CN112904005B - 用于鼻咽癌早期筛查的血浆外泌体蛋白标志物及其应用 - Google Patents
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Abstract
本发明公开了用于鼻咽癌早期筛查的血浆外泌体蛋白标志物及其应用。本发明筛选并进一步鉴定出鼻咽癌相关的6种早期特异性蛋白(血浆外泌体CA1、EPB41、ANK1、SPTA1、BLVRB和SELP)及所组成的分子分类器,通过分析其绝对蛋白表达量和相对丰度,实现鼻咽癌早期鉴定,其可适用于ELISA、化学发光、蛋白芯片等等一系列鼻咽癌相关的检测技术或检测试剂盒的开发,从而应用于临床鼻咽癌患者的早期筛查和发现。
Description
技术领域
本发明涉及分子标志物领域,具体涉及用于鼻咽癌早期筛查的血浆外泌体蛋白标志物及其应用。
背景技术
鼻咽癌是起源于鼻咽上皮细胞癌变的一种恶性肿瘤,好发于两广地区,发病隐匿,大多数患者确诊时已患有淋巴结转移。虽然放疗与化疗是鼻咽癌首选治疗方法,但随着发现时间的滞后,对于确诊时已经发生转移的患者,该治疗方法仍存在着高治疗失败率、高复发率和高死亡率的问题。因此,发现鼻咽癌早期特异性分子标志物,将有助于早期发现鼻咽癌患者,以尽可能早于转移前发现处于隐匿期的鼻咽癌,改善其临床治疗效果和生存期。
由于EB病毒(Epstein-Barr virus,EBV)感染是鼻咽癌的最主要致病因素之一,几乎100%的非角化型鼻咽癌具有EBV感染,因此,现有技术中主要通过检测EBV基因产物来协助鼻咽癌的诊断。关于鼻咽癌外周血标志物,主要以EBV EBNA-IgA和VCA-IgA作为NPC(非角化型鼻咽癌)早期筛查和诊断指标,但其存在着较为明显的缺陷,如EBV VCA-IgA阳性人群中NPC的检出率仅为3.19%,而且由于各个实验室EBV DNA检测技术、检测平台、阈值等不一致,目前没有标准化的流程,导致其诊断鼻咽癌的实用性、可重复性、推广性存在很大的困难。
近年来,有大量研究致力于肿瘤相关外泌体蛋白标志物的临床应用,例如:外泌体GPC1能很好地区分胰腺癌患者和非肿瘤患者,可作为一种非侵入性手段筛查和诊断适合实施手术的胰腺癌病人。但截至目前,尚未有鼻咽癌微环境来源的外泌体蛋白标志物及分子分类器的报道。因此,发现鼻咽癌微环境来源的血浆外泌体蛋白标志物及相应的分子分类器,将有助于进一步建立非侵入性、易操作性、高灵敏性和高特异性的鼻咽癌筛查或诊断检测方法,对鼻咽癌早发现、早诊断、早治疗产生较大现实意义。
发明内容
本发明的目的在于提供一种用于鼻咽癌早期筛查的标志物;
本发明的另一目的在于提供上述的标志物在鼻咽癌早期筛查中的应用;
本发明的另一目的在于提供一种鼻咽癌早期筛查试剂盒;
本发明的另一目的在于提供定量检测上述标志物的试剂在制备鼻咽癌早期筛查试剂中的应用。
本发明所采取的技术方案是:
本发明的第一个方面,提供:
一种用于鼻咽癌早期筛查的标志物,包括血浆外泌体CA1、EPB41、ANK1、SPTA1、BLVRB和SELP中的一种或多种。
更进一步地,上述标志物为血浆外泌体CA1、EPB41、ANK1、SPTA1、BLVRB和SELP的组合。
进一步地,上述血浆外泌体CA1、EPB41、ANK1、SPTA1、BLVRB和SELP中的至少一种在受试样品中与对照样品中表达差异。
更进一步地,上述血浆外泌体CA1、EPB41、ANK1、SPTA1、BLVRB和SELP中的至少一种在受试样品中与对照样品相比表达上调。
发明人发现,在正常人与处于鼻咽癌早期病患的研究中,血浆外泌体CA1、EPB41、ANK1、SPTA1、BLVRB、SELP可以作为用于区分NC(健康人)和E-NPC(鼻咽癌早期病患)的标志物。
本发明的第二个方面,提供:
上述的标志物在鼻咽癌早期筛查中的应用。
本发明的第三个方面,提供:
一种鼻咽癌早期筛查试剂盒,鼻咽癌早期筛查试剂盒包括定量检测上述标志物的试剂。
上述定量检测上述标志物的试剂可适用于ELISA、化学发光、蛋白芯片、微流控芯片、PCR等一系列本领域常规的蛋白检测试验。
