CN109680046B - 骨关节炎第一亚型的生物标记物以及用途 - Google Patents
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Abstract
本发明公布了骨关节炎第一亚型的生物标记物以及用途,主要是蛋白多糖代谢和/或硫酸软骨素代谢途径上的生物物质在诊断骨关节炎的分型上的用途。
Description
技术领域
本发明涉及一种基于转录组学对膝关节炎疾病分型的方法和疾病亚型标志性基因和通路的检测,特别的属于利用生物标记物来诊断骨关节炎的分型。
技术背景
骨关节炎(Osteoarthritis, OA)是全球所有国家遇到的最常见的肌肉骨骼系统疾病之一。在欧洲每一分半就有一例骨关节置换手术;全球有 50%的 65 岁以上人群患有骨关节炎;在中国,50 岁以 上人群有 25.3%患有骨关节炎。骨关节炎的临床症状是疼痛和关节功能障碍。关节功能的进行性衰退导致关节的活动性丧失以及功能受限;从而使患者活动受限,生活质量下降;约25%的OA患者无法完成正常生活中的主要活动。美国疾病防控中心和哈佛大学公共卫生部门的一项研究表明骨关节炎是引起老年人残疾的五个主要疾病之一。在发达国家,骨关节炎的治疗费占国内总生产值的 1.0%-2.5%。
目前对关节炎的发生机制假说有:“磨损” 理论,与衰老相关,软骨细胞对生长因子的反应性降低,线粒体功能障碍和 AGE (advanced glycation end products) 的异常累积;因此很多人认为,OA 是随着 机体老化自然产生的疾病;然而并不是所有的老年人都会患 OA。随着研究的深入,研究者发现软骨细胞外基质(如胶原或蛋白聚糖)可以诱导非酶胶原交联并 使蛋白聚糖分子缩短。胶原分子的过度交联影响软骨的生物力学性质,导致软骨僵硬、易碎;蛋白聚糖的缩短和降解导致其糖侧链缺失和亲水性丧失。 此外,AGEs 水平升高会导致合成代谢下降。但是这些现象并不能完全解释个体发生 OA 的风险和疾病进程,越来越多的研究表明 OA 的发生发展和遗传分子有紧密的联系。例如:(1)在蛋白编码基因方面:通过参与Wnt通路影响软 骨和骨发育的FRZB 基因已经被证明和 OA 有关。与胶原合成相关的基因COL2A1突变会导致OA,而COL1A1突变会减少女性患关节炎的风险。(2)表观遗传方面:甲基化的改变会影响基质降解基因(MMP13,ADAMTS4等)、 炎症因子基因(IL1b,IL6,IL8等)和其他 OA 相关转录因子(HOX,SOX9, DIO2 和 TGF-beta 等)的表达。组蛋白修饰的改变会影响胶原基因(COL2A1)和OA相关通路基因(SOX9,Sirtuin1 等)的表达。(3) 非编码基因方面:目前研究比较少,主要集中在小 RNA(mircoRNA)上。比如: miR-127-5p会抑 制 MMP-13表达,进而产生 IL-1b;如果抑制 miR-127-5p,MMP-13表达升高从而导致OA;miR-140 会靶向 ADAMTS5,IGFBP-5(insulin-like growth factor bindingprotein 5)等基因从而影响 OA 的发生。长非编码 RNA(long non-coding RNA, lncRNA)在骨关节炎中的报道也非常少,其中 Fu 发现一个lncRNA——UC.343 可能靶向HOXC8,影响IL-1b表达。
虽然有少数研究者对骨关节炎进行分类,但仅基于临床现象出发,所以现有的诊断骨关节炎的金标准依然是影像学;治疗也只能是止痛等保守的对症治疗或在病变部位采取手术方式。综上骨关节炎的分子研究现状可以发现,有很多研究已经揭示了骨关节炎的共性信号通路,对骨关节炎的治疗具有一定的启示作用;但没有能用于指导骨关节炎亚群诊断治疗的分子标记。而亚群解析研究对提升特定病人群体疗效具有重要意义并且已经建立了成功的实例,如肺癌中的表皮生长因子受体 (Epidermal growth factor receptor,EGFR)阳性亚群发现后,EFGR抑制剂给该亚型病人带来有效治疗效果。
这就需要更为准确的诊断OA的病例过程和分型,利于准确的治疗方案的,例如药物的使用。
发明内容
本发明从在基因层面研究骨关节炎的病理机制,对骨关节炎进行分型,找到有效和有针对性的诊断标志物,从而为治疗OA提供了新的标准。
本发明的目的是提供了一种基于转录组学对膝关节炎疾病分型的方法,首先利用OA患者软骨组织的转录组高通量测序数据,运用生物信息学方法对数据进行分析,找到四类骨关节炎亚型;并利用基因谱分析获得每种亚群的标志性基因。这些骨关节炎亚型的分型揭示了不同代谢路径的异常,从而为正确诊断不同亚型的关节炎提供解决方案,从而可以从根本上治疗骨关节炎的疾病,而不仅仅如传统的方法来使用止疼药。
一方面,本发明提供一种生物标记物质,这些生物标记物的与骨关节炎的分型或者类型直接关联。而这些生物标记物质与一些代谢途径相关联。
本发明的第一方面,本发明提供蛋白聚糖代谢和/或硫酸软骨素代谢途径上的生物物质在诊断骨关节炎的分型上的用途。在一些方式中,如果在一些蛋白聚糖代谢和/或硫酸软骨素代谢途径上的生物物质发生变化,例如增加或减少,则表示骨关节炎属于蛋白聚糖代谢和/或硫酸软骨素代谢类型的关节炎。优选的方式中,这些生物标记物质选自如下基因中的一种或者几种:蛋白聚糖代谢相关:PCOLCE2,S100B,ITM2C, ACAN,FBXO2,SOD3,SERPINA3,SSR3,DCN,WWP2,ITIH6,TCEAL2,FIBIN,FGFBP2,TSPAN6,PLA2G2A,SMOC2,TUBB2B,STC2,ACAN,DCN,FMOD,CDO1,PRELP,PAPSS2,B3GNT7,CHST3,CHST6,CSPG4,BGN,CSGALNACT1,CHPF;硫酸软骨素代谢相关基因:ITM2C,DCN,TMEM59,GALNT18,B3GNT7,CHST3,GALNT8,GALNT15,CSPG4,BGN,CSGALNACT1,CHPF,MAN1A2,ITM2A,EXTL1,RPN2,ITM2B,POFUT2,GOLGA2。在一些方式中,当这些基因的转录水平升高,则表示骨关节炎属于第一亚型的骨关节炎。
