CN101711281B - Cthrc1在肝癌诊断中的应用 - Google Patents
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Abstract
公开了CTHRC1蛋白或其核酸序列在制备肝癌诊断试剂或试剂盒中的用途,用其核酸探针检测肝癌的方法,含有抗-CTHRC1抗体或蛋白质特异性核酸探针、以及标签的试剂盒,以及检测特异性CTHRC1蛋白表达的方法。
Description
技术领域
本发明涉及分子生物学特别是基因诊断领域,尤其是CTHRC-1蛋白在诊断肝癌中的应用。
背景技术
肝细胞癌(Hepatocellular carcinoma,HCC)是世界上发生率和致死率较高的恶性肿瘤之一,全世界每年新发HCC病人约50%在我国[1]。HCC的发生发展和恶性转移机制是一个涉及多基因、多信号通路参与的复杂的网络调控体系。
哺乳动物CTHRC1(Collagen triple helix containing 1)基因最先是在+对正常大鼠动脉和球状损伤动脉的差异表达序列筛选中发现的,它包含一个N端信号肽、一段36个氨基酸的胶原三螺旋以及一个C端球状结构域[2]。在人和鼠中,该基因的氨基酸序列具有92%的同源性[3]。但未见该受体与人肝癌细胞有任何特异性的关联。
CTHRC1基因在大鼠动脉的损伤处,重塑的动脉外膜的成纤维细胞和新生内膜的平滑肌细胞中都有短暂表达。在大鼠成纤维细胞中该基因的高表达会促进细胞迁移并抑制I型胶原的合成[2],从而提示CTHRC1通过限制胶原基质沉积和促进细胞迁移参与了血管的损伤修复.而越来越多的证据表明,组织的修复与肿瘤发生之间有很紧密的关联[4-6]。
CTHRC1基因与人类肿瘤的关系首先是在乳腺癌中被报导的,cDNA芯片和原位杂交发现在乳腺癌的基质细胞中有该基因的mRNA的表达[7,8]。此后又有研究报导在黑色素瘤中该基因也存在异常表达,并且对癌细胞的侵袭和转移起作用[3]。已有许多报道证实,肿瘤微环境在肿瘤细胞的生长、侵袭和转移中有及其重要的作用[9-11]。当CTHRC1蛋白在纤维原细胞中表达上调时,可以抑制I型胶原的合成。CTHRC1可能是通过降低细胞外基质成分的合成,从而为肿瘤细胞的侵袭和转移创造合适的细胞外环境。因此,CTHRC1基因可能在癌症诊断和癌症转移复发的预防和治疗上具有良好的应用前景。
因此,本领域对于特定癌症的准确和特异性的检测有迫切的需求。
发明内容
因此,本发明的目的是提供能够准确诊断选自肝癌的诊断试剂盒、CTHRC-1基因的诊断试剂盒的用途,以及体外检测其表达量的方法。
在本发明的一个方面,提供了CTHRC1核酸序列或蛋白在制备肝癌诊断试剂或含有该诊断试剂的试剂盒中的用途。
在该方面的一个优选例中,肝癌诊断试剂是抗CTHRC1蛋白特异性抗体或CTHRC1蛋白特异性核酸探针。
在本发明的另一个方面,提供了一种检测肝癌的方法,包括下列步骤:
a)将与放射性核素偶联的CTHRC1核酸探针输给动物;
b)检测所述核酸探针在体内的聚集;
c)所述聚集表明肝癌的存在。
在该方面的一个优选例中放射性核素是α-32P。在另一个优选例中动物是人。
在本发明的另一个方面,提供了一种肝癌诊断试剂盒,该试剂盒含有容器,所述容器中含有抗CTHRC1抗体;以及标签,说明所述试剂盒用于诊断肝癌。
在本发明的还有一个方面,提供了一种肝癌诊断试剂盒,其特征在于,该试剂盒含有容器,其中含有含有CTHRC1特异性核酸探针;以及标签,所该签说明所述试剂盒用于诊断肝癌。
本发明的另一个方面,提供了一种体外检测特异性CTHRC1蛋白表达的方法,包括步骤:
用抗CTHRC1蛋白特异性抗体或CTHRC1特异性核酸探针与细胞样品反应,以正常肝细胞为对照;
比较抗体或探针的结合量,其中高于对照的量表明该细胞为肝癌细胞,低于或等于对照的量表明该细胞为正常细胞。
在该方面的一个优选例中,结合量是通过检测与探针或抗体偶联的可检测基团测得的。
在该方面的另一个优选例中,所述可检测基团选自生色团、化学发光基团、荧光团或同位素。
附图说明
图1显示了CTHRC1在肝癌细胞株中的表达情况。图1A是肝癌细胞株中的RT-PCR分析结果。图1B是对应于图1A的柱状图。
图2显示了CTHRC1在12例肝癌病人中的表达情况的分析。图2A是来自杭州的肝癌病人的癌组织和正常组织的表达mRNA水平的RT-PCR分析。图2B是CTHRC1在来自广西的肝癌病人中的mRNA水平的RT-PCR分析。其中:1,G139;2,G111;3,G116;4,G108;5,G83;6,G64;7,G114;8,G65。
图3显示了CTHRC1基因在人的多种正常组织中表达情况的分析。其中标号分别表示:1心脏;2大脑;3胎盘;4肺;5肝脏;6骨骼;7肾脏;8胰脏;9脾脏;10胸腺;11***;12睾丸;13卵巢;14小肠;15结肠;16外周血淋巴细胞。
图4是CTHRC1对MHCC97L细胞的迁移作用效果。
图5是CTHRC1对于MHCC97L细胞的侵袭作用效果。