进一步地,上述鼻咽癌早期筛查试剂盒还包括对照样品,上述对照样品为正常人外泌体。
正常人CA1、EPB41、ANK1、SPTA1、BLVRB和SELP含量低于早期鼻咽癌患者。
本发明的第四个方面,提供:
定量检测上述标志物的试剂在制备鼻咽癌早期筛查试剂中的应用。
进一步地,上述鼻咽癌早期筛查包括以下步骤:
采集受试样品;
用所述鼻咽癌早期筛查试剂检测受试样品和对照样品中的血浆外泌体CA1、EPB41、ANK1、SPTA1、BLVRB和SELP的蛋白表达量和蛋白相对丰度;
若受试样品中中的蛋白表达量或蛋白相对丰度多于对照样品,则有患早期鼻咽癌的风险。
本发明的有益效果是:
现有的鼻咽癌标志物专利在使用SELDI-TOF-MS技术时,并未做具体蛋白的鉴定,故不能直接用于鼻咽癌的临床筛查。本发明则针对于筛选出的鼻咽癌相关的6种早期特异性外泌体蛋白及所组成的分类器,可直接用于ELISA、化学发光、蛋白芯片等等一系列检测试剂盒的开发,检测其绝对表达量或者相对丰度,从而应用于临床鼻咽癌患者的早期筛查和发现。
附图说明
图1为外泌体蛋白的4D蛋白质组学分析技术路线;
图2为本发明中质谱数据结果基本统计图;
图3为不同比较组中差异表达蛋白数量分布柱状图;
图4为差异表达蛋白定量火山图;
图5为样品重复性检验;A:相对标准差(RSD);B:主成分分析(PCA);
图6为差异表达外泌体蛋白的富集分布气泡图;A:差异表达外泌体蛋白在KEGG通路中富集分布气泡图;B:差异表达外泌体蛋白在GO功能分类中富集分布气泡图;
图7为外泌体CA1、EPB41、ANK1、SPTA1、BLVRB、SELP区分NC和E-NPC的ROC曲线;
图8为蛋白相对表达水平箱状图;
图9为蛋白相对表达水平热图;
图10为标志物分子分类器区分NC和E-NPC的ROC曲线。A:外泌体CA1、EPB41、ANK1、SPTA1、BLVRB、SELP区分NC和E-NPC的ROC曲线;B:外泌体CA1和EPB41区分NC和E-NPC的ROC曲线。
具体实施方式
为了使本发明的发明目的、技术方案及其技术效果更加清晰,以下结合具体实施方式,对本发明进行进一步详细说明。应当理解的是,本说明书中描述的具体实施方式仅仅是为了解释本发明,并非为了限定本发明。
所使用的实验材料和试剂,若无特别说明,均为常规可从商业途径所获得的耗材和试剂。
蛋白的提取
采用本领域常规的外泌体蛋白的提取方法,提取健康人与确诊为鼻咽癌的患者血浆外泌体作为本发明的试验材料,健康人与确诊为鼻咽癌的患者人数满足统计学样本最低采集要求,因此,具有统计学意义上的代表性。
样本纳入标准:
健康人(NC):
①未患肿瘤;②无高脂血症、高血压;③无全身性炎症反应;④无囊肿、可疑性结节等。
早期鼻咽癌(E-NPC):
根据美国癌症联合会(AJCC)鼻咽癌临床分期Ⅰ-Ⅱ期。
实施例1蛋白的筛选与鉴定
选取早期鼻咽癌(E-NPC)、健康人(NC)血浆标本各10例,按年龄、性别匹配,分离血清中蛋白后用非标定量技术以及基于质谱的定量蛋白质组学技术有机结合,对样本进行定量蛋白组的研究(图1)。
本发明中,发明人一共鉴定到1431个蛋白,其中612个可定量(图2)。以1.5倍为差异表达变化阈值,以统计学检验t-test p-value<0.05为显著性阈值,E-NPC/NC比较组中,有184个蛋白上调,38个蛋白下调(图3、图4)。
采用主成分分析(PCA)和相对标准差(RSD)统计分析方法评估蛋白定量重复性。PCA显示重复样本间的聚集程度好,表明定量重复好(图5B);整体RSD值较小,也表明定量重复好(图5A)。
