CN114487201A - 鼻咽癌相关尿液标志物组合的检测试剂的应用 - Google Patents
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Abstract
本发明属于医学检验技术领域,具体涉及鼻咽癌相关尿液标志物组合的检测试剂的应用,该应用具体是在制备用于鼻咽癌诊断和/预后的产品中的应用,该标志物组合为Methyl(1‑(cyclohexylmethyl)‑1h‑indole‑3‑carbonyl)‑l‑valinate、Stachydrine、Decanoyl‑l‑carnitine和Octanoylcarnitine,CAS号依次为:1971007‑94‑9、471‑87‑4、1492‑27‑9和25243‑95‑2。通过定量检测受试者尿液中的该标志物,能很好预测鼻咽癌的发病,为鼻咽癌的早期诊断提供特异性的、迅速的、无创伤性的检测手段。
Description
技术领域
本发明属于医学检验技术领域,具体涉及鼻咽癌相关尿液标志物组合的检测试剂的应用。
背景技术
鼻咽癌的主要发病因素包括遗传易感性,饮食因素和EB病毒(Epstein-Barrvirus,EBV)感染,其发病机制的研究包括基因的突变、代谢组学、蛋白质组学、基因组学等方面。尽管如此,目前仍然难以全面理解鼻咽癌的发生、发展,这给其治疗带来了困难。
一般来说,鼻咽癌的诊断还是以鼻咽镜检查为主,但是价格贵,而且对人体有伤害;鼻咽癌的进一步确诊依然需要靠组织的病理诊断,但组织的活检具有侵袭性,患者比较痛苦。同时对于隐匿性鼻咽癌或者黏膜下型鼻咽癌有可能需要多次,如此反复的组织活检必然增加患者的经济负担和心理负担,也非常不利于大规模筛查。血浆EBV DNA检测在鼻咽癌早期诊断和筛查,复发和转移诊断,预后判断以及个体化治疗等方面具有较大的价值。据报道,鼻咽癌患者血浆中存在持续阳性的EBV DNA,其拷贝数与肿瘤的分期相关,有效治疗后迅速下降,复发和转移时再次升高。然而EB病毒检测敏感性和特异性均不高。因此,如何寻找到有效的、高效的、特异性、无创性的鼻咽癌肿瘤标志物(Bio-markers)对于提高患者的生存率至关重要。
发明内容
为了解决上述技术问题,本发明基于代谢组学技术,在尿液中筛选出能较好区分正常人对照和早期鼻咽癌的标志物组合,并提供了鼻咽癌相关尿液标志物组合的检测试剂在制备用于鼻咽癌诊断和/预后的产品中的应用,所述标志物组合为Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Stachydrine、Decanoyl-l-carnitine和Octanoylcarnitine,CAS号依次为:1971007-94-9、471-87-4、1492-27-9和25243-95-2。
进一步的,相对于正常健康人对照,鼻咽癌患者尿液中Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Stachydrine、Decanoyl-l-carnitine和Octanoylcarnitine的组合中,Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate和Stachydrine的量升高,而Decanoyl-l-carnitine和Octanoylcarnitine的量降低。
更进一步的,利用所述试剂检测所述标志物组合的方法,包括以下步骤:
1)获得受试者的尿液样本;
2)尿液样本采用超高效液相色谱系统HILIC色谱柱进行分离;
3)分离后的尿液样本,用质谱仪进行样本一级、二级谱图的采集,获得Wiff格式的原始数据,原始数据经ProteoWizard转换成.mzXML格式,然后采用XCMS软件进行峰对齐、保留时间校正和提取峰面积,得到受试者的尿液样本中Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Stachydrine、Decanoyl-l-carnitine和Octanoylcarnitine的定量结果。
更进一步的,步骤1)中,尿液样本在4℃环境下解冻后,取样本加入预冷的体积比为2:2:1的甲醇/乙腈/水溶液,涡旋混合,低温超声30min,-20℃静置10min,14000g,4℃离心20min,取上清真空干燥,质谱分析时加入100μL乙腈水溶液,乙腈和水的体积比为1:1,复溶,涡旋,14000g,4℃离心15min,待用。
更进一步的,步骤2)中,采用Agilent1290InfinityLC超高效液相色谱系统HILIC色谱柱进行分离;柱温25℃;流速0.5mL/min;进样量2μL;流动相组成A:水+25mM乙酸铵+25mM氨水,B:乙腈;梯度洗脱程序如下:0-0.5min,95%B;0.5-7min,B从95%线性变化至65%;7-8min,B从65%线性变化至40%;8-9min,B维持在40%;9-9.1min,B从40%线性变化至95%;9.1-12min,B维持在95%;整个分析过程中样品置于4℃自动进样器中。