本发明的第二方面,提供在胶原降解和/或胶原纤维形成的代谢途径上的生物物质在诊断骨关节炎的分型上的用途。在一些优选的方式中,这些生物标记物质选自如下基因中的一种或者几种:胶原降解代谢相关基因:COL6A,COL5A,COL3A,NBL,DKK,ADAMTS2,ABHD,FNDC,LMNA,S100A,MXRA,ANTXR1;胶原纤维形成相关基因:COL5A,COL3A,GJA,ADAMTS2,TGFB,SOX4,ITGA11,LTBP2,AEBP1。在一些方式中,当这些基因的转录水平升高,则表示骨关节炎属于第二亚型的骨关节炎。
本发明的第三方面,提供神经突触调节和/或离子通道上的生物物质在诊断骨关节炎的分型上的用途。在一些优选的方式中,这些生物标记物质选自如下基因中的一种或者几种:神经突触调节相关基因:GRID2,ADGRL3,NRXN1,LINGO2,IL1RAPL1,ADGRB3,PTPRD,LRRC4C,CTNNA2,MACROD2,PCDH15,SGCZ,CNTNAP2,GRID2, GALNTL6,MT-ND5, LRRTM4,CALN1,CTNNA3,AC007682.1,EYS, ADGRL3 ,GRM7,;离子通道相关基因:GRID2,CNTNAP2,KCNIP4,DPP10,GRIK2,DLG2,CACNA1A,CNTNAP5,CNTN5,KCNIP4,EYS,GRM7,ADGRL3;在一些方式中,当这些基因的转录水平升高,则表示骨关节炎属于第三亚型的骨关节炎。
本发明的第四方面,提供免疫反应和/或血管生成的生物物质在诊断骨关节炎的分型上的用途。在一些优选的方式中,这些生物标记物质选自如下基因中的一种或者几种:免疫反应相关基因:PECAM1,SYK,STOM,GMFG,TSPAN14,PTPRC,COTL1,ARPC5,CAP1,FCGR2B,CD36,VAMP8,CD14,RHOA,SDCBP,RAC1,CTSB,RAB10,RAP1B,PDXK,CYBA,GM2A,GRN,VCL,GNS,ADAM10,PSAP,ATP6AP2,ASAH1,ACTR2,FTL,PLD1,TCIRG1,NRAS,CST3,RAP2B,COLEC11,DUSP6,MPEG1,STOM,PLVAP,IFI16,ATP6V1B2,ADGRL4,PCDH18,APLNR,C1orf162,ZFP36L1,SLCO2B1,PTPRC,ACSL1,COTL1;血管生成相关基因:MCAM,SYK,APLNR,EGFL7,NOTCH3,CD34,MMRN2,ECSCR,NRP1,GPX1,JUN,CYP1B1,PLXND1,RHOA,GPNMB,C1GALT1,ENG,MYH9,EPAS1,GNA13,ATP5B,RBPJ,PDGFRA,PGF,MMP2,STAT1,TMSB4X,LAPTM5,LGALS3BP,YWHAB, ZEB2,EGFL7,GMFG,SYK,MCAM,MYL6,KCTD12。在一些方式中,当这些基因的转录水平升高,则表示骨关节炎属于第四亚型的骨关节炎。
在一方面,本发明提供关节液生物标记物质在诊断骨关节炎属于第一亚型上的用途,所述的生物标记物质选自于SCF,RANTES,IFN或IL18中的或者几种。本发明提供关节液中的生物标记物质在诊断骨关节炎属于第二亚型上的用途,所述的生物标记物质选自于VEGF和/或IL6。本发明提供关节液生物标记物质在诊断骨关节炎属于第四亚型上的用途,所述的生物标记物质选自于MIP,SDF或IL8。
这里可以说明,关节液中的标记物质的出现,也从另外一方面证明本发明的分型正确,也进一步说明这些标记物质的出现,也可以作为分型的一个确定的方法。并不是所有亚型的骨关节炎的炎症因子是一样的,而是不同的亚型的炎症因子是不同的,正好可以说明分型的重要性,与不同的代谢途径是相关联的。
另一方面,本发明提供一种诊断骨关节炎亚型的方法,所述的方法包括: 提供样本并对样本进行生物标记物的分析,如果分析的标记物属于蛋白多糖代谢和/或硫酸软骨素代谢途径上的标记物质,则认为该骨关节炎属于第一亚型的关节炎;如果分析的标记物属于胶原降解和胶原纤维形成的代谢途径上的标记物质,则认为该骨关节炎属于第二亚型的关节炎; 如果分析的标记物属于神经突触调节和离子通道上的生物物质,则认为该骨关节炎属于第三亚型的关节炎; 如果分析的标记物属于免疫反应和血管生成的生物物质,则认为该骨关节炎属于第四亚型的关节炎。
在一些方法中,提供样本的主体是骨关节炎患者。在另外一些方式中,所述的生物标记物质出现异常,从而根据异常来判断骨关节炎的分型。这的异常通常是与以上代谢物质相关的生物物质的含量高于正常人的水平(不是骨关节炎的患者)。这些的异常可以是蛋白、核酸水平的升高,例如一些基因表达升高,则表面某一些类型的骨关节炎的分型。
在一些优选的方式中,生物标记物质包括: 蛋白聚糖代谢相关:PCOLCE2,S100B,ITM2C, ACAN,FBXO2,SOD3,SERPINA3,SSR3,DCN,WWP2,ITIH6,TCEAL2,FIBIN,FGFBP2,TSPAN6,PLA2G2A,SMOC2,TUBB2B,STC2,ACAN,DCN,FMOD,CDO1,PRELP,PAPSS2,B3GNT7,CHST3,CHST6,CSPG4,BGN,CSGALNACT1,CHPF;和/或者与硫酸软骨素代谢相关基因:ITM2C,DCN,TMEM59,GALNT18,B3GNT7,CHST3,GALNT8,GALNT15,CSPG4,BGN,CSGALNACT1,CHPF,MAN1A2,ITM2A,EXTL1,RPN2,ITM2B,POFUT2,GOLGA2。在一些方式中,当这些基因的转录水平升高,则表示骨关节炎属于第一亚型的骨关节炎。
在一些优选的方式中,生物标记物质与胶原降解和/或胶原纤维形成的代谢途径相关。在一些优选的方式中,这些生物标记物质选自如下基因中的一种或者几种:胶原降解代谢相关基因:COL6A,COL5A,COL3A,NBL,DKK,ADAMTS2,ABHD,FNDC,LMNA,S100A,MXRA, ANTXR1;胶原纤维形成相关基因:COL5A,COL3A,GJA,ADAMTS2,TGFB,SOX4,ITGA11,LTBP2, AEBP1。在一些方式中,当这些基因的转录水平升高,则表示骨关节炎属于第二亚型的骨关节炎。