进行差异表达蛋白通路和功能分析。利用KEGG通路富集分析(图6A),差异表达蛋白与小细胞肺癌(Small cell lung cancer)、代谢(porphyrin and chlorophyllmetabolism)、局部黏附(Focal adhesion)、抗原提呈(Antigen processing andpresentation)、血小板激活(Platelet activation)、细胞骨架(regulation of actincytoskeleton)等通路密切相关。利用GO功能富集分析(图6B),差异表达蛋白与细胞底物连接组装(cell-substrate junction assembly)、正向调节蛋白结合(positive of proteinbinding)、细胞间黏附(homotypic cell-cell adhesion)、整连蛋白信号通路(integrin-mediated signaling pathway)、肌动蛋白细胞骨架组织(actin cytoskeletonorganization)、血小板激活(platelet activation)、正向调节细胞运动(positive ofcell motility)等功能显著相关。
根据KEGG通路富集分析、GO功能富集,选择倍数最高的18个差异表达蛋白(SwissProt蛋白编号):EPB41(P11171)、SELP(P16109)、ANK1(P16157)、CA1(P00915)、BLVRB(P30043)、SPTA1(P02549)、SPTB(P11277)、PKM(P14618)、ALDOA(P04075)、PRDX6(P30041)、CAP1(Q01518)、TLN1(Q9Y490)、SLC4A1(P02730)、ADAM10(O14672)、ZYX(Q15942)、WDR1(O75083)、LIMSI(P48059)、VASP(P50552)。在15个NC、10个E-NPC收受试样品中做进一步的定量蛋白组学分析(PRM)验证。
实施例2定量蛋白质组学分析(PRM)
采用本领域中常规的定量蛋白质组学分析方法测定EPB41(P11171)、SELP(P16109)、ANK1(P16157)、CA1(P00915)、BLVRB(P30043)、SPTA1(P02549)、SPTB(P11277)、PKM(P14618)、ALDOA(P04075)、PRDX6(P30041)、CAP1(Q01518)、TLN1(Q9Y490)、SLC4A1(P02730)、ADAM10(O14672)、ZYX(Q15942)、WDR1(O75083)、LIMSI(P48059)、VASP(P50552)在15个NC、10个E-NPC收受试样品中的表达差异。
结果发现,CA1、EPB41、ANK1、SPTA1、BLVRB、SELP区分NC和E-NPC的ROC曲线AUC值分别为0.9333、0.87、0.8267、0.8467、0.8633、0.75(图7),说明该标志物能较好的区分出NC和E-NPC。CA1、EPB41、ANK1、SPTA1、BLVRB、SELP一共6个蛋白表达差异显著,如蛋白相对表达水平箱状图(图8)和相对表达水平热图(图9)所示,可以作为E-NPC的蛋白标志物。CA1、EPB41、ANK1、SPTA1、BLVRB、SELP组合区分NC和E-NPC的灵敏度和特异度好,ROC曲线AUC为0.8516(图10A)。CA1和EPB41组合区分NC和E-NPC的灵敏度和特异度更高,ROC曲线AUC为0.9042(图10B)。
因此,CA1、EPB41、ANK1、SPTA1、BLVRB、SELP可以作为用于区分NC和E-NPC的标志物。
实施例3临床案例
在临床实践中,随机抽取NC和E-NPC患者进行验证检测,以下选择其中的NC和E-NPC患者各5例以展示本发明的实际效果。
健康人黄某某,女,47岁,PRM验证的蛋白相对丰度(relative abundance)为:CA1(0.07)、EPB41(0.12)、ANK1(0.06)、SPTA1(0.13)、BLVRB(0.13)、SELP(0.09);
健康人,男,62岁,PRM验证的蛋白相对丰度(relative abundance)为:CA1(0.07)、EPB41(0.14)、ANK1(0.05)、SPTA1(0.11)、BLVRB(0.10)、SELP(0.06);