更进一步的,步骤3)中,采用ABTripleTOF6600质谱仪进行样本一级、二级谱图的采集,得到原始Wiff格式的原始数据;样品经用Agilent1290InfinityLC超高效液相色谱系统分离后,用TripleTOF6600质谱仪进行质谱分析,分别采用电喷雾电离正离子和负离子模式进行检测,ESI源设置参数如下:雾化气辅助加热气1:60,辅助加热气2:60,气帘气:30psi,离子源温度:600℃,喷雾电压±5500V;一级质荷比检测范围:60-1000Da,二级子离子质荷比检测范围:25-1000Da,一级质谱扫描累积时间:0.20s/spectra,二级质谱扫描累积时间0.05s/spectra;二级质谱采用数据依赖型采集模式获得,并且采用峰强度值筛选模式,去簇电压:±60V,碰撞能量:35±15eV,IDA设置如下:动态排除同位素离子范围:4Da,每次扫描采集10个碎片图谱
本发明的有益效果如下:
本发明研究首次发现,相对正常健康人对照,鼻咽癌患者的尿液中Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Stachydrine、Decanoyl-l-carnitine和Octanoylcarnitine的组合的量发生显著变化,可作为尿液标志物预测鼻咽癌的发病,ROC分析发现该组合AUC值为0.98,准确度为92%,特异性为84%,该结果为鼻咽癌的早期诊断提供特异性的、迅速的、无创伤性的检测手段。
附图说明
图1为尿液样本PCA分析。
图2为基于随机森林(random forests)算法在训练组中5种显著差异的尿液代谢物ROC分析结果。
图3为基于随机森林(random forests)算法在测试组中尿液标志物组合(Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Stachydrine、Decanoyl-l-carnitine、Octanoylcarnitine)的ROC分析结果。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明,但不应理解为本发明的限制。如未特殊说明,下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段,下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
基于超高效液相色谱-串联飞行时间质谱联用(UHPLC-Q-TOF MS)的代谢组学技术,我们研究了27例鼻咽癌患者和27例正常健康人群(表1)的血清及尿液样本中的代谢图谱特征。通过数据库匹配、数据标准化、计量学以及统计学分析等发现,与正常健康人群相比,鼻咽癌患者血清中181种代谢物发生显著性变化(p<0.05),同时尿液中179种代谢组发生显著性变化(p<0.05)。接着取其交集,结果显示仅有7种代谢物在鼻咽癌患者血清和尿液中同时发生变化(表2)。按照VIP>1&FC>1.5&P<0.05的标准,筛选出6个差异代谢物,其中Dibucaine在鼻咽癌血清和尿液中上下调趋势相反,而剩余的5种代谢物(Decanoyl-l-carnitine、Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Octanoylcarnitine、Stachydrine和Paraxanthine)的变化趋势是一致的。最后利用随机森林(random forests)算法精准筛选出尿液标志物组合(Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Stachydrine、Decanoyl-l-carnitine、Octanoylcarnitine),中文名依次为(1-(环己基甲基)-1h-吲哚-3-羰基)-1-缬氨酸甲酯、水苏碱、癸酰左旋肉碱和辛酰肉碱,CAS号依次为:1971007-94-9、471-87-4、1492-27-9和25243-95-2。ROC分析发现尿液标志物组(AUC=0.98)准确度为92%左右。这些结果说明尿液标志物组能很好预测鼻咽癌的发病,可作为潜在的生物标志物,为鼻咽癌的早期诊断提供特异性的、迅速的、无创伤性的检测手段。
表1 27例鼻咽癌患者与27例正常健康人群基本信息
以下是对本发明的进一步描述:
一、尿液代谢组学分析
1)尿液样本在4℃环境下缓慢解冻后,取适量样本加入预冷甲醇/乙腈/水溶液(2:2:1,v/v),涡旋混合,低温超声30min,-20℃静置10min,14000g 4℃离心20min,取上清真空干燥,质谱分析时加入100μL乙腈水溶液(乙腈:水=1:1,v/v)复溶,涡旋,14000g 4℃离心15min,取上清液进样分析。
2)色谱条件:样品采用Agilent 1290Infinity LC超高效液相色谱系统(UHPLC)HILIC色谱柱进行分离;柱温25℃;流速0.5mL/min;进样量2μL;流动相组成A:水+25mM乙酸铵+25mM氨水,B:乙腈;梯度洗脱程序如下:0-0.5min,95%B;0.5-7min,B从95%线性变化至65%;7-8min,B从65%线性变化至40%;8-9min,B维持在40%;9-9.1min,B从40%线性变化至95%;9.1-12min,B维持在95%;整个分析过程中样品置于4℃自动进样器中。为避免仪器检测信号波动而造成的影响,采用随机顺序进行样本的连续分析。样本队列中插入QC样品,用于监测和评价系统的稳定性及实验数据的可靠性。
