CN105403701B - 膜联蛋白a2的血清检测方法、检测试剂盒及其应用 - Google Patents
膜联蛋白a2的血清检测方法、检测试剂盒及其应用 Download PDFInfo
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Abstract
本发明涉及膜联蛋白A2的血清检测方法、检测试剂盒及其应用，具体而言，涉及单独使用新的标志物膜联蛋白A2检测HCC或者与AFP联用使用检测HCC的方法、检测试剂盒及其应用。
Description
本申请为申请号为200810089657.4、发明名称为“膜联蛋白A2的血清检测方法、检测试剂盒及其应用”的发明申请的分案申请。
技术领域
本发明涉及生物学领域，具体而言，涉及单独使用新的标志物检测HCC或者与AFP联用使用检测HCC的方法、检测试剂盒及其应用。
背景技术
肝癌(Liver cancer)是一种严重危害人类健康的恶性肿瘤，2000年全世界新发病例约564,000人，在所有恶性肿瘤中排第五位。由于其恶性程度高、预后不良，五年生存率不足10％。中国是肝癌的高发区，集中了全世界约54％的新发病例，其中肝细胞肝癌(hepatocellular carcinomas,HCC)占原发性肝癌的90％以上[1,2]。据1991-2000年间，中国169,871人口的死因抽样调查显示，它的死亡率排在全部恶性肿瘤的第2位，年死亡率为54.7/100,000(男81.2，女29.0)[3]。
甲胎蛋白(alpha-fetoprotein,AFP)是目前临床上公认的HCC诊断标志物。20世纪50年代，Bergstrand等人首次在人类胎儿血清中发现了这种电泳迁移率相当于甲种球蛋白的蛋白质，在正常成人血清中并没有检测到它的存在[4]。1960年，Abelev G及其同事在小鼠的肝细胞癌中发现了这种甲种球蛋白，它不存在于正常小鼠的任何组织中，经证实它是胎鼠血清中的主要成分，在成年鼠肝细胞再生时会重新出现[5]。后来，HCC患者和畸胎瘤患者血清中胚胎特异性的甲种球蛋白也被检测出来[6,7]。因此，人们将这种主要存在于胎儿组织的蛋白命名为甲胎蛋白。
目前已知，AFP是一种分泌型糖蛋白，与白蛋白同属于白蛋白样基因家族。它主要存在于啮齿类动物和人胚胎期血清中，由胚胎发育时期卵黄囊内胚层和胎肝合成并分泌到血液中。在人类胚胎中AFP最高浓度可达3-4mg/ml，出生后，血清中AFP浓度迅速下降，新生儿期约为10-50μg/ml，正常成人一般在10ng/ml以下。怀孕期间母体血清中AFP浓度的变化可以提示多种胚胎发育缺陷[8]。而成人中，升高的血清AFP水平不仅存在于60％-70％的HCC病人，也可见于大约20％的慢性肝炎，20％-60％的肝炎肝硬化和某些胚胎性癌患者[9]。在有慢性肝炎和肝硬化背景的HCC患者中，AFP的诊断价值更低[10,11]。因此，AFP对于HCC患者的诊断灵敏度介于41％-97％之间，特异度在80％-95％，而阳性预测值徘徊在9％-58％之间[10-12]。更为重要的是，临床上有20％-30％的HCC患者血清AFP水平并不升高，从而大大限制了其在HCC临床检查中的应用价值。因此，目前迫切需要找到能与AFP联合使用，提高HCC诊断准确度的新标志物。
综上所述，由于目前尚缺乏有效的肝癌检测方法，因此，本领域迫切需要开发建立新的肝癌患者血清ELISA检测方法和可以方便快捷检测的试剂盒。