健康人,男,47岁,PRM验证的蛋白相对丰度(relative abundance)为:CA1(0.13)、EPB41(0.13)、ANK1(0.12)、SPTA1(0.14)、BLVRB(0.30)、SELP(0.35);
健康人,男,48岁,PRM验证的蛋白相对丰度(relative abundance)为:CA1(0.19)、EPB41(0.07)、ANK1(0.12)、SPTA1(0.14)、BLVRB(0.05)、SELP(0.17);
健康人,男,43岁,PRM验证的蛋白相对丰度(relative abundance)为:CA1(0.07)、EPB41(0.02)、ANK1(0.02)、SPTA1(0.06)、BLVRB(0.05)、SELP(0.04)。
患者陈某某,女,55岁,病理分期T1N0M0期,PRM验证的蛋白相对丰度(relativeabundance)为:CA1(3.64)、EPB41(2.13)、ANK1(2.35)、SPTA1(2.34)、BLVRB(2.83)、SELP(0.90);
患者许某某,男,51岁,病理分期T2N1M0期,PRM验证的蛋白相对丰度(relativeabundance)为:CA1(4.11)、EPB41(2.51)、ANK1(3.14)、SPTA1(3.08)、BLVRB(3.19)、SELP(0.96);
患者于某某,男,48岁,病理分期T2N1M0期,PRM验证的蛋白相对丰度(relativeabundance)为:CA1(4.48)、EPB41(2.58)、ANK1(3.46)、SPTA1(3.53)、BLVRB(3.79)、SELP(2.25);
患者熊某某,男,57岁,病理分期T2N1M0期,PRM验证的蛋白相对丰度(relativeabundance)为:CA1(4.06)、EPB41(3.38)、ANK1(3.35)、SPTA1(2.91)、BLVRB(4.36)、SELP(1.76);
患者游某某,男,47岁,病理分期T1N1M0期,PRM验证的蛋白相对丰度(relativeabundance)为:CA1(4.07)、EPB41(2.27)、ANK1(4.31)、SPTA1(4.50)、BLVRB(0.36)、SELP(2.56)。
从这部分案例中,由于病灶局限在鼻咽部的T1N0M0期(E-NPC)鼻咽癌在临床上不易被早期发现,而本发明中的CA1、EPB41、ANK1、SPTA1、BLVRB、SELP组成的鼻咽癌分子分类器对早期鼻咽癌有提示意义,因此,可以说明本发明的鼻咽癌分子分类器(标志物组合)对临床症状轻微不易被检出的早期鼻咽癌有较准确的提示作用,对实现鼻咽癌早发现、早治疗、提高生存率意义重大。
同理,基于本发明的标志物组合,进一步开发出的检测试剂、检测试剂盒也对对临床症状轻微不易被检出的早期鼻咽癌有较准确的提示作用,具有极高的医疗与实用价值。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (5)
1.一种用于鼻咽癌早期筛查的标志物,其特征在于,为外泌体CA1、EPB41、ANK1、SPTA1、BLVRB和SELP的组合或外泌体CA1和EPB41的组合;所述外泌体CA1、EPB41、ANK1、SPTA1、BLVRB和SELP在受试样品中与对照样品中表达上调;所述对照样品为正常人外泌体。
2.一种鼻咽癌早期筛查试剂盒,其特征在于,所述鼻咽癌早期筛查试剂盒包括定量检测权利要求1所述标志物的试剂。
3.根据权利要求2所述的鼻咽癌早期筛查试剂盒,其特征在于,所述鼻咽癌早期筛查试剂盒还包括对照样品,所述对照样品为正常人外泌体。
4.根据权利要求3所述的鼻咽癌早期筛查试剂盒,其特征在于,所述正常人外泌体携带有CA1、EPB41、ANK1、SPTA1、BLVRB和SELP。
5.定量检测权利要求1所述标志物的试剂在制备鼻咽癌早期筛查试剂盒中的应用。
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