3)Q-TOF质谱条件:采用AB Triple TOF 6600质谱仪进行样本一级、二级谱图的采集。样品经用Agilent 1290Infinity LC超高效液相色谱系统(UHPLC)分离后,用TripleTOF 6600质谱仪(AB SCIEX)进行质谱分析,分别采用电喷雾电离(ESI)正离子和负离子模式进行检测。ESI源设置参数如下:雾化气辅助加热气1(Gas1):60,辅助加热气2(Gas2):60,气帘气(CUR):30psi,离子源温度:600℃,喷雾电压(ISVF)±5500V(正负两种模式);一级质荷比检测范围:60-1000Da,二级子离子质荷比检测范围:25-1000Da,一级质谱扫描累积时间:0.20s/spectra,二级质谱扫描累积时间0.05s/spectra;二级质谱采用数据依赖型采集模式(IDA)获得,并且采用峰强度值筛选模式,去簇电压(DP):±60V(正负两种模式),碰撞能量:35±15eV,IDA设置如下:动态排除同位素离子范围:4Da,每次扫描采集10个碎片图谱。
4)数据分析流程:Wiff格式的原始数据经ProteoWizard转换成.mzXML格式,然后采用XCMS软件进行峰对齐、保留时间校正和提取峰面积。对XCMS提取得到的数据首先进行代谢物结构鉴定、数据预处理,然后进行实验数据质量评价,最后再进行数据分析。数据分析内容包括单变量统计分析、多维统计分析、差异代谢物筛选、差异代谢物相关性分析、KEGG通路分析等内容。
结果:在所有54例尿液样本中,正负离子模式合并后鉴定2509种代谢物。通过主成分分析(Principal Component Analysis,PCA)分析发现正离子模式和负离子模式下27例鼻咽癌患者尿液样本与27例正常健康人群尿液样本混在一起(图1)。进一步通过正交偏最小二乘判别分析(OPLS-DA)发现该模型能区分以上两组样本,以严格的VIP>1和p value<0.05为筛选标准,发现鼻咽癌患者尿液中有179种代谢物发生显著性变化。KEGG通路富集分析结果表明这些差异代谢物显著地富集(p value<0.05)在8条通路上,具体包括Caffeinemetabolism、Citrate cycle(TCA cycle)、ABC transporters、Glucagon signalingpathway、Nicotinate and nicotinamide metabolism、Central carbon metabolism incancer、Butanoate metabolism和Alanine,aspartate and glutamate metabolism。
二、血清代谢组学分析
血清样本中代谢物的提取、色谱条件、Q-TOF质谱条件、数据分析流程与尿液的相同。
在所有54例血清样本中,正负离子模式合并后鉴定1230种代谢物。通过主成分分析(Principal Component Analysis,PCA)分析发现正离子模式和负离子模式下27例鼻咽癌患者血清样本与27例正常健康人群血清样本有个明显的分开。进一步通过正交偏最小二乘判别分析(OPLS-DA)发现该模型也能区分以上两组样本,以严格的VIP>1和p value<0.05为筛选标准,发现鼻咽癌患者血清中有181种代谢物发生显著性变化。KEGG通路富集分析结果表明这些差异代谢物显著地富集(p value<0.05)在16条通路上,具体包括Aminoacyl-tRNA biosynthesis、Biosynthesis of unsaturated fatty acids、Fatty acidbiosynthesis、Aldosterone synthesis and secretion、Glutamatergic synapse、Arginine biosynthesis、Ovarian steroidogenesis、Long-term depression、GABAergicsynapse、Cholesterol metabolism、Basal cell carcinoma、Biosynthesis of aminoacids、Choline metabolism in cancer、Ferroptosis、D-Glutamine and D-glutamatemetabolism和Central carbon metabolism in cancer。
三、预测模型的建立和评估
结合血清和尿液代谢组学的结果发现仅有7种代谢物在鼻咽癌患者血清和尿液中同时发生变化(表2)。另外一方面Central carbon metabolism in cancer通路也是在鼻咽癌患者血清和尿液中同时显著性富集。按照Foldchange>1.5或Foldchange<0.67的筛选标准,最终表明5种代谢物在鼻咽癌患者血清和尿液中的变化趋势是一致的,具体包括:Decanoyl-l-carnitine、Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Octanoylcarnitine、Stachydrine和Paraxanthine。接着利用随机森林(randomforests)算法发掘生物标志物,将原始所有血清样本随机分为训练组和测试组,其中训练组用于建立预测模型,验证组用于测试模型的准确性。