CN111912985A - E3泛素化蛋白连接酶ubr5在制备肿瘤诊断或预后评估试剂盒中的应用 - Google Patents
E3泛素化蛋白连接酶ubr5在制备肿瘤诊断或预后评估试剂盒中的应用 Download PDFInfo
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Abstract
本发明涉及生物技术和医学诊断领域,具体是E3泛素化蛋白连接酶UBR5在制备肾透明细胞癌诊断或预后评估试剂或试剂盒中的应用,以及相应的检测试剂盒。本发明研究发现,肾透明细胞癌患者中E3泛素化蛋白连接酶UBR5分子的表达通常下调,且其表达水平与患者的预后显著相关,结合UBR5的表达能更好的预测肾透明细胞癌患者的总生存期(OS)与无进展生存期(PFS)。
Description
技术领域
本发明涉及生物技术和医学诊断领域,具体地说,是E3泛素化蛋白连接酶UBR5在制备肾透明细胞癌诊断或预后评估试剂盒中的应用。
背景技术
肾癌是全世界男女最常见的癌症之一。(Barata PC,Rini BI.Treatment ofrenal cell carcinoma:Current status and future directions.CA Cancer JClin.2017;67(6):507-524.)肾透明细胞癌(Clear Cell Renal Cell Carcinoma,ccRCC)是最常见的肾癌类型(Shuch B,Amin A,Armstrong AJ,et al.Understanding pathologicvariants of renal cell carcinoma:distilling therapeutic opportunities frombiologic complexity.Eur Urol.2015;67(1):85-97)。局限性ccRCC患者一般采用外科手术治疗,随着近年来的进步,其副作用明显减轻(Hsieh JJ,Purdue MP,Signoretti S,etal.Renal cell carcinoma.Nat Rev Dis Primers.2017;3:17009.)。尽管腹腔镜或机器人辅助腹腔镜等微创技术降低了手术引起功能损害的风险,但ccRCC术后复发、进展和转移通常会随之发生(Wang G,Zhang ZJ,Jian WG,et al.Novel long noncoding RNA OTUD6B-AS1 indicates poor prognosis and inhibits clear cell renal cell carcinomaproliferation via the Wnt/β-catenin signaling pathway.Mol Cancer.2019;18(1):15.)。因此,为了准确评价和监测ccRCC患者的病情发展和预后,有必要确定可靠的术后指标。
在临床上,ccRCC患者的具体手术方式或术后监测与随访在很大程度上取决于包括TNM分期在内的临床病理参数,TNM分期是预测ccRCC患者临床预后最普遍的预后因素之一(Barata PC,Rini BI.Treatment of renal cell carcinoma:Current status andfuture directions.CA Cancer J Clin.2017;67(6):507-524.)。此外,分期评分系统SSIGN(分期、大小、分级和坏死)将ccRCC患者分为不同风险的亚组,有助于预测术后预后(5.Ho TH,Kapur P,Eckel-Passow JE,et al.Multicenter Validation of Enhancer ofZeste Homolog 2 Expression as an Independent Prognostic Marker in LocalizedClear Cell Renal Cell Carcinoma.J Clin Oncol.2017;35(32):3706-3713.)。然而,为了更好的进行特殊治疗和指导精确的药物应用,上述这些因素对患者的分层仍显不足。