为此，本发明人进行了大量的研发工作，在前期工作中，发明人提取了三个肝癌细胞系HepG2、Hep3B和SK-HEP-1，及一个正常肝细胞系HL-7702的亚细胞组分，并经双向电泳分离(2-DE)，选取具有三倍以上差异的蛋白质点，胶内酶解后经MALDI-TOF-TOF质谱鉴定。在胞浆组分中，发明人意外地发现了一个在AFP阴性肝癌细胞系SK-HEP-1中特异性高表达的蛋白质点，经质谱鉴定为膜联蛋白2(Annexin A2，ANXA2)。
ANXA2属于annexins蛋白家族成员，是一种Ca2+离子结合蛋白，其C端核心结构域包含磷脂、F-actin和肝素的结合位点，而N端包含PKC(Ser-25)和Src(Tyr-23)的磷酸化调节位点[13-15]。ANXA2在细胞内以三种形式存在：单体、二聚体和四聚体。二聚体由一分子的ANXA2和一分子的3-磷酸甘油酸激酶组成。四聚体由两分子的ANXA2和两分子的S100A10(P11)组成。四聚体对单体的相对量随着细胞或组织的类型而不同，小肠上皮细胞中ANXA2的四聚体大约是100％，而培养的成纤维细胞中单体形式的ANXA2占了50％以上。目前认为，ANXA2可以中介细胞的胞泌、胞吞、actin依赖的囊泡转运、胞饮等过程，调节离子通道并影响细胞骨架重建、细胞间黏附和运动，而且，其在造血干细胞归巢、植入和维持骨髓微环境中发挥重要作用[16-34]。在各种转化细胞中，如v-src、v-H-ras、v-mos和SV40转化细胞等，ANXA2的表达均会被诱导。而且，其基因表达受多种生长因子，如胰岛素、IGF和EGF的调节。在多种人类肿瘤中如胰腺癌、高分化胶质瘤、胃癌、肾细胞癌、肺癌、HCC和急性早幼粒细胞白血病中ANXA2均表达上调，但在前列腺癌中表达下调[35-42]。因此表明，ANXA2的上调与细胞转化过程相关。另外，大约10％-15％的细胞内ANXA2与细胞核相关。ANXA2可以与DNA序列结合，是刺激DNA聚合酶α活性的引物识别复合物的组成成分[43-46]。ANXA2具有RNA结合能力，是体内mRNP的组成成分，目前已经发现，ANXA2可以直接和c-Myc mRNA的3’-UTR相互作用，将其特异的转运并定位到胞浆中的细胞骨架。细胞过表达ANXA2后，导致c-Myc蛋白表达水平同时上调[47,48]。在乳腺癌和头颈部肿瘤中的研究结果发现，ANXA2是潜在的纤溶酶原和组织型纤溶酶原激活蛋白(t-PA)受体，可能参与肿瘤侵袭和转移过程[49,50]。
Dreier R等人曾利用免疫组织化学染色研究了Annexin蛋白家族成员在人体各个正常器官、组织中的表达情况，发现肝脏内的肝细胞和Kupffer细胞均不表达Annexin A2[51]。Tucker CJ等人在动物模型中研究了砷酸诱发的HCC与对照正常肝脏之间的差异表达基因谱，其中鉴定到的一个差异表达基因就是Annexin A2，其在肿瘤组织中明显上调，realtime PCR结果发现，在正常对照成年雄性C3H小鼠中，ANXA2的表达水平为1.0±0.3，在诱发HCC的小鼠癌旁正常组织和肿瘤组织中Annexin A2的表达水平升高9倍和49倍[52]。Lim SO等人利用2-DE-MAILDI-TOF技术策略对比了正常、肝硬化和肝细胞肝癌组织蛋白表达谱的差异，鉴定到的差异蛋白之一就是Annexin A2，该蛋白在正常和肝硬化组织中的表达水平明显低于HCC肿瘤组织[53]。但是，这两篇研究仅仅从基因组和蛋白质组的角度筛查了HCC与健康肝脏之间的表达差异，并没有在临床样品上对Annexin A2进行任何验证，而且，目前没有任何Annexin A2可以在人类血清中检测到，并与人类疾病相关的报道。
发明内容