在一些优选的方式中,生物标记物质神经突触调节和/或离子通道上相关。在一些优选的方式中,这些生物标记物质选自如下基因中的一种或者几种:神经突触调节相关基因:GRID2,ADGRL3,NRXN1,LINGO2,IL1RAPL1,ADGRB3,PTPRD,LRRC4C,CTNNA2,MACROD2,PCDH15,SGCZ,CNTNAP2,GRID2, GALNTL6,MT-ND5, LRRTM4,CALN1,CTNNA3,AC007682.1,EYS, ADGRL3 ,GRM7,;离子通道相关基因:GRID2,CNTNAP2,KCNIP4,DPP10,GRIK2,DLG2,CACNA1A,CNTNAP5,CNTN5,KCNIP4,EYS,GRM7,ADGRL3;在一些方式中,当这些基因的转录水平升高,则表示骨关节炎属于第三亚型的骨关节炎。
在一些优选的方式中,生物标记物质免疫反应和/或血管生成的相关。在一些优选的方式中,这些生物标记物质选自如下基因中的一种或者几种:免疫反应相关基因:PECAM1,SYK,STOM,GMFG,TSPAN14,PTPRC,COTL1,ARPC5,CAP1,FCGR2B,CD36,VAMP8,CD14,RHOA,SDCBP,RAC1,CTSB,RAB10,RAP1B,PDXK,CYBA,GM2A,GRN,VCL,GNS,ADAM10,PSAP,ATP6AP2,ASAH1,ACTR2,FTL,PLD1,TCIRG1,NRAS,CST3,RAP2B,COLEC11,DUSP6,MPEG1,STOM,PLVAP,IFI16,ATP6V1B2,ADGRL4,PCDH18,APLNR,C1orf162,ZFP36L1,SLCO2B1,PTPRC,ACSL1,COTL1;血管生成相关基因:MCAM,SYK,APLNR,EGFL7,NOTCH3,CD34,MMRN2,ECSCR,NRP1,GPX1,JUN,CYP1B1,PLXND1,RHOA,GPNMB,C1GALT1,ENG,MYH9,EPAS1,GNA13,ATP5B,RBPJ,PDGFRA,PGF,MMP2,STAT1,TMSB4X,LAPTM5,LGALS3BP,YWHAB, ZEB2, EGFL7,GMFG,SYK,MCAM,MYL6,KCTD12。在一些方式中,当这些基因的转录水平升高,则表示骨关节炎属于第四亚型的骨关节炎。
在一些方式中,所述的样本为软骨组织,软骨下骨组织或滑膜组织中的一种或者几种。
另一方面,本发明的分型方法如下:基于转录组学对膝关节炎疾病分型的方法和疾病亚型标志性基因的检测步骤如下:
(1)收集病例样本;
(2)对样本进行cDNA建库,RNA-seq测序;
(3)利用无监督的方法对样本分群;
(4)挖掘亚型的标志性基因和功能;
(5)多组织分析验证分群;
(6)组织染色验证分群。
进一步地,所述步骤(1)的具体实现为:收集多中心病人样本,本研究收集了四个医院的骨关节置换手术病人的膝关节。提取了患者的软骨组织,软骨下骨组织和滑膜组织。
进一步地,所述步骤(2)的具体实现为:针对软骨组织的cDNA建库方法:选取每个样本的软骨,滑膜,软骨下骨,分别用研磨管及研磨仪进行组织研磨并提取RNA;逆转录后清洗,富集cDNA;Nanodrop测定RIN>7,总量>500ng的样本保留测序。
进一步地,所述步骤(3)的具体实现为:对获得的高通量转录组数据进行低质量数据去除,剩余的数据进行基因组映射,并统计样本中基因的表达量。基于基因表达谱利用非监督的方法对样本进行分群。此处用了SC3的聚类方法,我们发现分四群的结果有最好的Silhouette值。并且四群病人在表型上也有明显的不同,第一群患者蛋白多糖基质讲解严重(多糖降解型);第二群患者软骨旁骨赘增生严重(骨赘增生型);第三群患者年龄最小,表现出疼痛敏感(疼痛型);第四群患者关节间隙狭窄并有高炎症反应。
进一步地,所述步骤(4)的具体实现为:筛选p值小于0.001,AUROC大于0.9的基因作为亚型的标志性基因。
进一步地,所述步骤(6)的具体实现为:获取患者软骨下骨和滑膜组织的表达谱数据,并用配体受体关节分析整个关节中的多组织互做。发现蛋白多糖降解型患者膝关节的细胞外基质组成通路在多组织互做中活跃;胶原讲解型患者的胶原降解代谢通路在滑膜配体-软骨受体的互做表达显著;高炎型患者的骨分化和骨重建在下骨作为受体的互做中表达显著。这些发现证实了上面的亚型标志性通路与亚型表型的一致性。
进一步地,所述步骤(7)的具体实现为:Safarinin O &Fastgreen染色和组织免疫组化染色实验验证亚型代谢通路。本发明检测了4个OA亚群的目的基因表达水平。SO染色结果显示蛋白聚糖(Aggrecan)的表达水平, 比较四个群的染色结果,与正常样本相比,四群均有蛋白聚糖合成减少,C1呈现明显多糖代谢紊乱特征。最后,本发明选择了MMP13, 突触素(Synapotopsin),CD34三个标记物质来进行免疫组织化学染色实验,其中,MMP13是指征软骨基质降解的生物标记物质(marker), C2组化结果较其他三群显示明显MMP13表达增加的现象;Synapotopsin是与神经相关的marker, Synapotopsin在C3更高表达;CD34是指征炎症的重要生物标记物质,我们发现组化染色显示C4具有高炎症发生的特点。与分群结果一致,C1主要表现为多糖代谢紊乱,C2主要表现为软骨胶。由此可见,这些MMP13,Synapotopsin,CD34也可以作为炎症分型的重要指标之一,这是因为这些标记物的异常与分型是紧密联系的,也是不用代谢途径异常的重要的标记物质之一。
附图说明
图1是分析流程图。
图2是亚型标志性基因和标志性代谢通路的结果图。
图3是SO和组化染色验证。
图4是蛋白组学验证分析结果图。
具体实施方式
为了更为具体地描述本发明,下面结合附图及具体实施方式对本发明的技术方案进行详细说明。这些说明仅仅是表明本发明是如何实现的,并不能限定本发明的具体范围。
本发明基于转录组学对膝关节炎疾病分型的方法和疾病亚型标志性基因的发现具体步骤如下:
实施例子1:收集病例
收集多中心病人样本,本研究收集了四个医院的骨关节置换手术病人的膝关节。提取了患者的软骨组织,软骨下骨组织和滑膜组织。
实施例子2:对样本进行cDNA建库,RNA-seq测序
