CN104698192A - 与结直肠癌显著相关的血清多肽生物标志物及其应用 - Google Patents
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Abstract
本发明提供了与结直肠癌显著相关的血清多肽生物标志物,其由7个具有以下质荷比的人血清蛋白组成:1061.10m/z、1213.09m/z、1607.32m/z、1867.02m/z、1897.95m/z、2011.67m/z和5078.81m/z。本发明发现上述生物标志物联合使用对结直肠癌区分的灵敏度为96.3%-99.3%,特异度为95.7%-98.7%。
Description
技术领域
本发明涉及生物标志物检测,具体地说,涉及一种与结直肠癌显著相关的血清多肽生物标志物及其应用
背景技术
大肠癌早期诊断方法主要依赖于分子生物学的发展,对血液和粪便进行无创或微创检测。多种基因的表达或变异被发现与大肠癌发病相关,包括p53基因突变和p53蛋白质过度表达、K-ras基因突变、细胞色素D1表达、BCL-2基因重排、端粒酶家族基因hTERT过度表达有关;此外,APC和MCC基因突变、C-myc基因的过度表达、p16基因突变、CD44基因的异常表达等都与大肠癌有关。继基因表达和变异之后,多种蛋白质肿瘤标志物也被发现与大肠癌发病相关。
肿瘤标志物在肿瘤诊断和个性化医疗中起重要作用,然而既望的肿瘤标志物不能同时具有高敏感性和特异性。譬如,糖类抗原19-9(Casbohydrate Antigen CA19-9)是一种肿瘤相关抗原,对大肠癌的诊断敏感度56.7%,特异度89.7%。CA242是一种唾液酸化糖类抗原,其灵敏度60.0%,特异度95.4%。
发明内容
为了解决现有技术中存在的问题,本发明的目的是提供一种与结直肠癌显著相关的血清多肽生物标志物,其联合使用可以实现对结直肠较准确的诊断。
为了实现本发明的目的,本发明首先提供与结直肠癌显著相关的血清多肽生物标志物,所述生物标志物由7个具有以下质荷比的人血清蛋白组成:1061.10m/z、1213.09m/z、1607.32m/z、1867.02m/z、1897.95m/z、2011.67m/z和5078.81m/z。
本发明还提供了由前述生物标志物组成的质谱模型。
本发明还提供了所述质谱模型的制备方法,具体包括如下步骤:
1)收集多例临床确诊的结直肠癌患者血清和健康对照人员的血清作为两组血清标本,进行低温冷冻备用;
2)对血清蛋白进行质谱前预处理;
3)对预处理过的两组血清蛋白进行基质辅助激光解析电离飞行时间质谱测定,获得多肽质谱图;
4)分析多肽质谱图,筛选出具有下列质荷比峰的7个结直肠癌特征血清蛋白:1061.10m/z、1213.09m/z、1607.32m/z、1867.02m/z、1897.95m/z、2011.67m/z和5078.81m/z;
5)将上述7个结直肠癌特征血清蛋白作为结直肠癌标志物,建立用于检测结直肠癌的质谱模型。
进一步地,所述步骤2)包括使用磁珠纯化和稳定样品中的血清蛋白或多肽。
本发明还提供了前述生物标志物在建立检测结直肠癌的质谱模型中的应用。
本发明的有益效果在于:
本发明运用Clinprot技术(液体芯片-飞行时间质谱技术)对大肠癌患者和健康对照组的血清多肽谱进行分析,该系统由磁珠分离系统、质谱系统、分析软件和可选的液体样品自动处理系统四部分组成,可以对血液等液体中低分子量的蛋白/多肽有效富集和分析,筛选出差异具有统计学意义的多肽峰,敏感性、重复性和特异性均较好。筛选出的显著差异多肽对大肠癌临床诊断和治疗提供新的理想肿瘤标志物有重要的理论意义和实践价值。结果发现质荷比(m/z)分别为1061.10、1213.09、1607.32、1867.02、1897.95、2011.67和5078.81与结直肠癌显著相关,其联合使用对结直肠癌区分的灵敏度为96.3%-99.3%,特异度为95.7%-98.7%。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1
1、研究对象
研究中111例健康组人员来自在首都医科大学附属北京友谊医院进行常规体检的人群。选入标准为:(1)病历中无躯体疾病或精神异常史;(2)两周内无服药史。将明确诊断为严重的心血管、呼吸、泌尿、消化和血液系统具体疾病的个体予以排除。94例大肠癌患者组来自首都医科大学附属北京友谊医院明确诊断为大肠癌的病人。大肠癌的诊断以病理诊断为金标准,排除患有或曾患糖尿病、心脑血管、肝脏、肾脏、代谢性疾病以及其它肿瘤等重要疾病人群。所有研究对象均签署知情同意,本研究经首都医科大学伦理委员会的批准通过。
2、血浆样品
供分析使用的血浆样品根据自愿原则,静脉穿刺收集清晨空腹血液,37℃环境中半小时凝血。采集的血液以3000r/min离心15分钟分离血清并在-80℃下保存以供蛋白组学分析使用。
