在与6-OHDA一同注射miR4324的影响下的黑质的活神经元。通过H&E色法确认相同个体内的A(正常)和B(注射)中的病变。
图7示出在与6-OHDA一同注射miR4726-5的影响下的黑质的活神经元。通过H&E染色法确认相同个体内的A(正常)和B(注射)中的病变。
图8示出在6-OHDA一同注射miR6768-5的影响下的黑质的活神经元。通过H&E染色法确认相同个体内的A(正常)和B(注射)中的病变。
图9示出在与6-OHDA一同注射miR501-5p的影响下的黑质的活神经元。通过H&E染色法确认相同个体内的A(正常)和B(注射)中的病变。
实施发明的最佳方式
以下,通过实施例更加详细地说明本发明。这些实施例仅用于具体说明本发明,根据本发明的主旨,本发明的范围并不限定于这些实施例,这对本发明所属技术领域的普通技术人员而言是显而易见的。
实施例
材料及方法
1.培养SH-SY5Y细胞
在补充由为10%的加热灭活胎牛血清(GIBCO,MD,美国)的DMEM(Dulbecco’sModified Eagle’s Medium,Invitrogen,MD,美国)培养基中,在37℃的温度、5%的湿度的CO2腔室内培养C57BL/6 SH-SY5Y成神经细胞(Jang,S.-W.,Oh,M.-S.,Yang,S.I.&Cho,E.-M.Gene expression profiles of human neuroblastoma cells exposed to CuOnanoparticles and Cu ions.BioChip Journal 10,140-149(2016))。并且,在使用前将6-羟基多巴胺(Sigma-Aldrich,St.Louis,MO,美国)溶解于磷酸盐缓冲液来在-80℃的温度中保存,每当需要使用时,将溶液添加于板中并分批次使用试剂,尤其,注意了光,因为溶液是光敏性的。
2.细胞生存力试验
为了观察细胞的生存,使用了稳定的四唑盐、WST-1试验(Sigma-Aldrich,St.Louis,MO,美国),将SH-SY5Y细胞以各个孔5000个细胞的方式添加于96孔板,并用6-羟基多巴胺处理(6OHDA,25μM,24h)。与仅进行磷酸盐缓冲液处理的对照组进行比较,在实验结束2小时之前以10μl/孔添加WST-1溶液,并在37℃的腔室内培养细胞。在实验结束后,测定并确认在450nm处的颜色变化。
3.分离RNA
用60HDA处理(25μM,24h)的SH-SY5Y细胞系的总RNA通过使用Trizol试剂(reagent)来分离。其方法遵循制备公司的推荐方法(Invitrogen,California,美国),利用NanoDrop分光光度计(Nano Drop,Delaware,美国)以260/280nm(比例1.8至2.0)比例为基准测定分离的RNA的总量和纯度(Kim,G.W.e.a.Integrative analyses of differentialgene expression and DNA methylation of ethylbenzene-exposed workers.BioChipJournal 9,259-267(2015))。
4.分析miRNA表达
为了miRNA表达谱分析,使用Affymetrix miRNA 4.0阵列(Lee,S.E.e.a.Identification and characterization of MicroRNAs in acrolein-stimulated endothelial cells:Implications for vascular disease..BioChipJournal 9,144-155(2015)),利用基因表达综合(Gene Expression Omnibus,GEO)数据库分析微阵列数据。
5.靶预测及基因本体分析
为了根据miRNA表达谱预测miRNA靶基因,利用把扫描6.2数据库(TargetScan6.2DB),并遵循米兰达算法(miRanda algorithm)(Kim,G.W.et al.Integrative analyses ofdifferential gene expression and DNA methylation of ethylbenzene-exposedworkers.BioChip Journal 9,259-267(2015))。并且,利用基因本体(Gene Ontology,GO)类别(http://www.geneontology.org/)再次确认呈现频率最高且表达量高的变化的靶miRNA(Cho,H.et al.A relationship between miRNA and gene expression in themouse Sertoli cell line after exposure to bisphenol A.BioChip Journal 4,75-81(2010);Jeong,S.I.et al.MicroRNA microarray analysis of human umbilical veinendothelial cells exposed to benzo(a)pyrene.BioChip Journal 6,191-196(2012);Park,H.R.,Lee,S.E.,Yang,H.,Son,G.W.