组织样本的获取和处理(实施例子1的样本):本发明使用外科环钻从直接在先前检查的侵蚀和完整软骨下面的区域获得0.5cm直径,1cm深的软骨下骨小梁骨芯。去除残留的软骨组织。立即在液氮中快速冷冻骨,并使用液氮冷却研磨机进行低温研磨。软骨取磨损的全层软骨组织,在液氮中快速冷冻并使用液氮冷却研磨机进行低温研磨。取磨损软骨附近的滑膜,同样冷冻并进行研磨。
RNA分离和逆转录:使用RNeasy Fibrous Tissue Mini Kit(Qiagen,Duesseldorf,Germany)按照制造商的说明从匀浆组织中分离总RNA。通过Nano-Drop(NanoDrop Technologies,DE,USA)测定RNA浓度和纯度在Bioanalyzer 2100(Agilenttechnologies,CA,USA)中评估RNA的完整性,以便随后仅使用RIN(RNA完整性数值)等于或大于6的那些样品。
将总RNA与1μl锚定的oligo-dT引物(一种随机引物)和1μl的dNTP混合物(10mM,Fermentas)混合,在72℃变性5分钟。 7微升第一链反应混合物(含有0.50μlSuperScriptII逆转录酶(Invitrogen),0.25μlRNA酶抑制剂(Clontech),2μlSuperscriptII First-Strand Buffer(Invitrogen),0.25μlDTT(Invitrogen),1μl将甜菜碱(Sigma),0.9μlMgCl2(Sigma)和0.1μl无核酸酶水(Gibco)加入每个样品中。通过在42℃温育90分钟,然后进行10个循环(50℃2分钟,42℃2分钟)进行逆转录反应。最后,通过在70℃温育15分钟使逆转录酶失活。然后在加入10μl第二链合成缓冲液2μl,酶混合物1μl,cDNA 100ng和水至最终反应体积为20μl后进行第二链合成。将反应物在16℃温育150分钟,然后用AMPure XP珠纯化。
转录组文库的制备:将5纳克cDNA用Nextera DNA样品制备试剂盒(Illumina)进行的标记反应,加入2.5μl2×Tagment DNA缓冲液和1.25μlTagmentDNA酶,最终体积为5μl。将标记反应在55℃下孵育10分钟。然后将整个体积用于限制性循环富集PCR,以及3.75μl的Nextera PCR引物混合物(NPM),1.25μl的Index 1引物(N7xx)和1.25μl的PCR引物混合物(PPC)。如下进行第二轮扩增:72℃3分钟,98℃30秒,然后5个循环(98℃10秒,63℃30秒,72℃3分钟)。用1:1比例的AMPure XP磁珠进行纯化,并将样品上样到High-Sensitivity DNA芯片上以检查文库的质量,同时用Qubit高灵敏度DNA试剂盒(Invitrogen)进行定量。将文库稀释至2nM的终浓度并合并,并在Illumina HiSeq 2500上对10pmol进行测序。
实施例子3:利用无监督的方法对样本分群
对获得的高通量转录组数据进行低质量数据去除:1)用FASTX_TOOLKIT去除低质量的数据,去除质量大于20的碱基的含量小于80%的序列。2)选择总的序列读数在三十万到两百万之间的样本。剩余的数据进行基因组映射,并统计样本中基因的表达量。选择在大于10个样本中出现的基因。基于这些基因的表达谱,利用非监督的方法对样本进行分群。此处用了SC3的聚类方法,我们发现分四群的结果有最好的轮廓系数(Silhouette)值。
3.1 挖掘亚型的标志性基因
筛选p值小于0.0001,AUROC大于0.9的基因作为亚型的标志性基因。本发明发现显著的标志性基因主要有(具体见图2A和2B.1-4)
第一亚型:PCOLCE2,S100B,ITM2C,C2orf40,ACAN,FBXO2,SOD3,SERPINA3,SSR3, DCN,WWP2,ITIH6,TCEAL2,FIBIN,FGFBP2,TSPAN6,PLA2G2A,SMOC2,TUBB2B,STC2。
第二亚型:TGFB,COL6,NBL,DKK,GJA,COL5,ABHD,FNDC,LMNA,S100A,COL3,S100A, MXRA。
第三亚型:LRRC4C,CTNNA2,MACROD2,PCDH15,SGCZ,CNTNAP2,GRID2,CNTNAP5, CNTN5,GALNTL6,MT-ND5,KCNIP4,LRRTM4,CALN1,CTNNA3,AC007682.1,EYS,GRM7,ADGRL3。
第四亚型:TMSB4X,PECAM1,LAPTM5,LGALS3BP,COLEC11,DUSP6,MPEG1,STOM, YWHAB,PLVAP,IFI16,ZEB2,ATP6V1B2,EGFL7,GMFG,SYK,MCAM,MYL6,KCTD12,ADGRL4, PCDH18,APLNR,C1orf162,ZFP36L1,SLCO2B1,PTPRC,ACSL1,COTL1。
分别对这些不同亚型的基因对应的功能进行分析,发现不同的亚型对应不同的代理途径。对他们的功能进行分析发现不同亚型的标志性功能通路是:第一亚型:蛋白多糖代谢或硫酸软骨素代谢;第二亚型:胶原降解或胶原纤维组成;第三亚型:神经突触调节或离子通道;第四亚型:免疫反应或血管生成。
从以上基因的转录水平进行分析,发现显著区分的四个亚型,则从基因水平上进行了骨关节炎的不同类型的划分。另外,不同的基因对应不同的代谢途径或者生物体内的生物物质循环路径不同。所以,这些基因可以作为分型的生物标记物质,当发现患者是骨关节炎的时候,分析这些基因是否在关节软骨组织中存在以上基因的高度表达或者转率水平升高,则可以按照上述标准来进行亚型的区分。