3、血浆的磁珠预处理
根据ClinProtTM供应商(Bruker Daltonics,Billerica,USA)提供的说明,将所有血浆样品使用弱阳离子交换磁珠(MB-WCX)进行分离。通过结合、洗涤和洗脱三个步骤对样品进行纯化和分离。首先,在试管中加入10μl磁珠、10μl MB-WCX结合液(BS)和5μl血样,充分混合并放置五分钟。第二步,将试管放置在磁珠分离装置上(BrukerDaltonics,Billerica,USA)一分钟,使磁珠吸附在管壁上。然后取出上清,加入100μl磁珠洗涤液(WS)充分混匀。洗涤三次后取出上清,加入5μl磁珠洗脱液(ES),在分离装置上洗脱管壁上的磁珠两分钟。将上清转移到一个新的试管中,加入5μl磁珠固定液(SS)充分混合。由此产生的洗脱液在-20℃下储存。
4、AnchorChip靶芯片点样和蛋白质分析
用2:1乙醇和丙酮溶液当天配置0.3g/L的α-氰基-4-羟基肉桂酸基质溶液,用该基质溶液按1:10比例稀释洗脱的样品。例如,在10μl的基质溶液中加入1μl的洗脱液,取出1μl混合液点在基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)样品靶上(AnchorChipTM,Bruker Daltonics,Billerica,USA),在室温下干燥后进行分析。使用美国布鲁克道尔顿公司的Autoflex飞行时间设备进行基质辅助激光解析电离飞行时间质谱测定。出于质量控制的目的,用11个平均分子质量偏差不超过100ug/g的肽作为外部标准制剂,数据采集前该标准制剂在每第八个样品上进行了再次校准。另外,用13个参考血样作为外部标准,变异系数小于30%即表示系统性能可接受。每个样品平均激光照射400次,每个靶位轰击100下,轰击四个不同的区域,检测范围为600-10000Da,获得多肽质谱图。
5、统计分析
用ClinProTools2.0软件对样本原始质谱图(使用总离子流)正式分析前,先进行基线消减、校正和归一化,确定检测范围为600-10000Da,设置信噪比(S/N)大于5,质量范围不大于0.1%。以峰面积作为定量标准,将两组间相关峰值水平的比较,筛选出大肠癌血清显著差异多肽峰。统计学分析使用SPSS13.0软件,符合正态分布的数据分析使用独立样本t检验,不符合正态分布的连续数据使用Mann-Whitney秩和检验。采用Logistic回归分析向前条件似然比法(forward conditional),以选入界值α≤0.05、剔除界值α≥0.10为标准,分析性别、年龄以及筛选出的115个显著差异多肽峰中对发生大肠癌的危险因素。P<0.05为差异有统计学意义。
采用多元Logistic回归建立诊断模型(回归方程为LogitP=-34.444+0.998x2-0.086x10+0.185x16-0.454x23+0.430x26+0.068x28-0.050x31-0.221x79,其中自变量分别为质荷比(m/z)1061.10、1213.09、1607.32、1867.02、1897.95、2011.67和5078.81的多肽),该对结直肠癌区分的灵敏度为96.3%-99.3%,特异度为95.7%-98.7%(见表1)。
表1:诊断模型对结直肠癌的诊断效果
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (5)
1.与结直肠癌显著相关的血清多肽生物标志物,其特征在于,所述生物标志物由7个具有以下质荷比的人血清蛋白组成:1061.10m/z、1213.09m/z、1607.32m/z、1867.02m/z、1897.95m/z、2011.67m/z和5078.81m/z。
2.由权利要求1所述的生物标志物组成的质谱模型。
3.权利要求2所述质谱模型的制备方法,其特征在于,所述制备方法包括如下步骤:
1)收集多例临床确诊的结直肠癌患者血清和健康对照人员的血清作为两组血清标本,进行低温冷冻备用;
2)对血清蛋白进行质谱前预处理;
3)对预处理过的两组血清蛋白进行基质辅助激光解析电离飞行时间质谱测定,获得多肽质谱图;
4)分析多肽质谱图,筛选出具有下列质荷比峰的7个结直肠癌特征血清蛋白:1061.10m/z、1213.09m/z、1607.32m/z、1867.02m/z、1897.95m/z、2011.67m/z和5078.81m/z;
5)将上述7个结直肠癌特征血清蛋白作为结直肠癌标志物,建立用于检测结直肠癌的质谱模型。
4.根据权利要求3所述的制备方法,其特征在于,所述步骤2)包括使用磁珠纯化和稳定样品中的血清蛋白或多肽。
5.权利要求1所述的生物标志物在建立检测结直肠癌的质谱模型中的应用。
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