图6是CTHRC1的mRNA序列(gi|34147546|ref|NM_138455.2|)和氨基酸序列(gi|19923989|ref|NP_612464.1|)。这两条序列可以在GenBank中以上述参考号找到。
具体实施方式
发明人采用基因芯片技术,通过比较肝癌组织和相应的癌旁肝组织基因表达谱的差异,发现CTHRC1在肝癌细胞中有特别高的特异性表达。
如本文所用,术语“CTHRC1”指在大鼠动脉和球状损伤动脉的差异表达序列筛选中发现的一种富含胶原三螺旋结构的基因。本发明所指的CTHRC1基因包括其完整的DNA编码序列,其RNA序列,其突变体,以及其功能上活性的片段。需理解的是,当编码相同的氨基酸时,密码子中的核苷酸的取代是可接受的。另外需理解的是,由核苷酸取代而产生的保守的氨基酸取代时,核苷酸的变换也是可被接受的。
在得到了CTHRC1的核酸片段的情况下,可根据核苷酸序列来设计特异性探针。核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法,可根据本发明所公开的有关核苷酸序列,尤其是开放阅读框序列来设计引物,并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板,扩增而得有关序列。当序列较长时,常常需要进行两次或多次PCR扩增,然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列,就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外,还可用人工合成的方法来合成有关序列,尤其是片段长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。
目前,已经可以完全通过化学合成来得到编码本发明蛋白(或其片段,衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明中,CTHRC1多核苷酸序列可***到重组表达载体中。总之,只要能在宿主体内复制和稳定,任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含CTHRC1的DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上,以指导mRNA合成。转化载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外,表达载体优选地包含一个或多个选择性标记基因,以提供用于选择转化的宿主细胞的表型性状,如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP),或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体,可以用于转化适当的宿主细胞,以使其能够表达蛋白质。
宿主细胞可以是原核细胞,如细菌细胞;或是低等真核细胞,如酵母细胞;或是高等真核细胞,如哺乳动物细胞。代表性例子有:大肠杆菌,链霉菌属的细菌细胞;真菌细胞如酵母;植物细胞;昆虫细胞;动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时,能吸收DNA的感受态细胞可在指数生长期后收获,用CaCl2法处理,所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要,转化也可用电穿孔的方法进行。当宿主是真核生物,可选用如下的DNA转染方法:磷酸钙共沉淀法,常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养,表达本发明的基因所编码的多肽。根据所用的宿主细胞,培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后,用合适的方法(如温度转换或化学诱导)诱导选择的启动子,将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要,可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于:常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
在获得了核酸序列后,可根据核酸序列设计特异性核酸探针。设计探针的方法是本领域常规的,可见Sambrook等人,分子克隆实验室手册(New York:ColdSpring Harbor Laboratory Press,1989)中所述。检测生物样品中是否存在DKK-1蛋白或核酸的示范性方法包括获得测试受试者的生物样品,使该生物样品接触能与DKK-1mRNA或基因组DNA杂交的标记的核酸探针。该核酸探针可以是,例如人的核酸或及一部分,如长至少15、30、50、100个核苷酸并能在严谨条件下与DKK-1mRNA或基因组DNA充分杂交的核酸探针。用于本发明诊断试验的其它探针如本文所述。