例如,当在软骨组织检测发现PCOLCE2,S100B,ITM2C,C2orf40,ACAN,FBXO2,SOD3,SERPINA3,SSR3,DCN,WWP2,ITIH6,TCEAL2,FIBIN,FGFBP2,TSPAN6,PLA2G2A,SMOC2,TUBB2B,STC2中的一个基因或者多个基因的转率水平升高,则表示该关节炎属于蛋白多糖代谢或/和硫酸软骨素代谢问题引起的关节炎。可以理解,当在软骨组织检测发现TGFB,COL6,NBL, DKK,GJA,COL5,ABHD,FNDC,LMNA,S100A,COL3,S100A,MXRA中的一个基因或者多个基因的转录水平升高,则表示该关节炎属于胶原降解和/或胶原纤维组成路径出现问题而引起的关节炎。更可以理解,当在软骨组织检测发现LRRC4C,CTNNA2,MACROD2,PCDH15,SGCZ,CNTNAP2,GRID2,CNTNAP5,CNTN5,GALNTL6,MT-ND5,KCNIP4,LRRTM4,CALN1,CTNNA3,AC007682.1,EYS,GRM7,ADGRL3中的一个基因或者多个基因的转录水平升高,则表示该关节炎属于神经突触调节和离子通道出现了问题而引起的关节炎。还可以理解,更可以理解,当在软骨组织检测发现TMSB4X,PECAM1,LAPTM5,LGALS3BP,COLEC11,DUSP6,MPEG1,STOM,YWHAB,PLVAP,IFI16,ZEB2,ATP6V1B2,EGFL7,GMFG,SYK,MCAM,MYL6,KCTD12,ADGRL4,PCDH18,APLNR,C1orf162,ZFP36L1,SLCO2B1,PTPRC,ACSL1,COTL1中的一个基因或者多个基因的转录水平升高,则表示该关节炎属于免疫反应和血管生成出现了问题而引起的关节炎。
具体分型数据见下表:
表1: 第一亚型相关的基因
标志基因 | 特征曲线下面积AUROC | 矫正后p值 | 描述 | 生物学过程(GO) | 分类 |
PCOLCE2 | 1.21E-14 | 0.95 | 前胶原C-内肽酶增强剂 | GO:0016504:肽酶激活剂活性;GO:0010559:糖蛋白生物合成过程的调节;GO:0030198:细胞外基质组成 | 蛋白聚糖代谢 |
S100B | 9.52E-14 | 0.94 | S100钙结合蛋白B. | GO:0005509:钙离子结合;GO:0005539:糖胺聚糖结合 | 蛋白聚糖代谢 |
ITM2C | 1.55E-13 | 0.94 | 整合膜蛋白2C | GO:1903510:粘多糖代谢过程;GO:0030204:硫酸软骨素代谢过程 | 硫酸软骨素代谢 |
ACAN | 4.76E-13 | 0.93 | 硫酸软骨素蛋白多糖核心蛋白 | GO:0030246:碳水化合物结合;GO:0006022:氨基多糖代谢过程;GO:0030203:糖胺聚糖代谢过程 | 蛋白聚糖代谢;硫酸软骨素代谢 |
FBXO2 | 1.68E-12 | 0.92 | F-Box蛋白2 | GO:0030246:碳水化合物结合;GO:1903510:粘多糖代谢过程 | 蛋白聚糖代谢 |
SOD3 | 1.96E-12 | 0.92 | 超氧化物歧化酶3 | GO:0051216:软骨发育;GO:0010559:糖蛋白生物合成过程的调节 | 蛋白聚糖代谢 |
SERPINA3 | 2.12E-12 | 0.92 | Serpin家族蛋白3 | GO:0005578:蛋白质细胞外基质;GO:0006027:糖胺聚糖分解代谢过程 | 蛋白聚糖代谢 |
DCN | 4.79E-12 | 0.92 | 核心蛋白聚糖 | GO:0006022:氨基多糖代谢过程;GO:0009100:糖蛋白代谢过程;GO:0030204:硫酸软骨素代谢过程 | 蛋白聚糖代谢;硫酸软骨素代谢 |
WWP2 | 4.79E-12 | 0.92 | 含有E3泛素蛋白连接酶的WW结构域 | GO:0001190:转录激活因子活性;GO:0001085:RNA聚合酶II转录因子结合 | 蛋白聚糖代谢 |
ITIH6 | 4.97E-12 | 0.92 | Inter-Alpha抑制剂H5样蛋白 | GO:0006022:氨基多糖代谢过程;GO:0009100:糖蛋白代谢过程; | 蛋白聚糖代谢 |
SMOC2 | 8.36E-11 | 0.90 | 分泌的模块化钙结合蛋白 | GO:0030198:细胞外基质组成;GO:0050654:硫酸软骨素蛋白多糖代谢过程 | 蛋白聚糖代谢 |
STC2 | 1.16E-10 | 0.90 | Stanniocalcin相关蛋白2 | GO:0070482:对氧气水平的反应;GO:0005539:糖胺聚糖结合 | 蛋白聚糖代谢 |
FMOD | 2.41E-10 | 0.89 | 角蛋白硫酸盐蛋白多糖纤维调节素 | GO:0008194:UDP糖基转移酶活性;GO:0009100:糖蛋白代谢过程;GO:0030204:硫酸软骨素代谢过程 | 蛋白聚糖代谢;硫酸软骨素代谢 |
表2: 第二亚型相关的基因
TGFBI | 1.35E-05 | 0.89 | 转化生长因子β诱导 | GO:0005578:蛋白质细胞外基质;GO:0005518:胶原结合 | 胶原纤维形成 |
COL6A | 1.80E-05 | 0.89 | 胶原蛋白VI型Alpha | GO:0030574:胶原蛋白分解代谢过程;GO:0032963:胶原代谢过程;GO:0001503:骨化 | 胶原降解代谢;胶原纤维形成 |
NBL | 2.86E-05 | 0.88 | DAN家族BMP拮抗剂 | GO:0007178:跨膜受体蛋白丝氨酸/苏氨酸激酶信号通路 | 胶原降解代谢 |
DKK | 3.94E-05 | 0.88 | Dickkopf WNT信号通路抑制剂 | GO:0071559:对转化生长因子β的反应 | 胶原降解代谢 |
GJA1 | 6.00E-05 | 0.88 | 间隙连接蛋白Alpha | GO:0007517:肌肉器官发育 | 胶原纤维形成 |
COL5A | 0.000186051 | 0.86 | 胶原蛋白V型Alpha | GO:0030574:胶原蛋白分解代谢过程;GO:0030199:胶原原纤维组成 | 胶原降解代谢;胶原纤维形成 |
LMNA | 0.000227153 | 0.86 | Lamin A/C | GO:0010463:间充质细胞增殖 | 胶原降解代谢 |
COL3A | 0.000412635 | 0.85 | 胶原蛋白III型Alpha | GO:0031589:细胞基底粘附;GO:0030199:胶原原纤维组成;GO:0044420:细胞外基质成分 | 胶原降解代谢;胶原纤维形成 |
S100A10 | 0.000470572 | 0.85 | S100钙结合蛋白A. | GO:0007160:细胞基质粘附 | 胶原降解代谢;胶原纤维形成 |