因此,许多研究报道了包括癌基因、抑癌基因和癌干细胞(CSC)标记在内的其他指标可用于预测患者术后预后(Wang M,Li X,Zhang J,et al.AHNAK2is a Novel Prognostic Markerand Oncogenic Protein for Clear Cell Renal Cell Carcinoma.Theranostics.2017;7(5):1100-1113.;Li X,Ma X,Chen L,et al.Prognostic value of CD44 expression inrenal cell carcinoma:a systematic review and meta-analysis.Sci Rep.2015;5:13157.)。然而,以往的研究主要集中在肿瘤细胞的特性上。
自从1975年泛素被发现以来,大量的研究表明泛素系统通过控制广泛的细胞功能对维持体内平衡至关重要。泛素与底物蛋白的赖氨酸(Lys)残基共价结合,分别作为信号分子介导其降解或调节其生物学功能。泛素结合是一个多步反应,涉及三种酶的后续作用:E1,泛素激活酶;E2,泛素结合酶;E3,泛素连接酶。整个过程始于泛素分子的C末端甘氨酸残基的ATP依赖性激活,并通过泛素与底物蛋白的结合而终止。单个泛素(单泛素化)或多个Ub分子(多泛素化)可以连接到目标蛋白的Lys残基上。此外,通过七个赖氨酸残基中的一个或N-末端蛋氨酸残基(M1)连接多个泛素部分,形成不同的链。最后,底物结合的Ub分子也可以被其他ptm修饰,例如SUMOylation、磷酸化和乙酰化,增加泛素代码的复杂程度。值得注意的是,泛素化是一个可逆的过程。泛素分子自身之间以及泛素与其底物之间的连接可以通过氘化酶(DUBs)的作用水解。综上所述,泛素化表现出广泛的多样性。
泛素系统作为一种有前途的药物靶点正受到人们的关注。存在大量的底物蛋白和泛素连接酶(E3)(人类有600多个)负责底物识别。其次,底物蛋白的命运在很大程度上取决于多种泛素结合,如单泛素化、多泛素化和至少有八种不同连接的多泛素化。第三,泛素化可以被约100种去泛素化酶(DUBs)调节,这些去泛素酶可以去除或修剪基质蛋白上的泛素链。
UBR5是HECT结构域E3泛素蛋白连接酶家族的29个成员之一,据报道是多能性发展和维持所必需的(Koyuncu S,Saez I,Lee HJ,et al.The ubiquitin ligase UBR5suppresses proteostasis collapse in pluripotent stem cells from Huntington'sdisease patients.Nat Commun.2018;9(1):2886.)。UBR5在DNA损伤后泛素介导的信号转导的内稳态中发挥了关键作用(Li CG,Mahon C,Sweeney NM,et al.PPARγInteractionwith UBR5/ATMIN Promotes DNA Repair to Maintain Endothelial Homeostasis.CellRe.2019;26(5):1333-1343.e7.)。UBR5与多种癌症有关。来自癌症基因组图谱(TCGA)的数据表明,UBR5基因扩增在许多癌症类型中是一种常见的改变(Shearer RF,Iconomou M,Watts CK,et al.Functional Roles of the E3 Ubiquitin Ligase UBR5 in Cancer.MolCancer Res.2015;13(12):1523-1532.)。研究表明,UBR5促进包括结肠癌、乳腺癌、卵巢癌、宫颈癌在内的恶性肿瘤的生长和进展(Ji SQ,Zhang YX,Yang BH.UBR5promotes cellproliferation and inhibits apoptosis in colon cancer by destablizingP21.Pharmazie.2017;72(7):408-413.;Liao L,Song M,Li X,et al.E3 UbiquitinLigase UBR5 Drives the Growth and Metastasis of Triple-Negative BreastCancer.Cancer Res.2017;77(8):2090-2101.;Matsuura K,Huang NJ,Cocce K,etal.Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitinligase and its role in ovarian cancer resistance to cisplatin.Oncogene.2017;36(12):1698-1706.;Subbaiah VK,Zhang Y,Rajagopalan D,et al.E3 ligase EDD1/UBR5is utilized by the HPV E6 oncogene to destabilize tumor suppressorTIP60.Oncogene.2016;35(16):2062-2074.)。另一方面,UBR5错义突变也发生在一些癌症中,如具有复发性UBR5突变的地幔细胞淋巴瘤(Meissner B,Kridel R,Lim RS,et al.TheE3 ubiquitin ligase UBR5 is recurrently mutated in mantle celllymphoma.Blood.2013;121(16):3161-3164.)。然而,UBR5在ccRCC中的表达及其临床意义尚未被证实。
发明内容
本发明的目的在于寻找到可有效用于肿瘤诊断、肿瘤治疗方案选择、肿瘤预后评估的UBR5分子,本发明的另一目的在于提供该UBR5分子在临床相应的检测试剂盒中的应用。
本发明从基因芯片中筛选出了肿瘤(尤其是透明细胞肾细胞癌)表达相关性及其对透明细胞肾细胞癌生长的具有调控作用的分子—E3泛素化蛋白连接酶UBR5,由此本发明进一步提供了UBR5分子在用于对象中肿瘤诊断、肿瘤治疗方案选择、肿瘤预后评估中的新用途,并提供了相应的检测试剂盒。
本发明将UBR5表达与现有临床指标(TNM分期或SSIGN评分)相结合,提供ccRCC患者更精确的危险分层以及预后预测,有利于指导ccRCC患者的精确个性化治疗。
本发明的第一方面,提供E3泛素化蛋白连接酶UBR5在制备肾透明细胞癌预后评估试剂或试剂盒中的应用。
进一步的,所述的E3泛素化蛋白连接酶UBR5,Gene ID:51366。
进一步的,所述的肾透明细胞癌选自:原发性肾透明细胞癌、或转移性肾透明细胞癌。
进一步的,所述的试剂或试剂盒检测E3泛素化蛋白连接酶UBR5在肾透明细胞癌患者肿瘤组织中的表达量。
进一步的,所述的试剂或试剂盒采用免疫组化方法或生物芯片检测法检测E3泛素化蛋白连接酶UBR5在肾透明细胞癌患者肿瘤组织中的表达量。所述的肿瘤组织选自:获自对象的新鲜组织、福尔马林固定或石蜡包埋组织等。
更进一步的,所述的试剂或试剂盒是将E3泛素化蛋白连接酶UBR5作为分子标记,利用E3泛素化蛋白连接酶UBR5单克隆抗体或多克隆抗体,以及免疫组化试剂,分析E3泛素化蛋白连接酶UBR5在肾透明细胞癌患者肿瘤组织中的表达量,预测肾透明细胞癌患者的预后。
进一步的,所述的E3泛素化蛋白连接酶UBR5单克隆抗体,如兔UBR5单克隆抗体(NB100-1591,Novus Biologicals,Littleton,CO,USA)。
进一步的,所述的免疫组化试剂包括二甲苯,乙醇,3%H2O2溶液,1%BSA封闭液,DAB显色试剂,苏木素和辣根过氧化物酶标记的羊抗鼠IgG。
本发明发现E3泛素化蛋白连接酶UBR5高表达的肾透明细胞癌患者预后更好,E3泛素化蛋白连接酶UBR5低表达的肾透明细胞癌患者预后较差。根据上述方法检测肾透明细胞癌组织中E3泛素化蛋白连接酶UBR5表达情况,进行评分,H-score低于130分为低表达,H-score等于或高于130分为高表达,肾透明细胞癌组织中E3泛素化蛋白连接酶UBR5高表达的肾透明细胞癌患者预后较好。
肾透明细胞癌组织中E3泛素化蛋白连接酶UBR5低表达是一个显著独立的预测肾透明细胞癌患者较低总体生存时间和无进展生存时间的危险因素,与临床指标如TNM分期、SSIGN评分等相比具有更好的预测价值。
进一步的,所述的检测E3泛素化蛋白连接酶UBR5在肾透明细胞癌患者肿瘤组织中的表达量的方法,包括以下步骤:
(a)利用免疫组化试剂二甲苯,乙醇,3%H2O2溶液,1%BSA封闭液,DAB显色试剂,苏木素和辣根过氧化物酶标记的羊抗鼠IgG将肾透明细胞癌组织切片进行免疫组化染色;
(b)利用显微镜和成像装置拍摄为数码照片;
(c)根据免疫组化染色结果H-score评分原则,给出评分。
更进一步的,所述的方法具体步骤如下:
(1)制备肾透明细胞癌组织石蜡切片,60℃烤箱过夜;
(2)切片脱腊至水;
(二甲苯Ⅰ①10min→二甲苯Ⅱ②10min→二甲苯Ⅲ③10min→100%乙醇5min→95%乙醇5min→85%乙醇5min→75%乙醇5min→双蒸水5min)
(3)3%H2O2溶液,室温放置20min;
(4)双蒸水洗5min×3;
(5)抗原修复:切片放入0.01M柠檬酸盐缓冲液(pH 6.0)中煮沸30min;
(6)自然冷却至室温,双蒸水洗5min×3;
(7)1%BSA封闭30min,37℃;
(8)甩去封闭液,不洗,直接加一抗(兔UBR5抗体,购自Novus Biologicals公司,稀释比例1:500)。置入湿盒中4℃冰箱过夜16小时;
(9)4℃取出,室温复温15min,然后0.01M PBS洗5min×4;
(10)滴加二抗(辣根过氧化物酶标记羊抗鼠IgG,购自中国福州迈新生物公司,即用型,无需稀释)45min,37℃;
(11)0.01M PBS洗5min×4,DAB显色2-10min,镜下观察;
(12)双蒸水终止显色,苏木素复染10秒;
(13)分化后自来水返蓝,蒸馏水浸泡;
(14)脱水透明,盖玻片覆盖;
(15)显微镜下观察阳性染色,于肾透明细胞癌组织随机选取3个视野并拍照;
(16)采用高倍镜下综合染色强度和阳性细胞所占比例进行半定量测定。
具体H-score评分细则如下:
1,染色强度评分标准:阴性0分,弱阳性1分,中等阳性2分,强阳性3分;
2,阳性细胞所占比例评分标准:分别评估弱阳性、中等阳性、强阳性细胞的百分比;
3,染色强度评分与对应评分的阳性细胞百分比相乘,得出H-score评分,范围0-300分。
根据上述方法检测肾透明细胞癌组织中UBR5表达情况,进行评分,H-score低于130分为低表达,H-score等于或高于130分为高表达,肾透明细胞癌组织中UBR5高表达的肾透明细胞癌患者预后较好。
本发明的第二方面,提供一种评估肾透明细胞癌患者预后的试剂盒,所述的试剂盒中包含检测E3泛素化蛋白连接酶UBR5在肾透明细胞癌患者肿瘤组织中的表达量的试剂。
进一步的,所述的检测E3泛素化蛋白连接酶UBR5表达量的试剂,选自:对UBR5具有检测特异性的IgG抗体等。
进一步的,所述的试剂盒中包含:
(i)检测生物样品中E3泛素化蛋白连接酶UBR5表达量的一种或多种试剂;
(ii)选自下组的一种或多种物质:二甲苯,乙醇,3%H2O2溶液,1%BSA封闭液,DAB显色试剂,苏木素和辣根过氧化物酶标记的羊抗鼠IgG。
进一步的,所述的检测试剂盒,检测E3泛素化蛋白连接酶UBR5的方法选自以下任一:免疫组化、生物芯片检测法。
进一步的,所述的试剂还带有可检测标记,优选的可检测标记选自:放射性同位素、荧光团、化学发光部分、酶、酶底物、酶辅因子、酶抑制剂、染料、金属离子、配体(如,生物素或半抗原)。
优选的,所述的试剂盒中包含:E3泛素化蛋白连接酶UBR5单克隆抗体或多克隆抗体,以及免疫组化试剂;所述的免疫组化试剂包括二甲苯,乙醇,3%H2O2溶液,1%BSA封闭液,DAB显色试剂,苏木素和辣根过氧化物酶标记的羊抗鼠IgG。
本发明的第三方面,提供E3泛素化蛋白连接酶UBR5在制备肾透明细胞癌诊断试剂或试剂盒中的应用。
进一步的,所述的试剂或试剂盒检测E3泛素化蛋白连接酶UBR5在肿瘤组织中的表达量。
本发明优点在于:
1、本发明研究发现,肾透明细胞癌患者中E3泛素化蛋白连接酶UBR5分子的表达通常下调,且UBR5表达水平与患者的预后显著相关,结合UBR5的表达水平能更好的预测肾透明细胞癌患者的总生存期(OS)与无进展生存期(PFS)。
2、本发明的E3泛素化蛋白连接酶UBR5与肾透明细胞癌相关性的发现,为预测肾透明细胞癌患者预后提供了全新的生物标志物,E3泛素化蛋白连接酶UBR5高表达的肾透明细胞癌患者预后较好。