核酸探针与扩增的标记序列接触。该探针优选连接到一种发色团,但可被放射标记。在另一个实施例中,探针连接到一种结合伴侣上,如抗体或生物素,或另一种携带可检测结构域的结合伴侣上。
在传统的方法中,检测可通过Southern印迹以及与标记的探针杂交来进行。Southern印迹所涉及的技术是本领域技术人员所熟知的(参见Sambrook等,1989)。常规的检测还有生物芯片、荧光显影技术、细胞流式计数等。
另一方面,本发明还包括对CTHRC1 DNA或是其片段编码的多肽具有特异性的多克隆抗体和单克隆抗体,尤其是单克隆抗体。这里,“特异性”是指抗体能结合于CTHRC1基因产物或片段。较佳地,指那些能与DKK-1基因产物或片段结合但不识别和结合于其它非相关抗原分子的抗体。本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。
本发明不仅包括完整的单克隆或多克隆抗体,而且还包括具有免疫活性的抗体片段,如Fab’或(Fab)2片段;抗体重链;抗体轻链;遗传工程改造的单链Fv分子;或嵌合抗体。
抗CTHRC1蛋白的抗体可用于免疫组织化学技术中,检测活检标本中的CTHRC1蛋白,还可以作为用于预防肝癌转移和侵袭的特异性治疗剂。
血样或尿液中的CTHRC1的直接测定可以作为肿瘤的辅助诊断和愈后的观察指标,也可作为肿瘤早期诊断的依据。
抗体可以通过ELISA、Western印迹分析,或者与检测基团偶联,通过化学发光、同位素示踪等方法来检测。
本发明也包括试剂盒,以进行这里描述的任何方法。在一个非限制的实例中,所述试剂盒将以适当的容器形式包含这些试剂中的一种或多种。所述试剂盒也可包含用于RNA分离、扩增细胞中RNA的纯化的试剂、标记等。
试剂盒的组分可以以水介质的形式或以冻干的形式来包装。试剂盒中适当的容器通常至少包括一种小瓶、试管、长颈瓶、宝特瓶、针筒或其它容器,其中可放置一种组分,并且优选地,可进行适当地等分。在试剂盒中存在多于一种的组分时,试剂盒中通常也将包含第二、第三或其它附加的容器,其中分离地放置附加的组分。然而,不同组合的组分可被包含在一个小瓶中。本发明的试剂盒通常也将包括一种用于容纳反应物的容器,密封以用于商业销售。这种容器可包括注模或吹模的塑料容器,其中可保留所需的小瓶。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆实验室手册(New York:Cold SpringHarbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。
实施例1.Northen印迹分析
1.1主要试剂Trizol购自Invitrogen公司,Hybond-N+膜购自Amersham公司,正常人多组织Northern膜片(MTN blot)和ExpressHyb杂交液购自Clontech公司,NEBlot试剂盒购自NEB公司;真核表达载体pCMV-3Tag购买自stratagene公司;限制性内切酶及T4连接酶购自Promega公司;real-time PCR试剂盒购自美国ABI公司;CTHRC1及管家基因β-肌动蛋白的Taqman-MGB探针及引物由ABI公司设计合成;血液/细胞/组织基因组DNA提取试剂盒购自北京天为时代公司;引物设计采用Primer 3软件,引物合成由上海生工生物工程技术服务有限公司完成;Hyclone特级胎牛血清购自Hyclone公司;高糖型DMEM购自Gibco公司;质脂体LipofectamineTM Reagent购自Invitrogen公司;Transwell小室(内径6.5mm,孔径8.0mm)购自Corning公司;MatrigelTM购自Corning公司;其余试剂均为国产分析纯。
1.2肝癌病人癌组织和癌旁肝组织样品、肝癌细胞株的收集
肝癌患者的癌组织及癌旁肝组织来源于广西医科大学及浙江大学医学院附属第一医院。低转移肝癌细胞株MHCC97L和高转移肝癌细胞株HCCLM3由复旦大学中山医院肝癌研究所提供,其它细胞株为本室保存(肝癌细胞HepG2、肝癌细胞Hep3B、肝癌细胞MHCC97L、肝癌细胞HCCLM3、肝癌细胞HuH7)。手术后组织标本立即置液氮中冷冻,随后保存于-80℃超低温冰箱。
1.3细胞培养
5种细胞株HepG2、Hep3B、MHCC97L、HCCLM3、HuH7均培养在含10%胎牛血清的DMEM培养基中,加100U/ml青霉素和100g/ml链霉素,在37℃、5%CO2培养箱中培养。
1.4 RT-PCR分析以及Northern印迹分析
采用RT-PCR方法检测肝癌细胞株中CTHRC1的表达状况。用Trizol抽提8种肝癌细胞株的总RNA,各取5μg进行逆转录,条件如下:65℃ 5min,50℃50min,85℃ 5min,37℃ 20min。所得产物取1μL稀释5倍后作为模板进行PCR扩增,在PCR反应到达平台期前取扩增产物电泳检测,以β-actin作为内对照,引物序列如下:上游引物为5’-TGGATGGAATTCAGTTTCTCGCATCA-3’,下游引物为5’-GCTTCAATCAAAAGTGGTTTCAA-3’。PCR反应条件为:94℃预变性2min;94℃变性30s,58℃退火30s,72℃延伸45s,循环30次;72℃延伸5min。