MXRA5 | 0.000955334 | 0.84 | 基质重塑相关蛋白 | GO:0071559:对转化生长因子β的反应 | 胶原降解代谢;胶原纤维形成 |
SOX4 | 0.001684379 | 0.84 | SRY(性别决定区域Y)-Box 4 | GO:0001501:骨架系统发育 | 胶原纤维形成 |
ANTXR1 | 0.001737043 | 0.84 | ANTXR细胞粘附分子 | GO:0030198 细胞外基质组成;GO:0005518 胶原结合 | 胶原降解代谢;胶原纤维形成 |
ITGA11 | 0.001906274 | 0.83 | 整合素亚基Alpha | GO:0007160:细胞基质粘附;GO:0001503:骨化 | 胶原纤维形成 |
LTBP2 | 0.003962057 | 0.82 | 潜在转化生长因子β结合蛋白 | GO:0005578:蛋白质细胞外基质;GO:0071559:对转化生长因子β的反应 | 胶原纤维形成 |
AEBP1 | 0.005507051 | 0.82 | AE结合蛋白 | GO:0001501:骨架系统发育 | 胶原纤维形成 |
表3: 第三亚型相关的基因
LRRC4C | 1.25E-05 | 1.00 | 富含亮氨酸的重复区域4C | GO:0098794:突触后;GO:0097060:突触膜 | 神经突触调节 |
CTNNA2 | 3.89E-05 | 1.00 | 钙粘蛋白相关蛋白 | GO:0050807:调节突触组成;GO:0030424:轴突;GO:0048813:树突形态发生 | 神经突触调节 |
PCDH15 | 5.92E-07 | 1.00 | Protocadherin相关蛋白 | GO:0098742 通过质膜粘附分子的细胞 - 细胞粘附 | 神经突触调节 |
GRID2 | 4.85E-05 | 1.00 | 谷氨酸离子型受体Delta型亚基2 | GO:0051965:正突触组装的正调控;GO:0007416:突触组装 | 神经突触调节;离子通道 |
CNTNAP | 9.99E-05 | 1.00 | 接触蛋白相关蛋白 | GO:0034705:钾通道复合物;GO:0034702:离子通道复合体;GO:0030424:轴突 | 离子通道 |
LRRTM4 | 2.46E-06 | 0.99 | 富含亮氨酸重复跨膜神经元 | GO:0045211:突触后膜 | 神经突触调节;离子通道 |
KCNIP4 | 0.000123233 | 0.99 | 钾电压门控通道相互作用蛋白4 | GO:0034705:钾通道复合物;GO:0034703:阳离子通道复合体;GO:0099106:离子通道调节剂活性 | 离子通道 |
GRM7 | 1.06E-05 | 0.99 | 谷氨酸代谢受体 | GO:0042734:突触前膜;GO:0035249:突触传递,谷氨酸能 | 神经突触调节;离子通道 |
表4: 第四亚型相关的基因
PECAM1 | 3.87E-07 | 0.99 | 血小板和内皮细胞粘附分子 | GO:0042119:嗜中性粒细胞活化;GO:0002446:嗜中性粒细胞介导的免疫;GO:0002576:血小板脱粒 | 免疫反应;血管生成 |
TMSB4X | 3.89E-07 | 0.99 | 胸腺素β4X连锁 | GO:0003779:肌动蛋白结合;GO:0030335:细胞迁移的正调控 | 血管生成 |
LAPTM5 | 1.48E-06 | 0.97 | 溶酶体蛋白质跨膜5 | GO:0005774:液泡膜;GO:0110053:肌动蛋白丝组织的调节;GO:0031589:细胞 - 基底粘附 | 血管生成 |
LGALS3BP | 1.52E-06 | 0.97 | 凝集素半乳糖苷结合可溶性3-结合蛋白 | GO:0034774:分泌颗粒流明;GO:0060205:细胞质囊泡腔;GO:0072562:血微粒 | 免疫反应;血管生成 |
COLEC11 | 1.61E-06 | 0.97 | 聚集亚家族成员 | GO:0006898:受体介导的内吞作用 | 免疫反应 |
DUSP6 | 2.35E-06 | 0.97 | 双特异性磷酸酶 | GO:0001933:蛋白质磷酸化的负调节;GO:0070371:ERK1和ERK2级联 | 免疫反应 |
STOM | 2.60E-06 | 0.97 | 红细胞膜蛋白带 | GO:0042119:嗜中性粒细胞活化;GO:0002446:嗜中性粒细胞介导的免疫 | 免疫反应 |
YWHAB | 2.71E-06 | 0.96 | 酪氨酸3-单加氧酶/色氨酸5-单加氧酶活化蛋白β | GO:0045296:钙粘蛋白结合;GO:0061013:mRNA分解代谢过程的调节;GO:0050839:细胞粘附分子结合 | 免疫反应;血管生成 |
PLVAP | 1.55E-06 | 0.96 | Plasmalemma囊泡相关蛋白 | GO:0098589:膜区域;GO:0030335:细胞迁移的正调控;GO:0034612:对肿瘤坏死因子的反应 | 免疫反应 |
IFI16 | 4.35E-06 | 0.96 | 干扰素γ诱导蛋白16 | GO:0045088:调节先天免疫反应;GO:0031349:防御反应的积极调节 | 免疫反应 |
ZEB2 | 5.37E-06 | 0.96 | 锌指Homeobox | GO:0022604:调节细胞形态发生;GO:0001667:ameboidal型细胞迁移;GO:0010631:上皮细胞迁移 | 血管生成 |
EGFL7 | 5.30E-06 | 0.95 | 血管内皮素 | GO:0001525:血管生成;GO:0001935:内皮细胞增殖 | 血管生成 |
ATP6V1B2 | 6.36E-06 | 0.95 | ATP酶H +转运V1亚基B2 | GO:1901652:对肽的反应;GO:0005774:液泡膜 | 免疫反应 |
SYK | 2.96E-06 | 0.95 | 脾相关酪氨酸激酶 | GO:0042119:嗜中性粒细胞活化;GO:0001525:血管生成;GO:0007596:血液凝固 | 免疫反应;血管生成 |