&Park,Y.S.Functional screening of alteredmicroRNA expression in 3-methylcholanthrene-treated human umbilical veinendothelial cells.BioChip Journal 8,260-268(2014);Park,J.H.et al.Expressionprofiles of miRNAs during ethanol-induced differentiation of neural stemcells.BioChip Journal 6,73-83(2012))。并以根据log2倍数变化示出表达水平的增减的顺序筛选miR。
6.制备帕金森病的动物模型
为了使6-OHDA(Sigma,St.Louis,美国)引起的毒性仅影响多巴胺能神经元,在注入6-OHDA的30分钟前,向实验动物的腹腔注射作为去甲肾上腺素转运阻断剂的地昔帕明(12.5mg/kg;Sigma,St.Louis,美国)。通过腹腔注射氯胺酮(40mg/kg)和甲苄噻嗪(5mg/kg)混合物,使实验动物处于深度麻醉状态下,之后在呼吸麻醉下固定于脑立体定向装置(David KOPF instrument.,CA,USA),切开头部皮肤来露出头骨后,确认前囟点后,并在以上述前囟点为基准向后侧移动1.1mm、向右侧移动1.2mm的位置利用牙钻穿小孔。插入26号针,使其通过孔到达后面5.0mm位置,使得其位于前脑内侧束(medial forebrain bundle;MFB)上。利用输液泵(Harvard Apparatus.,USA)利用5μL的Hamilton注射器以0.5μL/min的速度注射2μL的6-OHDA溶解于0.1%的抗坏血酸的溶液(2.5μg/μL)。利用另一个5μL的Hamilton注射器以0.5μL/min的速度注射5uL的实验miRNA,注射完成5分钟之后,去除Hamilton注射器并缝合皮肤。左半球为了用作内部对照而未注入任何物质。利用氯胺酮(70mg/kg)和甲苄噻嗪(8mg/kg)混合物麻醉实验动物后,通过心脏向动物灌注4%的多聚甲醛(在0.1M磷酸盐缓冲液中,pH7.4),然后固定,切除脑组织。
结果
1.PD模型的细胞的凋亡期间下调的miR
图1示出上述表达的上调和下调的miRNA中在PD模型细胞凋亡期间,表达的高度下调的miR。特别是,尚未报道miR494-3p与帕金森病相关,其在每次实验中反复表现出显著的减少。此外,在图1列出了以对数比为基准示出-2.0(30%以上)以上的表达下调的miR。特别是,生物信息数据分析显示,miR-1244靶向蛋白质“TBC1结构域家族成员2B”。重要的是,miRNA作为基本调节剂,靶向针对在帕金森病中的神经元凋亡期间特异性表达的基因之一(A Network View on Parkinson′s Disease,Comput Struct Biotechnol J.2013;7:e201304004)。
2.PD模型的细胞的凋亡期间上调的miR
图2示出上述表达的上调和下调的miRNA中在PD模型细胞凋亡期间,表达的高度上调的miR。
3.miR序列
上述表达的上调和下调的miRNA的序列信息如下:
在PD模型的细胞的凋亡步骤中,表达的高度下调的miRNA为hsa-miR-494-3p(序列1:UGAAACAUACACGGGAAACCUC)、hsa-miR-1244(序列2:AAGUAGUUGGUUUGUAUGAGAUGGUU)、hsa-miR-6768-5p(序列3:CACACAGGAAAAGCGGGGCCCUG)、hsa-miR-4324(序列4:CCCUGAGACCCUAACCUUAA)、hsa-miR-4726-5p(序列5:AGGGCCAGAGGAGCCUGGAGUGG)及hsa-miR-501-5p(序列6:AAUCCUUUGUCCCUGGGUGAGA)。
在PD模型的细胞的凋亡期间,表达的高度上调的miRNA为hsa-miR-1226-5p(序列7:GUGAGGGCAUGCAGGCCUGGAUGGGG)、hsa-miR-4767(序列8:CGCGGGCGCUCCUGGCCGCCGCC)及hsa-miR-3064-5p(序列9:UCUGGCUGUUGUGGUGUGCAA)。
4.H&E染色
在脑的功效评价中,通过H&E染色确认并比较发生帕金森病的主要病变的黑质致密部的细胞生存力。
1)组织的固定:使细胞的酶失活,通过凝结或沉淀使组织内的成分转换为不溶性状态,将组织浸渍于固定液并在4℃温度条件下固定一天或一天以上,以很好地保存结构。
2)脱水及清洗:去除水分并去除用于去除水分的溶剂。这是为了使石蜡容易渗透至亚组织中。
3)制备石蜡块:利用石蜡对组织的内外部进行定型,由此使组织或细胞结构不变形。
4)制备载玻片:用切片刀将石蜡块切成10μm厚度的连续冠状切片以制备硅烷涂层载玻片。
5)染色:处理后,去除石蜡而保存组织,并利用哈里斯苏木精对细胞核进行染色,并利用伊红Y进行对比染色。