对8例肝癌患者(G139、G111、G116、G108、G83、G64、G114、G65)的癌组织及癌旁肝组织进行real-time PCR分析,RNA抽提及反转录过程同RT-PCR,real-time PCR反应条件为:50℃ 2min,95℃ 10min;95℃ 15s,60℃ 1min,循环40次。PCR反应过程于ABI7300仪上进行,实时监测每个循环中延伸阶段的荧光值。数据分析由ABI7300***软件自动完成。
实验结果如图1所示。图1显示了CTHRC1在肝癌细胞株中的表达情况。图1A是荧光定量PCR的结果,图B是相对应的RT-PCR结果。结果显示,在5种肝癌细胞株中,CTHRC1在HepG2、Hep3B、MHCC97L、HCCLM3、HuH7中有较高的表达水平。
对上述5种人类肝癌细胞株进行了Northern印迹分析。人肝癌细胞株的总RNA使用Trizol抽提。每份样品各取10mg总RNA于1%甲醛变性胶电泳后转移至Hybond-N+膜。837bp的CTHRC1基因探针(GenBank号NM_138455.2序列第58至894bp)用[α-32P]dCTP进行标记,核素标记采用NEBlot试剂盒,随后使用ExpressHyb杂交液进行杂交反应。Northern印迹的结果(用β-肌动蛋白作为对照),显示在这5种肝癌细胞中CTHRC1都有特异性的表达。
实施例2.CTHRC1在肝癌病人组织中的表达情况
以β-肌动蛋白作为内对照,共做了14对肝癌及相应的癌旁组织(其中HK组来自杭州的病人,而G组是来自广西的病人),其中用Northern印迹分析了6对来自杭州病人的肝癌和相应的癌旁组织,荧光定量PCR分析了8对来自广西病人的肝癌和相应的癌旁组织。结果显示其中11对样品中CTHRC1在癌组织中的表达高于癌旁组织(P<0.01),其余3对没有统计学差别(图2)。该结果说明,在大多数肝癌病人体内,CTHRC1是在肝癌组织中有特异性表达的。
实施例3人正常组织中CTHRC1表达的Northern印迹分析
用Northern blot分析了16种人类正常组织中CTHRC1的表达情况,进行灰度扫描后作统计图(图3)。结果显示,在大部分人类的正常组织中,CTHRC1没有特异性表达。
实施例4损伤修复实验
4.1pCMV-3Tag-CTHRC1真核表达载体的构建
从人胎盘cDNA克隆CTHRC1基因的完整编码区序列,上游引物为5’-AAGGAAAAAAGCGGCCGCGCCACCATGCGACCCCAGGGC-3’,下游引物5’-CCGCTCGAGATTTTGGTAGTTCTTC-3’,PCR反应条件为:94℃预变性2min;94℃变性30s,52℃退火30s,72℃延伸1min,循环35次;72℃延伸5min。PCR产物经1%琼脂糖凝胶电泳鉴定,纯化回收约765bp大小的目的片段.pCMV-3Tag质粒和CTHRC1基因的PCR扩增片断分别进行NotI和XhoI双酶切,纯化回收酶切后的pCMV-3Tag载体和CTHRC1片断。用T4DNA连接酶连接目的片段和线性化的空载体pCMV-3Tag-9,摩尔比为3∶1,16℃反应过夜。连接产物转化TOP10感受态细菌,将转化的菌液涂在含有氨苄青霉素的LB平板上培养12h,挑取阳性克隆进行少量扩增、质粒小抽,以ApaI酶切鉴定阳性细菌克隆。送上海鼎安生物科技有限公司测序,结果证实pCMV-3Tag-CTHRC1真核表达载体构建成功。
4.2细胞划痕实验
将生长状态良好的MHCC97L细胞种于12孔板,3×105个细胞/孔。10%FBS的DMEM培养基,37℃,5%CO2条件下培养。待细胞长满后进行转染。取LIPOFECTAMINE 2000 2ml(1mg/ml)和pCMV-3Tag-CTHRC1质粒DNA(或空载体pCMV-3Tag)0.5mg分别用DMEM稀释到100ml,二者混合,室温放置20min。用无血清DMEM洗细胞2次,将DNA和脂质体的混合物用无血清DMEM补足到1ml,转染细胞。5h后,用1ml 10%FBS的DMEM换液。24h后用200μl枪头在孔内划痕,用无血清DMEM培养基漂洗3次,加入2%FBS的DMEM培养基继续培养24h。实验终止后将孔内细胞进行固定,用结晶紫染色,显微镜记数并拍照。实验组和对照组各设3个平行样本,每个样本观测2个视野(物镜,×10),迁移的细胞个数分别为:16.0+2.65,3.33+1.16(图4)。差异具有统计学意义(P<0.01)。
实施例5体外侵袭实验