GMFG | 6.76E-06 | 0.95 | 胶质细胞成熟因子Gamma | GO:0002446:嗜中性粒细胞介导的免疫;GO:0003779:肌动蛋白结合 | 免疫反应;血管生成 |
MCAM | 6.62E-06 | 0.95 | 黑色素瘤细胞粘附分子 | GO:0005925:局灶性粘连;GO:0001525:血管生成;GO:0030335:细胞迁移的正调控 | 血管生成 |
MYL6 | 7.85E-06 | 0.95 | 肌球蛋白轻链 | GO:0015629:肌动蛋白细胞骨架 | 血管生成 |
以上也充分说明,我们可以用这些标记物质的转录水平的升高来诊断具体关节炎是属于哪一类型的关节炎,属于什么亚型的关节炎,找到发病的机理和根本生理原理后,从而为后续治疗提供有力的帮助和支持。
可以理解,以上发现的基因水平仅仅是这些4个不同生理路径上的部分基因水平的变化,则并不说明仅仅是这些基因水平的变化而导致关节炎的分型不同。可以理解的是,这4种不同生理路径发生了异常的变化,则说明这些变化属于引起关节炎的因素之一,例如蛋白多糖代谢或/和硫酸软骨素代谢异常、或者胶原降解和/或胶原纤维组异常、神经突触调节和/或离子通道出现异常、或者,免疫反应和/或血管生成异常,则这些异常中出现一种或者几种,则是骨关节炎的致病机理。这里的异常一般是与这些代谢途径相关的直接或者检测的生物物质的水平异常而表现出来的,例如这些代谢途径先关的蛋白、基因水平的变化而表现出来的。可以理解,这些的异常是指与正常非骨关节炎患者相比较而言的。这些基因的转录水平的升高,会直接影响不同代谢途径的相关生物物质的变化,一般是数量和活性的变化,这些变化表明与之对应的代谢途径出现异常,从而导致骨关节炎的不同分型,即不同的致病机理,为后期有效诊断提供准确的诊断结果。
所以,这些不同生理过程的生物物质,不仅包括基因水平,还是蛋白水平(包括酶的水平)或者其他物质的升高,或者一些物质的降低,都是诊断关节炎分型的一种生物标记物质。这容易理解,基因水平出现异常,一般都会导致基因转录水平的异常(例如比正常人升高),从而导致蛋白水平的异常。蛋白水平的异常通常是酶的活性或者数量的异常,或者前体物质的异常,这些对于表现异常的生物物质都是出现在某些物质的代谢途径上,从而表现为某些物质的代谢异常。 当然,蛋白多糖或者硫酸软骨素本身水平的异常(例如浓度水平升高)也是说明骨关节炎的成病机理。也就是说,与蛋白多糖代谢或/和硫酸软骨素代谢异常、或者胶原降解和/或胶原纤维组异常、神经突触调节和/或离子通道出现异常、或者,免疫反应和/或血管生成异常先关的生物物质,例如直接相关的物质(酶,基因转录水平)或者间接相关的物质(代谢途径的前体物质等等)都可以作为关节炎分型的标记物质,利用这些标记物质来诊断关节炎的分型。这解决了传统的认识,传统的技术都没有找到关节炎的病因也谈不上分型,更谈不上有效的治疗,所以通常采用止痛药来进行缓解疼痛,达不到根本治疗的目的。这一点是容易理解的。所以,这些生物物质不仅包括本发明证实的具体基因转录水平的升高,还包括对应的蛋白(例如酶)的水平的异常,还可以包括前体物质的水平的异常。这里的“水平”可以是数量、活性或者两者兼有的意思。
另外,通过药物治疗或者减轻关节炎疼痛的也从改变这些异常出发,从而有效治疗关节炎。这里的异常一般与正常相对而言,针对骨关节炎而言,正常是指没有骨关节炎的人群为正常人员,所以,这里的异常是指具有关节炎的人群,从而通过不同的异常指标来判断具体处于什么类型或者亚型的关节炎,从而利于后期对症进行药物治疗。治疗则选择恰当的方式来让异常的代理途径恢复正常,从而可以从根本上达到治疗骨关节炎或者缓解疼痛,而不仅仅是采用止痛药来解决。
实施例子4:蛋白组学验证分群
我们利用ELISA检测患者滑膜中的蛋白因子表达量,确认是否有四个OA亚型的炎症反应相同。具体步骤如下:
根据制造商的说明书,通过多重测定技术ProcartaPlex Human Cytokine Panel(eBioscience,Inc.,San Diego,CA,USA)测定OA患者的滑液。通过Luminex LX100 TM(Luminex,Austin,TX,USA)多重测定检测系统读取测定。测量下列细胞因子:BDNF,Eotaxin/ CCL11,EGF,FGF-2,GM-CSF,NGFβ,IFNα,IL-1β,IL-1α,IL-2,IL-4,IL-5, IL-7,IL-8 /CXCL8,IL-9,IL-10,IL-12 p70,IL-13,IL-15,IL-17A,IL-21,IL-22,IL-23,IL-27 ,IL-31,IP-10 / CXCL10,MIP-1α/ CCL3,RANTES / CCL5,TNFα,TNFβ/ LTA,PDGF-BB,PLGF,VEGF-D。具体见图4.1-4.6所示。
结果分析:我们发现在第一群患者中RANTES(图4.3), IFNγ和 SCF(图4.3)等高表达,这些蛋白功能都是与诱导中性粒细胞相关。第二群患者中VEGFA, IL6等高表达,说明第二群患者的免疫反应和B细胞相关。第三群因为和神经相关所以没有特别的表现出免疫反应的特点。第四群患者MIP1α, SDF1α和 IL8 高表达,说明第四群患者的免疫反应和T细胞相关。其中,C1表示第一亚型的患者的炎症反应图,C2表示第二亚型的患者的炎症反应图, C3表示第三亚型的患者的炎症反应图,C4表示第四亚型的患者的炎症反应图。(具体见图4.1-4.6)
实施例子5:组织染色验证分群
组织免疫组化染色实验:
1)切片用二甲苯脱蜡,经梯度乙醇至水化:二甲苯(I)60min→二甲苯(II)60min→二甲苯:乙醇(1:1)30min→100%(I)乙醇90sec→100%(II)乙醇90sec→90%的乙醇90sec→80%乙醇90sec→70%乙醇90sec→50%乙醇90sec→蒸馏水洗2min;
2)切片浸没于抗原修复液进行热修复,65℃,过夜(>12h);
3)自然冷却到室温,PBS洗3次,每次5min;
4)0.3% H2O2覆盖样本,常温灭活10min,PBS洗3次,每次5min;
5)0.2% TritonX-100覆盖样本,室温15min破膜,PBS洗3次,每次5min;
6)加2% BSA封闭液,室温封闭90min;
7)1%BSA稀释一抗。加入一抗,覆盖样本,湿盒中室温放置半小时后置于4℃过夜(不加抗体作为阴性对照),第二天冰箱取出后复温半小时;