鉴于ANXA2在肿瘤发生发展中的重要作用，发明人对其在肝癌组织中的表达情况进行了大样本量的验证分析，结果显示，在19例配对肝癌组织的Western blot结果中，ANXA2在肿瘤组织中的表达上调率为68.4(13/19)，而40例配对HCC组织的免疫组织化学染色结果表明，其在肿瘤组织中的表达阳性率为60％(24/40)，而在癌旁正常组织中的表达阳性率仅为10％，经过Chi-square检验证明，ANXA2是一种在肝癌组织中高表达的蛋白质(p<0.0001)。因此，发明人认为ANXA2蛋白的表达与肝癌具有十分重要的联系，可以作为肝癌相对特异性的敏感标志蛋白。虽然ANXA2不具有信号肽，但是，文献报道，它存在于外体(exosome)中，我们推测，它可以经非经典分泌途径分泌入血[54,55],在肝癌患者血清/血浆中可检测到，并作为肝癌早期血清学检测的特异性标志物。在上述发现的基础上，发明人首次建立了人类ANXA2的血清间接双夹心ELISA检测方法，并对其检测结果进行了评价。进而本发明提供了提高HCC诊断准确度的新的标志物Annexin A2的血清ELISA检测方法、检测试剂盒及其应用。具体而言，本发明包括以下几个方面。
一方面，本发明提供了下述获得作为中间结果的信息的方法，所述方法为检测待测生物样品中的Annexin A2水平是否异常，进而获得作为中间结果的信息的方法，所述方法包括步骤：
1)测定待检生物样品中的Annexin A2水平；
2)比较待检生物样品中的Annexin A2水平和正常生物样品对照的Annexin A2水平；
3)获得作为中间结果的信息，
4)根据比较结果确定待检生物样品是否有可能是HCC样品。
2.上述1的方法，进一步包括与AFP联合检测。
3.上述1或2的方法，其中步骤1)的生物样品是已经脱离人体或者动物体的组织、体液或者排泄物。
4.上述1或者2的方法，其中步骤1)的生物样品是血清。
5.上述1或2的方法，其中步骤1)的测定是血清ELISA法。
6.使用Annexin A2检测HCC的方法，所述方法包括步骤：
1)测定待检生物样品中的Annexin A2水平；
2)比较待检生物样品中的Annexin A2水平和正常生物样品对照的Annexin A2水平；
3)根据比较结果确定待检生物样品是否是HCC样品。
7.上述6的方法，进一步包括与AFP联合检测。
8.上述6的方法，其中步骤1)的生物样品是血清。
9.上述6的方法，其中步骤1)的测定是血清ELISA法。
10.Annexin A2作为HCC的标志物的用途。
11.上述10的用途，其中Annexin A2与AFP联合应用。
12.用于检测Annexin A2的HCC检测试剂盒。
13.上述12的HCC检测试剂盒，其中包括：作为标准的Annexin A2、抗Annexin A2抗体。
14.用于联合检测Annexin A2和AFP的HCC检测试剂盒。
附图说明：
图1为血清AFP、ANXA2和两个指标联合使用的受试者检测曲线。图中虚线代表AFP，点虚线代表ANXA2，实线代表两个指标联合使用。A表示曲线下面积。
具体实施方式
为了进一步清楚地阐述本发明，下面提供了本发明的具体实施例，不过本发明的内容并不局限于所述实施例，任何本法的方法和产品的等价的变形和修饰等都包括在本发明的范围内。
实施例1：采用人类ANXA2的血清间接双夹心ELISA检测方法
为了对生物样品进行检测，进行了如下的ELISA检测方法，所述方法的具体步骤为：
1.包被：用pH9.6的50mM碳酸盐缓冲液(15mM Na2CO3；35mM NaHCO3)作为稀释液，将山羊抗人ANXA2抗体(Santa Cruz Biotechnology公司,Cat.No.sc-1924)稀释为2μg/ml。在聚苯乙烯的96孔酶标板的微孔中加入50μl包被液，用保鲜膜封好，在4℃冰箱中放置过夜(>16h)。次日，用350μl 150mM的PBST缓冲液(137mM NaCl，2mM KH2PO4,10mM Na2HPO4，加入0.05％的Tween-20,调节至pH7.4)浸泡式洗板，3min×3次。