3、本发明利用免疫组化技术、显微镜拍照和H-score评分半定量测定肿瘤组织中E3泛素化蛋白连接酶UBR5的表达量,并结合术后随访信息,确定E3泛素化蛋白连接酶UBR5表达量与肾透明细胞癌患者的生存时间存在相关性,E3泛素化蛋白连接酶UBR5能用于作为判断肾透明细胞癌患者预后的生物标志物。对于肾透明细胞癌患者,可以利用穿刺获得肾透明细胞癌组织进行UBR5表达水平的检测,从而评估患者的预后。
附图说明
图1为310例肾透明细胞癌患者癌组织及癌旁正常组织中UBR5的免疫组化染色的代表性图,可见肿瘤组织中UBR5表达高低情况。
图2为训练组186例肾透明细胞癌患者的ROC分析图,得出UBR5预测ccRCC患者预后的最佳截断值为130。
图3为肾透明细胞癌患者UBR5低表达组和UBR5高表达组的生存分析图,可见UBR5高表达组中的患者预后更好。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
实施例1:
选取310例肾透明细胞癌患者的肾透明细胞癌组织石蜡切片(肾透明细胞癌组织切片来自上海长海医院310例,由2名病理科医生确诊为肾透明细胞癌),采用免疫组化方法检测E3泛素化蛋白连接酶UBR5在肾透明细胞癌患者肿瘤组织及癌旁正常组织中的表达量并计算免疫组化评分,具体步骤如下:
(1)制备肾透明细胞癌组织石蜡切片,60℃烤箱过夜;
(2)切片脱腊至水;
(二甲苯Ⅰ①10min→二甲苯Ⅱ②10min→二甲苯Ⅲ③10min→100%乙醇5min→95%乙醇5min→85%乙醇5min→75%乙醇5min→双蒸水5min)
(3)3%H2O2溶液,室温放置20min;
(4)双蒸水洗5min×3;
(5)抗原修复:切片放入0.01M柠檬酸盐缓冲液(pH 6.0)中煮沸30min;
(6)自然冷却至室温,双蒸水洗5min×3;
(7)1%BSA封闭30min,37℃;
(8)甩去封闭液,不洗,直接加一抗(兔UBR5抗体,购自Novus Biologicals公司,稀释比例1:500)。置入湿盒中,4℃冰箱过夜16小时;
(9)4℃取出,室温复温15min,然后0.01M PBS洗5min×4;
(10)滴加二抗,45min,37℃;
(11)0.01M PBS洗5min×4,DAB显色2-10min,镜下观察;
(12)双蒸水终止显色,苏木素复染10秒;
(13)分化后自来水返蓝,蒸馏水浸泡;
(14)脱水透明,盖玻片覆盖;
(15)显微镜下观察阳性染色,于肾透明细胞癌组织随机选取3个视野并拍照;
(16)采用H-score评分方法,即高倍镜下综合染色强度和阳性细胞比例进行半定量分析,具体评分标准如下:
1,染色强度评分标准:阴性0分,弱阳性1分,中等阳性2分,强阳性3分。
2,阳性细胞所占比例评分标准:分别评估弱阳性、中等阳性、强阳性细胞的百分比。
3,染色强度评分与对应评分的阳性细胞百分比相乘,得出H-score评分,范围0-300分。
采用以上免疫组化方法的试验步骤检测E3泛素化蛋白连接酶UBR5在310例肾透明细胞癌患者癌组织及癌旁正常组织中表达情况,并依据评分标准进行评分。结果表明:81.34%的患者癌组织UBR5表达水平相对癌旁正常组织低,提示UBR5在肾透明细胞癌组织中是低表达的(见图1)。
实施例2:
结合预后信息,把实施例1中的310例肾透明细胞癌患者3:2比例随机分成训练组和验证组。利用训练组的186例患者的E3泛素化蛋白连接酶UBR5的H-score评分根据5年生存时间为标准进行接受者操作特征曲线(Receiver Operating Characteristic Curve,ROC)分析,数据分析采用R软件(3.5.3版本),得出UBR5预测ccRCC患者预后的最佳截断值(cut-off value)为130见图2)。
实施例3:
结合预后信息,根据实施例2中得到的cut off值把训练组186例、验证组124例患者分为UBR5高表达组(H-score大于或等于130)和UBR5低表达组(H-score小于130),进行病理特征统计分析,数据分析采用SPSS软件(25.0版本),结果表明UBR5高表达组具有更低的TNM分期、WHO/ISUP核分级以及SSIGN评分(见表1-2)。