6)细胞染成蓝色,对比染色部分为粉红色。
5.miRNA的神经元凋亡抑制效果
如图3至图9所确认,在仅注射6-OHDA的内部对照(图3)中,不仅发生神经元凋亡,还由于组织的变形其大小过于收缩,相反,在接受miR494-3p(图4)、miRl244(图5)、miR4324(图6)、miR4726-5p(图7)、miR6768-5p(图8)或miR501-5p(图9)的实验组中,确认到神经元仍存活。由此,证明了本发明的miRNA具有卓越的预防神经元凋亡的效果。
尽管详细记述了本发明的特定部分,但是对于本技术领域的普通技术人员而言显而易见的是,这种具体技术仅为优选实施方案,并不意图限制本发明的范围。因此,本发明的实质范围由所附权利要求和其等同技术方案限定。
Claims (11)
1.一种提供用于诊断帕金森病的信息的方法,包括:
(a)对选自从对象获取的样品中所包含的miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324及miR-4726-5p中的一种或多于一种的miRNA的表达水平与正常样品中所选miRNA的表达水平进行比较;以及
(b)在对象的样品中所选miRNA的表达水平比正常样品中所选miRNA的表达水平减少的情况下,诊断为对象患有帕金森病。
2.根据权利要求1所述的方法,还包括:
(a)对选自从对象获取的样品中所包含的miR-1226-5p、miR-4767及miR-3064-5p中的一种或多于一种的miRNA的表达水平与正常样品中所包含的所选miRNA的表达水平进行比较;以及
(b)在对象的样品中所选miRNA的表达水平比正常样品中所选miRNA的表达水平增加的情况下,诊断为对象患有帕金森病。
3.一种提供用于诊断帕金森病的信息的方法,包括:
(a)对选自从对象获取的样品中所包含的miR-1226-5p、miR-4767及miR-3064-5p中的一种或多于一种的miRNA的表达水平与正常样品中所选miRNA的表达水平进行比较;以及
(b)在对象的样品中所选miRNA的表达水平比正常样品中所选miRNA的表达水平增加的情况下,诊断为对象患有帕金森病。
4.一种用于帕金森病的诊断或预后分析的试剂盒,包含能够与选自miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324、miR-4726-5p、miR-1226-5p、miR-4767及miR-3064-5p中的一种或多于一种的miRNA特异性结合的核酸。
5.一种筛选诱发帕金森病物质的方法,包括:
(i)用诱发帕金森病候选物质处理表达选自miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324及miR-4726-5p中的一种或多于一种的miRNA的细胞,并量化在经处理的细胞中所选miRNA的表达水平;以及
(ii)在经处理的细胞中所选miRNA的表达水平下调的情况下,判断为诱发帕金森病物质。
6.一种筛选诱发帕金森病物质的方法,包括:
(i)用诱发帕金森病候选物质处理表达选自miR-1226-5p、miR-4767及miR-3064-5p中的一种或多于一种的miRNA的细胞,并量化在经处理的细胞中所选miRNA的表达水平;以及
(ii)在经处理的细胞中所选miRNA的表达水平上调的情况下,判断为帕金森病诱发物质。
7.一种筛选帕金森病治疗剂的方法,其特征在于,包括:
(i)用帕金森病治疗剂候选物质处理表达选自miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324及miR-4726-5p中的一种或多于一种的miRNA的细胞,并量化在经处理的细胞中所选miRNA的表达水平;以及
(ii)在经处理的细胞中所选miRNA的表达水平上调的情况下,判断为帕金森病治疗剂。
8.一种筛选帕金森病治疗剂的方法,其特征在于,包括:
(i)用帕金森病治疗剂候选物质处理表达选自miR-1226-5p、miR-4767及miR-3064-5p中的一种或多于一种的miRNA的细胞,并量化在经处理的细胞中所选miRNA的表达水平;以及
(ii)在经处理的细胞中所选miRNA的表达水平下调的情况下,判断为帕金森病治疗剂。
9.一种用于预防或治疗帕金森病的药剂学组合物,包含选自miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324及miR-4726-5p中的一种或多于一种的miRNA作为活性成分。
10.一种用于预防或改善帕金森病的食品组合物,包含选自miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324及miR-4726-5p中的一种或多于一种的miRNA作为活性成分。
11.一种用于治疗帕金森病的方法,包括向对象施用有效量的选自miR-494-3p、miR-501-5p、miR-1244、miR-6768-5p、miR-4324及miR-4726-5p中的一种或多于一种的miRNA。
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