2.封闭：每孔加入300μl的2％BSA(Sigma-Aldrich公司,Cat.No.A7906)，室温封闭4h。用350μl PBST缓冲液浸泡式洗板，1min×3次。
3.抗原孵育：在每个反应孔中加入50μl的HCC患者血清(用PBST缓冲液1:10稀释)或梯度稀释的人重组ANXA2蛋白(Abnova公司，Cat.No.H00000302-P02)，37℃孵育1h，同时，用洗涤缓冲液替代抗原作为阴性对照。每个样品设置3个平行孔。孵育结束后，弃去抗原，用350μl PBST缓冲液浸泡式洗板，3min×3次。
4.检测抗原反应：在每个反应孔中加入50μl用PBST缓冲液稀释的1μg/ml的小鼠抗人ANXA2抗体(Santa Cruz Biotechnology公司,Cat.No.sc-28385)作为检测抗体，室温孵育1h。而后弃去，用350μl PBST缓冲液浸泡式洗板，3min×3次。
5.二抗孵育：在每个反应孔中加入50μl用PBST缓冲液1:5000稀释的辣根过氧化物酶标记的山羊抗小鼠IgG(Jackson ImmunoReasearch公司，Cat.No.115-005-003)，室温孵育1h。而后弃去，用350μl PBST缓冲液浸泡式洗板，3min×3次。
6.TMB(3,3’,5,5’-四甲基联苯胺)底物显色：称取10mg TMB(Sigma-Aldrich公司，Cat.No.15053)溶于5ml无水乙醇中，配置成TMB贮存液。而后取0.5ml TMB贮存液加入10ml磷酸柠檬酸底物缓冲液(51.4mM Na2HPO4，24.3mM柠檬酸，pH5.0)中，并加入32μl 0.75％的H2O2混匀，配置成TMB显色溶液。在每个反应孔中加入100μl TMB显色溶液，轻轻混匀，室温孵育30min。
7.终止：待颜色满意并稳定以后，每孔加入100μl 2M H2SO4溶液终止反应，并轻轻混匀。
8.OD值的测定：立即将酶标板置于Model 680酶标仪(Bio-Rad公司)中，OD450/570双波长读数。
9.结果的判读：首先确定3个阴性对照孔的读数，取平均值作为本底读数。
然后对于每一例血清样品或重组ANXA2标准品，先计算3个平行孔的孔间变异系数(CV)，对于那些CV值小于15％的样品，取3次重复的平均值作为该样品的OD值；CV值大于15％的样品，去除一个异常值后，重新计算孔间变异(孔间变异＝两孔OD值之差/两孔OD值之和)，如果该变异值<15％，认为该样品为有效病例，并取两孔平均值作为该样品的OD值，否则，舍弃该例样品。而后，利用梯度稀释的重组ANXA2标准品绘制标准曲线，从而计算出每一例有效病例的血清ANXA2浓度。注：上述实验方法所用材料，除特殊注明以外，均为国产分析纯试剂。
实施例2：人类ANXA2的血清间接双夹心ELISA检测方法的临床检测应用
利用实施例1的上述方法，对年龄、性别相匹配的30例健康成人(平均年龄51岁，最大年龄70岁，最小年龄33岁)和98例HCC患者(平均年龄54岁，最大年龄83岁，最小年龄15岁)的血清ANXA2浓度进行了测定。试验结果和标准曲线的浓度梯度和扣除本底的OD值见表1，两者的线性相关系数为0.971，表明这一浓度梯度基本落在检测反应的线性范围内。同样表明所建立的这一系统比较稳定，对所有病例结果均有效，绝大多数样品的CV值控制在10％以内。
表1：ANXA2标准曲线的设定和检测的OD值
