CN112858551B - 用于诊断食管鳞癌的联合型代谢生物标志物的应用及试剂盒 - Google Patents
用于诊断食管鳞癌的联合型代谢生物标志物的应用及试剂盒 Download PDFInfo
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Abstract
本发明涉及血清样本中小分子代谢产物次黄嘌呤、2‑酮异己酸、谷氨酸及天冬氨酸作为联合标志物在制备用于区分受试者中的食管鳞癌及食管上皮非典型增生患者的试剂盒中的新应用。本发明还涉及检测受试者中的食管鳞癌患者的试剂盒,通过检测来自受试者的血清样本中上述联合标志物的相对浓度,基于线性回归方程计算所述联合标志物变量P,再基于确定的阈值判断所述受试者是否患有食管鳞癌。所述试剂盒可实现高灵敏、高效检测本发明涉及的几种代谢物,具有检测成本低,重复性好的特点。本发明可应用于临床辅助诊断食管鳞癌,具有诊断灵敏度高,可将食管鳞癌与其癌前病变进行有效区分的特点,具有较好的应用前景。
Description
技术领域
本发明涉及分析化学、生物化学及临床医学领域。
背景技术
食管癌(esophageal cancer,EC)是我国高发的一种恶性肿瘤,我国每年的发病例数约占世界发病总数的一半,食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)为我国最为常见的食管癌类型,其术后整体5年生存率不足25%。食管鳞癌具有病程进展快、恶性程度高、预后差的特点。食管鳞癌的发生与吃腌制食品、吃过热食物等不良的饮食习惯密切相关,该病的发生往往伴随着食管相关病理变化的发生,其中食管上皮非典型增生(esophageal squamous dysplasia,ESD)是最为常见的食管鳞癌癌前病变,其临床症状与食管鳞癌相似,二者的区分对于食管鳞癌的防治及高危人群病理状态的监测具有重要的意义。然而,当前临床上尚无可对二者进行有效区分的生物标志物,只有通过病理检测才能将二者进行区分,极大地限制了食管鳞癌的早检早诊及高危人群的监测。
近年来,代谢组学作为新兴的系统生物学科学被广泛地用于疾病生物标志物的发掘研究中。色谱-质谱联用技术是代谢组学研究的主要技术手段,其检测小分子代谢物在疾病诊断中的应用已有成功的案例,包括通过检测多种脂酰肉碱筛查新生儿先天性缺陷、通过检测固醇水平判断肝脏功能状态等。由于体液中单一代谢物的诊断效能所受影响因素较多,因此从血清代谢组中筛选到少数代谢物进一步通过回归模型整合成“联合型标志物”,并利用计算的“判别可能性”P值(Probability)可显著增强代谢物对疾病诊断的灵敏度和特异度。考虑到有机酸、核苷酸等中大极性代谢物在肿瘤发生发展过程中的作用,本发明采用基于气相色谱-质谱联用的代谢组学技术及方法,检测血清中的中大极性代谢物。利用统计学方法筛选得到目标代谢物,旨在在食管鳞癌及其癌前病变食管上皮非典型增生的判别中进行应用。
本发明基于气相色谱-质谱联用采集食管鳞癌及食管上皮非典型增生患者的血清代谢轮廓数据,经多变量筛选及验证,确定了基于次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸的联合使用,以作为一种联合型的代谢标志物在食管鳞癌及食管上皮非典型增生的判别中的新用途。该组代谢物参与体内重要生理功能及生命活动。次黄嘌呤是黄嘌呤氧化反应的重要下游产物,其被发现在抗氧化应激导致的细胞损伤等方面发挥重要作用。2-酮异己酸是支链氨基酸异常代谢的中间产物,具有一定的细胞毒性,该物质的慢性升高可导致槭糖尿病的发生。谷氨酸是二十种蛋白合成氨基酸之一,在神经递质合成及冲动传导等细胞代谢过程中发挥重要作用。天冬氨酸也是二十种蛋白合成氨基酸之一,其在尿素循环及DNA代谢过程中发挥重要作用。目前尚无将该四种代谢产物联合用于食管鳞癌诊断的报道。
发明内容
本发明的目的是针对食管鳞癌与食管上皮非典型增生难以区分的临床实际问题,提供了一种基于新的小分子代谢物的组合对食管鳞癌及食管上皮非典型增生进行有效判断的新用途,并提供用于上述小分子代谢物检测的方法。
该代谢物联合标志物包括下列四种代谢物:次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸,其中次黄嘌呤用气相色谱质谱联用检测到离子碎片包括:m/z45、m/z73、m/z147、m/z193、m/z206、m/z265、m/z280,2-酮异己酸的碎片包括m/z73、m/z89、m/z189、m/z200、m/z216,谷氨酸的碎片包括:m/z73、m/z147、m/z156、m/z230、m/z246,天冬氨酸的碎片包括:m/z 45、m/z 59、m/z 73、m/z 117、m/z 130、m/z 160、m/z 245。
上述四种物质的检测试剂盒如下:
标准品:包括次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸,所述标准品分别用于对应的血清代谢物次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸的定性;用于预处理来自受试者的血清样本的提取液;包含内标物十三酸(10μg/mL)的甲醇溶液,甲氧胺吡啶溶液(20mg/mL)、甲基三甲基甲硅烷基三氟乙酰胺(N-methyl-N-(trimethylsilyl)trifluoroacetamide,MSTFA)。
计算受试者血清样本中的联合标志物变量P的方法如下:
1)受试者样本处理:用血清样本的提取液处理来自受试者的血清样本,然后进一步提取中大极性代谢物。
2)以气相色谱对经1)处理的血清样本进行分离,并由质谱检测。
3)经气相色谱-质谱联用分析后,所得的色谱峰强度分别与内标比较,获得上述四种物质的相对浓度。
4)进一步将次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸回归为联合标志物变量P,线性回归方程如下:
P=1/(1+e-(1.731-3.52*10-6*a+2.22*10-5*b-9.38*10-7*c-4.39*10-6*d))
其中,a为血清中次黄嘌呤的相对含量,b为血清中2-酮异己酸的相对含量,c为血清中谷氨酸的相对含量,d为血清中天冬氨酸的相对含量,e为欧拉数,即自然对数函数的底数。所得变量P在食管鳞癌患者中降低,该变量值可用于辅助区分食管鳞癌及其癌前病变食管上皮非典型增生。本发明确定的联合标志物的对食管鳞癌判断的最佳截点值(cut-off值)设为0.814,低于该截点值的则可能为食管鳞癌。也可以根据实验者的实际结果通过回归得到新的方程,并定义该实验室的最佳截点值。截点值的确定依据为根据联合标志物变量P值作受试者工作特征曲线(ROC曲线),取灵敏度及特异度之和最大的P值作为最佳截点值,大于等于最佳截点值为食管上皮非典型增生,小于最佳截点值为食管鳞癌。
该联合型代谢标志物用途:将食管鳞癌与食管上皮非典型增生进行有效区分,适用于食管上皮非典型增生患者的常规体检监测。
本发明原理如下:
(1)利用气相色谱-质谱联用的代谢组学分析技术,对食管鳞癌及食管上皮非典型增生血清样本进行代谢轮廓分析,获得可用于定性及定量分析的代谢组数据。
(2)基于统计学数据分析方法对标志物进行筛选及验证,具体实施方式为:
a)多变量分析:基于训练集数据,建立偏最小二乘-判别分析(PLS-DA)模型,代谢物挑选条件:变量重要性投影(VIP)值大于1。
b)单变量分析:基于训练集数据,利用T检验根据p<0.05筛选出在食管鳞癌和食管上皮非典型增生组间有显著统计学差异的代谢物。
c)利用线性回归优选出次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸这一联合标志物组合。
(3)应用另一批次验证集中食管鳞癌和食管上皮非典型增生患者血清样本对候选代谢物进行验证。
本发明具有如下效果:
血清中的次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸构成的联合标志物及其衍生的联合标志物变量P,能够对食管鳞癌与食管上皮非典型增生进行很好的区分,本发明所涉及的检测试剂盒,对上述代谢物组合的检测,具有简便、快速、重复性好的优点,适用于食管鳞癌的诊断与疑似病症区分。所述试剂盒可实现高灵敏、高效检测本发明涉及的几种代谢物,具有检测成本低,重复性好的特点。本发明可应用于临床辅助诊断食管鳞癌,具有诊断灵敏度高,可将食管鳞癌与其癌前病变进行有效区分的特点,具有较好的应用前景。
附图说明
图1.次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸在训练集与验证集两组中的含量变化(中位数±四分位间距表示)。*表示两组间具有显著性差异,*:0.01<p<0.05,**:p<0.01。
图2.(A)实例1(训练集)中联合标志物用于判别食管鳞癌和食管上皮非典型增生患者的ROC曲线图。(B)实例2(验证集)中联合标志物用于判别食管鳞癌和食管上皮非典型增生的ROC曲线图。
图3.联合标志物用于训练集及验证集中食管鳞癌和食管上皮非典型增生的判别散点图。截点值为0.814。
具体实施方式
实施例1
1.血清样本收集
本发明中涉及到的所有志愿者在血清样本采集前均签署了知情同意书。
在相同采集条件下收集40例食管鳞癌及33例食管上皮非典型增生患者血清样本,设定为训练集。所有患者均经过组织病理学确证。采集得到的血样静置30分钟后以4000转离心10分钟,取上层血清并置于-80℃冰箱中保存。
2.分析方法
2.1血清样本预处理