结果表明以上5种差异的血清代谢物在训练组中的重要性从高至低依次为Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Stachydrine、Decanoyl-l-carnitine、Octanoylcarnitine和Paraxanthine,前四种代谢物组合的AUC达到最高值(图2):0.9208;在测试组中尿液标志物组合(Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Stachydrine、Decanoyl-l-carnitine、Octanoylcarnitine)ROC分析发现AUC值为0.98,准确度为92%,特异性为84%(图3)。
表2在鼻咽癌患者血清和尿液中同时发生显著性变化的7种代谢物
Foldchange*代表鼻咽癌患者组与正常健康人群组的比值。
以上结果表明:Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Stachydrine、Decanoyl-l-carnitine和Octanoylcarnitine的组合可作为早期鼻咽癌的诊断标志物,为鼻咽癌的早期诊断提供特异性的、迅速的、无创伤性的检测手段。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (6)
1.鼻咽癌相关尿液标志物组合的检测试剂在制备用于鼻咽癌诊断和/预后的产品中的应用,其特征在于,所述标志物组合为Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Stachydrine、Decanoyl-l-carnitine和Octanoylcarnitine,CAS号依次为1971007-94-9、471-87-4、1492-27-9和25243-95-2。
2.根据权利要求1所述的应用,其特征在于,相对于正常健康人对照,鼻咽癌患者的尿液中的Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate和Stachydrine的量升高,而Decanoyl-l-carnitine和Octanoylcarnitine的量降低。
3.根据权利要求2所述的应用,其特征在于,利用所述试剂检测所述标志物组合的方法,包括以下步骤:
1)获得受试者的尿液样本;
2)尿液样本采用超高效液相色谱系统HILIC色谱柱进行分离;
3)分离后的尿液样本,用质谱仪进行样本一级、二级谱图的采集,获得Wiff格式的原始数据,原始数据经ProteoWizard转换成.mzXML格式,然后采用XCMS软件进行峰对齐、保留时间校正和提取峰面积,得到受试者的尿液样本中Methyl(1-(cyclohexylmethyl)-1h-indole-3-carbonyl)-l-valinate、Stachyd rine、Decanoyl-l-carnitine和Octanoylcarnitine的定量结果。
4.根据权利要求3所述的应用,其特征在于,步骤1)中,尿液样本在4℃环境下解冻后,取样本加入预冷的体积比为2:2:1的甲醇/乙腈/水溶液,涡旋混合,低温超声30min,-20℃静置10min,14000g,4℃离心20min,取上清真空干燥,质谱分析时加入100μL乙腈水溶液,乙腈和水的体积比为1:1,复溶,涡旋,14000g,4℃离心15min,待用。
5.根据权利要求4所述的应用,其特征在于,步骤2)中,采用Agilent1290InfinityLC超高效液相色谱系统HILIC色谱柱进行分离;柱温25℃;流速0.5mL/min;进样量2μL;流动相组成A:水+25mM乙酸铵+25mM氨水,B:乙腈;梯度洗脱程序如下:0-0.5min,95%B;0.5-7min,B从95%线性变化至65%;7-8min,B从65%线性变化至40%;8-9min,B维持在40%;9-9.1min,B从40%线性变化至95%;9.1-12min,B维持在95%;整个分析过程中样品置于4℃自动进样器中。
6.根据权利要求5所述的应用,其特征在于,步骤3)中,采用ABTripl eTOF6600质谱仪进行样本一级、二级谱图的采集,得到原始Wiff格式的原始数据;样品经用Agilent1290InfinityLC超高效液相色谱系统分离后,用TripleT OF6600质谱仪进行质谱分析,分别采用电喷雾电离正离子和负离子模式进行检测,ESI源设置参数如下:雾化气辅助加热气1:60,辅助加热气2:60,气帘气:30psi,离子源温度:600℃,喷雾电压±5500V;一级质荷比检测范围:60-1000Da,二级子离子质荷比检测范围:25-1000Da,一级质谱扫描累积时间:0.20s/spectra,二级质谱扫描累积时间0.05s/spectra;二级质谱采用数据依赖型采集模式获得,并且采用峰强度值筛选模式,去簇电压:±60V,碰撞能量:35±15eV,IDA设置如下:动态排除同位素离子范围:4Da,每次扫描采集10个碎片图谱。
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