将生长状态良好的MHCC97L细胞种于12孔板,3×105个细胞/孔。10%FBS的DMEM培养基,37℃,5%CO2条件下培养。待细胞长满后进行转染。取LIPOFECTAMINE 2000 2ml(1mg/ml)和pCMV-3Tag-CTHRC1质粒DNA(或空载体pCMV-3Tag)0.5mg分别用DMEM稀释到100ml,二者混合,室温放置20min。用无血清DMEM洗细胞2次,将DNA和脂质体的混合物用无血清DMEM补足到1ml,转染细胞。5h后,用1ml 10%FBS的DMEM换液。24h后用200μl枪头在孔内划痕,用无血清DMEM培养基漂洗3次,加入2%FBS的DMEM培养基继续培养24h。实验终止后将孔内细胞进行固定,用结晶紫染色,显微镜记数并拍照。实验组和对照组各设3个平行样本,每个样本观测中心视野(物镜,×10),共计3个视野,发生侵袭的细胞个数分别为:49.3+6.66,8+1.87。图5A显示了一组实验和对照样本的视野照片。图5B是三个样本平均值的柱状图。差异具有统计学意义(P<0.01)。实验结果表明,MHCC97-L CTHRC1(实验组)细胞的侵袭能力明显高于对照组(P<0.05),证明了过表达CTHRC1可以提高肝癌细胞MHCC97-L的侵袭能力。
综上所述,CTHRC1基因与肝癌密切相关,并且在肝癌组织和细胞中有显著的特异性。而且,观察到被该基因转染的低转移性肝癌细胞株MHCC97L的转移和侵袭性都大大增强,说明该基因与肝癌的转移性和侵袭性密切相关,可作为预防和治疗转移和侵袭性肝癌的靶标。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
[参考文献]
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[4]TANG L,DAI DL,SU M,等,Aberrant expression of collagen triplehelix repeat containing 1 in human solid cancers.Clin Cancer Res 2006 Jun15;12(12):3716-3722.
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[6]BEACHY PA,KARHADKAR SS,BERMAN DM.Tissue repair and stem cellrenewal in carcinogenesis.Nature.2004 Nov 18;432(7015):324-331.
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Claims (6)
1.CTHRC1核酸序列或蛋白在制备肝癌诊断试剂或含有所述诊断试剂的试剂盒中的用途。
2.如权利要求1所述的用途,其特征在于,所述肝癌诊断试剂是抗CTHRC1蛋白特异性抗体或CTHRC1蛋白特异性核酸探针。
3.如权利要求2所述的用途,其特征在于,所述核酸探针与放射性核素偶联。
4.如权利要求3所述的用途,所述放射性核素是α-32P。
5.一种肝癌诊断试剂盒,其特征在于,该试剂盒含有容器,所述容器中含有抗CTHRC1抗体;以及标签,所述标签说明所述试剂盒用于诊断肝癌。
6.一种肝癌诊断试剂盒,其特征在于,该试剂盒含有容器,所述容器中含有CTHRC1特异性核酸探针;以及标签,所述标签说明所述试剂盒用于诊断肝癌。
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US20050147602A1 (en) * | 2000-10-19 | 2005-07-07 | Maine Medical Center Research Institute | Compositions, methods and kits relating to CTHRC1, a novel modulator of collagen matrix |
WO2007032631A1 (en) * | 2005-09-12 | 2007-03-22 | Daewoong Co., Ltd. | Markers for diagnosis of cancer and its use |
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US20050147602A1 (en) * | 2000-10-19 | 2005-07-07 | Maine Medical Center Research Institute | Compositions, methods and kits relating to CTHRC1, a novel modulator of collagen matrix |
WO2007032631A1 (en) * | 2005-09-12 | 2007-03-22 | Daewoong Co., Ltd. | Markers for diagnosis of cancer and its use |
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