从-80℃中取出的血清样本置于4℃条件下解冻,涡旋10秒后取50μL血清,加入200μL含有10μg/mL十三酸(又可称之为十三烷酸)的冷甲醇(0℃),涡旋1分钟后置于冰水浴中10分钟。接下来将样本进行高速离心(12000g,4℃)15分钟,并取200μL上清液置于新的样本管中。将所得样本进行冻干。冻干后的样本中加入50μL甲氧胺吡啶(20mg/mL)并涡旋1分钟,置于37℃空气浴中肟化反应90分钟,再加入40μL甲基三甲基甲硅烷基三氟乙酰胺在37℃空气浴硅烷化反应1小时,衍生溶液取出后以12000g、4℃离心15分钟,取上清置于玻璃进样瓶中待分析。
2.2气相色谱-质谱联用分析
(1)气相色谱分析条件:采用7890A系列气相色谱系统(Agilent,USA),色谱柱为安捷伦DB-5MS(30m×0.25mm×0.25μm),氦气为载气,进样体积为1μL,洗针液为二氯甲烷,线速为40cm/sec,分流比为10:1,柱温箱初始温度为80℃,保持1min,以30℃/min的程升速率升至210℃,再以20℃/min的梯度升至320℃保持4min。
(2)质谱分析条件:采用5977A系列四级杆质谱分析系统(Agilent,USA),溶剂延迟时间为2.8分钟,增益因子为4,质量范围为33至600Da,离子源温度为230℃,四级杆温度为150℃,离子源为电子轰击离子源,能量为70eV。
3.血清测试结果及诊断分析
利用MassHunter定性分析软件对气相色谱质谱联用数据进行解卷积并通过与NIST数据库比对得到样本定性表,进一步利用MassHunter定量分析软件对数据进行积分、峰匹配并得到了相对含量数据矩阵。根据质荷比为265.0±0.5Da,保留时间为6.88±0.1分钟的标准提取次黄嘌呤,根据质荷比为216.0±0.5Da,保留时间为4.13±0.1分钟的标准提取2-酮异己酸,根据质荷比为246.0±0.5Da,保留时间为6.04±0.1分钟的标准提取谷氨酸,根据质荷比为160.0±0.5Da,保留时间为5.20±0.1分钟的标准提取天冬氨酸的峰面积,并定量分析次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸在训练集中食管鳞癌及食管上皮非典型增生组的血清含量,结果如图1所示。与食管上皮非典型增生组相比,食管鳞癌患者血清中的次黄嘌呤、谷氨酸及天冬氨酸水平显著升高,2-酮异己酸水平显著降低。
使用数据统计软件SPSS,进一步将训练集样本血清中次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸的含量回归为联合标志物变量P。回归方程如下:
P=1/(1+e-(1.731-3.52*10-6*a+2.22*10-5*b-9.38*10-7*c-4.39*10-6*d))
其中,a为血清中次黄嘌呤的相对含量、b为血清中2-酮异己酸的相对含量、c为血清中谷氨酸的相对含量、d为血清中天冬氨酸的相对含量。所得变量P在食管鳞癌患者中降低,该变量值可用于辅助区分食管鳞癌和食管上皮非典型增生。
图2A中,该联合标志物通过判断变量P用于判定食管鳞癌及食管上皮非典型增生时,基于训练集数据得到ROC曲线的曲线下面积为0.826,具有较好的灵敏度与特异性,当灵敏度有特异性之和最大时,可获得当前的最佳截点值,即0.814。图3实例1图中,当使用该截点值时,联合标志物可有效用于食管腺癌与食管上皮非典型增生患者的判别。以上结果表明该联合标志物具有较好的判别食管腺癌及食管上皮非典型增生的潜能。
实施例2
1.血清样本收集
样品收集方法同实例1。实施例2包括了34例食管鳞癌及39例食管上皮非典型增生,这些样本设定为验证集。
2.分析方法
同实施例1。
3.血清验证结果及诊断潜力分析
实施例2验证结果与实施例1训练集数据基本吻合。具体结果如图1所示。利用线性回归将次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸组合使用,结果分别如图1、图2B、图3所示,其曲线下面积、灵敏度及特异度均较高,各组判别效果较好,具有较好的应用前景。
本发明还涉及了检测受试者中的食管鳞癌患者的试剂盒,通过检测来自受试者的血清样品中上述联合标志物各自的相对浓度,基于线性回归方程计算所述联合标志物变量P,再基于确定的截点值,判断所述受试者是否患有食管鳞癌,所述试剂盒可实现高灵敏、高效检测本发明涉及的几种小分子代谢物,且具有检测成本低、重复性好的特点。本发明可应用于食管鳞癌与其早期病变的辅助判别,具有较好的应用前景。
Claims (2)
1.一种联合型代谢标志物在制备用于区分食管鳞癌及食管上皮非典型增生疾病的试剂盒的用途,联合型代谢标志物是由次黄嘌呤、2-酮异己酸、谷氨酸及天冬氨酸共同构成的。
2.按照权利要求 1所述的用途,通过患者的血清样品,区分食管增生患者中食管鳞癌患者与食管上皮非典型增生患者。
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