实验结果发现，健康成人血清ANXA2的浓度为17.58μg/ml，检测区间为4.81～29.29μg/ml。而HCC患者的血清ANXA2平均浓度是28.68μg/ml，最小值为13.45，最大值为230.66μg/ml。经过Mann-Whitney Rank Sum检验，两者差异具有显著性(p<0.0001)。同时，健康对照中的血清ANXA2浓度与其年龄、性别之间没有明显的相关性(p＝0.9462，p＝0.3646)。AFP阳性和阴性的HCC患者，血清ANXA2表达水平没有明显差异(p＝0.1595)。而且，我们发现，低分化HCC患者的血清ANXA2明显高于中高分化患者(p<0.05)。
实施例3：采用对照方法进行的平行检测
利用电化学发光方法(临床上常规使用的方法)测定了前述实施例2的所有ELISA样品的血清AFP浓度，结果表明，健康成人和HCC患者的血清AFP平均浓度分别是3.5875ng/ml和31.415ng/ml。分别绘制这两个蛋白用于HCC检测的受试者曲线(ROC曲线)(图1)，结果发现，AFP的曲线下面积为0.81，ANXA2的曲线下面积为0.78，这两个蛋白的诊断价值基本相同(卡方检验,p＝0.6096)。此时，获得最佳诊断效果的血清AFP浓度是11ng/ml，诊断灵敏度为93.3％，特异度为63.5％。而ANXA2的阈值为27.93μg/ml，此时的诊断灵敏度和特异度分别为96.7％和52.1％。当联合使用这两个血清学指标时，ROC曲线下面积增加到0.87，诊断灵敏度和特异度增加到100％和72.9％。
上述结果表明，所建立的血清ANXA2ELISA检测方法具有重复性好、灵敏度高的特点。将其用于128例健康成人和HCC患者的血清ANXA2测定发现，这一指标具有很高的检测灵敏度，表明它可能是一个很有潜力的血清早期筛查指标。而且，其与AFP的诊断效力基本相同，具体而言，单独使用ANXA2可以独立对HCC进行检测，其与目前临床常用的AFP具有近似的诊断价值，而且更加灵敏(ANXA2的诊断灵敏度为96.7％，而AFP为93.3％)，特别适合于肿瘤的人群筛查。如果联合使用这两个血清学指标，则可以100％的检出HCC患者。
实施例4：检测ANXA2的肝癌检测试剂盒
本发明还提供了一种肝癌检测试剂盒，其中含有重组的人ANXA2作为蛋白标准，含有抗人ANXA2抗体(包被抗体：工作浓度2μg/ml，稀释缓冲液为50mM碳酸盐缓冲液，pH 9.0；检测抗体：工作浓度1μg/ml，稀释缓冲液为150mM的PBST缓冲液，pH 7.4)，以及实施例1中其它的检测试剂。
使用本发明的试剂盒采用实施例1的方法已完成临床试验128例，检测阳性率为100％，灵敏度高达96.7％。根据文献检索，目前国内外尚无相同工作的报道。
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Claims (4)
1.测定Annexin A2水平的试剂在制备诊断HCC的试剂盒中的用途，所述试剂盒通过包括下述步骤的方法，通过检测待测血清样品中Annexin A2水平而诊断HCC：
1)测定待检血清样品中的Annexin A2水平；
2)比较待检血清样品中的Annexin A2水平和正常血清样品对照的Annexin A2水平；
3)根据比较结果确定待检血清样品是否是HCC样品。
2.权利要求1的用途，所述试剂盒进一步包括用于AFP检测的试剂。
3.权利要求1的用途，其中步骤1)的测定是血清ELISA法。
4.权利要求1-3任一项的用途，其中所述试剂盒包括：作为标准的Annexin A2、和抗Annexin A2抗体。
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