8)PBS洗三次,每次5min;
9)稀释二抗。加入二抗,覆盖样本,室温2h;
10)PBS洗三次,每次5min;
11)配置DAB混合液。加入DAB混合液,覆盖标本,显微镜下观察显色;
12)PBS洗三次,每次5min;
13)苏木精染核。苏木精染色2min30s,自来水充分水洗后,1%盐酸酒精分色1sec;
14)自来水充分水洗, 0.1%氨水返蓝30sec,置于自来水中
15)常规脱水,透明,封片:70%乙醇3-4sec→80%乙醇3-4sec→90%乙醇3-4sec→100%乙醇(I)2min→100%乙醇(II)2min→二甲苯:乙醇(1:1)5min→二甲苯(I)10min→二甲苯(II)10min→中性树脂封固。
Safarinin O &Fastgreen染色
1)患者软骨组织的切片用二甲苯脱蜡,经梯度乙醇至水化:二甲苯(I)60min→二甲苯(II)60min→二甲苯:乙醇(1:1)30min→100%(I)乙醇90sec→100%(II)乙醇90sec→90%的乙醇90sec→80%乙醇90sec→70%乙醇90sec→50%乙醇90sec→蒸馏水洗2min;
2)苏木精染核。苏木精染色2min30sec,自来水充分水洗后,1%盐酸酒精分色1sec;
3)自来水充分水洗, 0.1%氨水返蓝30sec,置于自来水中
4)0.02%Fastgreen 染色8min;
5)自来水洗去多余染料,吸去多余水分;
6)1%醋酸中分色,3-4sec;
7)自来水彻底水洗>2min;
8)0.1% Safrain-O染色6min;
9)自来水洗去多余染料;
10)常规脱水,透明,封片:70%乙醇3-4sec→80%乙醇3-4sec→90%乙醇3-4sec→100%乙醇(I)2min→100%乙醇(II)2min→二甲苯:乙醇(1:1)5min→二甲苯(I)10min→二甲苯(II)10min→中性树脂封固。
结果(具体见图3A-3B):
通过番红O染色(safranin O staining,SO染色)和免疫组织化学染色(Immunohistochemical staining,IHC staining)检测了4个骨关节炎(Osteoarthritis,OA)亚群的目的基因表达水平。其中,SO染色可通过使软骨基质着上红色检测软骨的基质蛋白多糖合成水平。红色越深说明蛋白多糖水平越高,指征软骨基质越正常。免疫组织化学染色是通过带显色剂标记的特异性抗体在组织细胞原位通过抗原抗体反应和组织化学的呈色反应,对相应抗原进行定性、定位、定量测定,染色结果呈现黄褐色,黄褐色一般呈现在细胞核的周边,黄色越深说明该标记物表达越高。在该研究中,我们通过对正常组和骨关节炎四个亚群进行SO染色,骨关节炎四个亚群SO染色皆较正常组浅,说明骨关节炎的切片样本的软骨基质遭受破坏。其中一群(C1)染色结果最浅,说明一群多糖代谢紊乱。同时我们选择了基质金属蛋白酶13(Matrix metallopeptidase 13, MMP13), Synapotopsin,CD34三个标记物来进行免疫组织化学染色实验, 其中MMP13是指征软骨胶原降解的标记物, MMP13染色越深,说明其表达越高,即软骨胶原降解越严重,二群(C2)免疫组织化学染色结果较其他三群显示明显MMP13表达增加的现象;Synapotopsin是与神经相关的标记物,Synapotopsin染色深说明该群与神经相关,Synapotopsin在三群(C3)中更高表达;CD34是指征炎症的重要标记物,我们发现组化染色显示四群(C4)具有高炎症发生的特点。与我们的分群结果一致,C1主要表现为多糖代谢紊乱,C2主要表现为软骨胶原降解,C3是神经相关,C4是炎症相关。由此可见,本发明收集多中心临床样本,发明了一种基于转录组学对膝关节炎疾病分型的方法和疾病亚型标志性基因和通路的检测。
上述对实施例的描述是为了便于本技术领域的普通技术人员能够理解和应用本发明。熟悉本领域的人员显然可以容易地对上述实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,对于本发明做出的改进和修改都应该在本发明的保护范围之内。
在缺少本文中所具体公开的任何元件、限制的情况下,可以实现本文所示和所述的发明。所采用的术语和表达法被用作说明的术语而非限制,并且不希望在这些术语和表达法的使用中排除所示和所述的特征或其部分的任何等同物,而且应该认识到各种改型在本发明的范围内都是可行的。因此应该理解,尽管通过各种实施例和可选的特征具体公开了本发明,但是本文所述的概念的修改和变型可以被本领域普通技术人员所采用,并且认为这些修改和变型落入所附权利要求书限定的本发明的范围之内。
本文中所述或记载的文章、专利、专利申请以及所有其他文献和以电子方式可得的信息的内容在某种程度上全文包括在此以作参考,就如同每个单独的出版物被具体和单独指出以作参考一样。申请人保留把来自任何这种文章、专利、专利申请或其他文献的任何及所有材料和信息结合入本申请中的权利。
Claims (3)
1.软骨组织中生物标记物质在制备诊断骨关节炎是否属于硫酸软骨素代谢类型的试剂上的用途; 所述的生物标记物质由下列基因组成:PCOLCE2,S100B,ITM2C,C2orf40, ACAN,FBXO2,SOD3,SERPINA3,SSR3,DCN,WWP2,ITIH6,TCEAL2,FIBIN,FGFBP2,TSPAN6, PLA2G2A,SMOC2,TUBB2B和STC2;其中,如果这些基因的转录水平升高,则表示骨关节炎属于硫酸软骨素代谢亚型的骨关节炎。
2.一种非诊断目的的确定主体骨关节炎分型的方法,该方法包括:检测软骨组织中硫酸软骨素代谢途径上的生物物质是否上调,如果出现上调,则表示属于硫酸软骨素代谢亚型的骨关节炎, 所述的生物物质由下列基因组成:PCOLCE2,S100B,ITM2C,C2orf40,ACAN, FBXO2,SOD3,SERPINA3,SSR3,DCN,WWP2,ITIH6,TCEAL2,FIBIN,FGFBP2,TSPAN6,PLA2G2A, SMOC2,TUBB2B和STC2。
3.根据权利要求2所述的方法,其中该主体为患有骨关节炎。
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