表1训练组中肾透明细胞癌患者UBR5表达与临床病理特征的相关性研究
表2验证组中肾透明细胞癌患者UBR5表达与临床病理特征的相关性研究
实施例4:
结合预后信息,把实施例3的患者(训练组186例、验证组124例患者)进行生存分析,数据分析采用R软件(3.5.3版本),生存曲线分析使用Kaplan-Meier法,两组之间比较用log-rank检验。结果表明:UBR5高表达组患者的预后更好(见图3)。
实施例5:
结合预后信息,把实施例3的患者(训练组186例、验证组124例患者)进行影响肾透明细胞癌预后的危险因素的单因素和多因素Cox回归分析,数据分析采用R软件(3.5.3版本),结果表明UBR5低表达是一个显著独立的预测肾透明细胞癌患者较低总体生存时间和无疾病进展生存时间的危险因素(见表3-4)。
表3训练组的单因素和多因素Cox回归分析
表4验证组的单因素和多因素Cox回归分析
实施例6:
结合预后信息,把实施例3的患者(训练组186例、验证组124例患者)进行一致性指数C-index分析,数据分析采用R软件(3.5.3版本),结果表明UBR5与临床指标如TNM分期、SSIGN评分等相比具有更好的预测价值(见表5)。
表5训练组和验证组的一致性指数C-index分析
由以上试验结果可知,通过采用免疫组化的方法检测E3泛素化蛋白连接酶UBR5分子表达量,结果表明E3泛素化蛋白连接酶UBR5的表达量与肾透明细胞癌患者的预后具有相关性,因此,以E3泛素化蛋白连接酶UBR5作为分子标志物,对其表达量进行检测能指导肾透明细胞癌诊断或预后评估。相应的,特异性抗E3泛素化蛋白连接酶UBR5的抗体,包括单克隆抗体和多克隆抗体,能用于制备判定肾透明细胞诊断或预后评估的试剂或试剂盒,这对于本领域技术人员来说是显而易见的。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (9)
1.E3泛素化蛋白连接酶UBR5在制备肾透明细胞癌预后评估试剂或试剂盒中的应用。
2.根据权利要求1所述的E3泛素化蛋白连接酶UBR5在制备肾透明细胞癌预后评估试剂或试剂盒中的应用,其特征在于,所述的肾透明细胞癌选自:原发性肾透明细胞癌、或转移性肾透明细胞癌。
3.根据权利要求1所述的E3泛素化蛋白连接酶UBR5在制备肾透明细胞癌预后评估试剂或试剂盒中的应用,其特征在于,所述的试剂或试剂盒检测E3泛素化蛋白连接酶UBR5在肾透明细胞癌患者肿瘤组织中的表达量。
4.根据权利要求3所述的E3泛素化蛋白连接酶UBR5在制备肾透明细胞癌预后评估试剂或试剂盒中的应用,其特征在于,所述的试剂或试剂盒采用免疫组化方法或生物芯片检测法检测E3泛素化蛋白连接酶UBR5在肾透明细胞癌患者肿瘤组织中的表达量。
5.根据权利要求4所述的E3泛素化蛋白连接酶UBR5在制备肾透明细胞癌预后评估试剂或试剂盒中的应用,其特征在于,所述的试剂或试剂盒是将E3泛素化蛋白连接酶UBR5作为分子标记,利用E3泛素化蛋白连接酶UBR5单克隆抗体或多克隆抗体,以及免疫组化试剂,分析E3泛素化蛋白连接酶UBR5在肾透明细胞癌患者肿瘤组织中的表达量,预测肾透明细胞癌患者的预后。
6.根据权利要求5所述的E3泛素化蛋白连接酶UBR5在制备肾透明细胞癌预后评估试剂或试剂盒中的应用,其特征在于,所述的免疫组化试剂包括二甲苯,乙醇,3%H2O2溶液,1%BSA封闭液,DAB显色试剂,苏木素和辣根过氧化物酶标记的羊抗鼠IgG。
7.一种评估肾透明细胞癌患者预后的试剂盒,其特征在于,所述的试剂盒中包含检测E3泛素化蛋白连接酶UBR5在肾透明细胞癌患者肿瘤组织中的表达量的试剂。
8.根据权利要求7所述的评估肾透明细胞癌患者预后的试剂盒,其特征在于,所述的试剂盒中包含:
(i)检测生物样品中E3泛素化蛋白连接酶UBR5表达量的一种或多种试剂;
(ii)选自下组的一种或多种物质:二甲苯,乙醇,3%H2O2溶液,1%BSA封闭液,DAB显色试剂,苏木素和辣根过氧化物酶标记的羊抗鼠IgG。
9.E3泛素化蛋白连接酶UBR5在制备肾透明细胞癌诊断试剂或试剂盒中的应用。
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