CN1673734A - New method for detecting esophageal cancer haemocyanin fingerprint - Google Patents

Abstract

Translated fromChinese

本发明涉及一种检测食管癌血清蛋白指纹的方法。该蛋白指纹由采集人血清、血清与弱阳离子亲和蛋白芯片结合、用质谱仪读取血清蛋白图谱、以及用蛋白质指纹分析软件对质谱数据的分析而获得。食管癌血清蛋白指纹由12个不同质荷比(M/Z)的蛋白质组成。本发明提供的食管癌血清蛋白指纹能够对食管癌进行早期检测，其敏感性和特异性均达到80％以上，为食管癌的早期发现、早期治疗和对高危人群的预防提供了新的方法。The invention relates to a method for detecting serum protein fingerprints of esophageal cancer. The protein fingerprint is obtained by collecting human serum, combining the serum with a weak cationic affinity protein chip, reading the serum protein map with a mass spectrometer, and analyzing the mass spectrum data with protein fingerprint analysis software. The serum protein fingerprint of esophageal cancer consists of 12 proteins with different mass-to-charge ratios (M/Z). The serum protein fingerprint of esophageal cancer provided by the invention can detect esophageal cancer early, and its sensitivity and specificity reach more than 80%, which provides a new method for early detection, early treatment and prevention of high-risk groups of esophageal cancer.

Description

Translated fromChinese

一种检测食管癌血清蛋白指纹的新方法A new method for the detection of serum protein fingerprints in esophageal cancer

技术领域technical field

本发明涉及一种检测食管癌血清蛋白指纹的方法，使用该方法可确定食管癌血清蛋白质指纹标准的灵敏度、特异性指标，对食管癌的早期发现、早期诊断和早期治疗提供有益的帮助。The invention relates to a method for detecting the serum protein fingerprint of esophageal cancer. The sensitivity and specificity index of the serum protein fingerprint standard of esophageal cancer can be determined by using the method, which provides beneficial help for early discovery, early diagnosis and early treatment of esophageal cancer.

背景技术Background technique

随着全球性基因组计划尤其是人类基因组计划规模空前、速度惊人的推进，科学家现在已经把关注的焦点转移到了人类蛋白质组(proteome)——人体组织和细胞制造的各种各样的蛋白质。虽然人类基因组包含了我们身体的全部遗传信息，但那也只是制造蛋白质的原材料，而构成细胞并发挥各种功能则是由蛋白质来完成的。蛋白质也是机体各种不同类型细胞之间差异的决定因素，尽管所有的细胞都拥有同样的基因组，但在不同的细胞内会有不同的基因处在活跃状态，从而合成不同的蛋白质。同样，病变细胞和正常细胞的差异在很大程度上也是由功能基因及其合成蛋白质的不同来决定的。With the unprecedented scale and speed of global genome projects, especially the Human Genome Project, scientists have now turned their attention to the human proteome—the wide variety of proteins made by human tissues and cells. Although the human genome contains all the genetic information of our body, it is only the raw material for making proteins, and it is proteins that make up cells and perform various functions. Protein is also the determinant of the differences between different types of cells in the body. Although all cells have the same genome, different genes are active in different cells, thus synthesizing different proteins. Similarly, the difference between diseased cells and normal cells is largely determined by the difference in functional genes and their synthetic proteins.

在各大公司和学术界的科学家正试图列出所有的蛋白质种类，并揭示他们之间相互作用的关系。实现这个目的并非易事，研究蛋白质要比研究基因困难得多，生物技术公司都在全力开发最好的技术和实验仪器，这方面的竞争已经全面展开。同时，各国政府的科研计划也在为学术界提供经费，支持对癌细胞、血清和其它液体成份蛋白质组的研究。其最终目的是揭示包括癌症在内的人类各种疾病的特征性蛋白及其组成，即具有疾病特征的“蛋白质指纹”(biomarkerpatterns)，进而发明疗效更高、副作用更小的新药。Scientists in major companies and academia are trying to list all the protein species and reveal the relationship between them. Achieving this goal is no easy task. It is much more difficult to study proteins than to study genes. Biotechnology companies are all striving to develop the best technology and experimental equipment. The competition in this regard is already in full swing. At the same time, government research programs are providing funding to academia to support research on the proteome of cancer cells, serum and other fluid components. Its ultimate goal is to reveal the characteristic proteins and their composition of various human diseases including cancer, that is, "protein fingerprints" (biomarker patterns) with disease characteristics, and then invent new drugs with higher efficacy and fewer side effects.

近年来，美国Ciphergen Biosystems公司创建了一种蛋白质芯片(ProteinChip)技术，它不仅克服了某些传统蛋白质组分析技术的局限性，集分离与质谱仪于一身，从小量血液、尿液的体液样本和组织、细胞裂解物等粗提样品中，不经纯化高效、快速和准确地直接分析差异蛋白质。伴随着这一技术的出现，形成了一种以分析疾病相关的特殊蛋白质指纹为基础的蛋白组组成模式(pattern)的疾病辅助诊断和预后疗效判断的新思路。In recent years, Ciphergen Biosystems of the United States has created a protein chip (ProteinChip) technology, which not only overcomes the limitations of some traditional proteome analysis techniques, but also integrates separation and mass spectrometers, and can be used from a small amount of blood and urine body fluid samples. In crude samples such as tissues and cell lysates, differential proteins can be directly analyzed efficiently, quickly and accurately without purification. With the emergence of this technology, a new idea of disease-aided diagnosis and prognostic efficacy judgment has been formed based on the analysis of special protein fingerprints related to diseases based on proteome composition patterns.

食管癌是危害我国人民健康的常见肿瘤，世界上70％以上的食管癌发生在我国，1995年部分地区统计资料显示食管癌致死占恶性肿瘤死因的第四位。大部分食管癌患者确诊时已属中晚期，以手术为主治疗后的五年生存率一直徘徊在25-30％。但是，早期食管癌手术治疗后，五年生存率可达80％以上。因此，早诊早治是目前改善食管癌患者长期生存的最佳途径。显然，如果能够找到一些在影像学方法检查出食管占位性病变之前就能发现早期食管癌的血清蛋白标志物，对于食管癌的治疗、延长患者术后生存期具有重要意义。Esophageal cancer is a common tumor that endangers the health of our people. More than 70% of esophageal cancers in the world occur in my country. Statistics in some regions in 1995 show that esophageal cancer is the fourth cause of death from malignant tumors. Most patients with esophageal cancer are already in the advanced stage when they are diagnosed, and the five-year survival rate after surgery-based treatment has been hovering at 25-30%. However, after surgical treatment of early esophageal cancer, the five-year survival rate can reach more than 80%. Therefore, early diagnosis and treatment is currently the best way to improve the long-term survival of patients with esophageal cancer. Obviously, if we can find some serum protein markers that can detect early esophageal cancer before imaging methods detect esophageal space-occupying lesions, it will be of great significance for the treatment of esophageal cancer and prolonging the postoperative survival of patients.

肿瘤的发生发展是正常细胞经过多基因、多步骤的变化，逐渐演变成具有恶性增殖特性的癌细胞并不断转移播散的过程。肿瘤细胞与增殖、凋亡、分化和转移相关的多种基因的表达都发生了改变，变化最终表现为细胞内各种蛋白质的表达和功能改变，从而影响细胞的生命活动。在癌变过程中发生改变的蛋白质和小肽可以经肿瘤细胞代谢、分泌释放到血液或其它体液中。因此，通过研究食管癌血清蛋白质组的改变，有可能发现与食管癌相关或特异的蛋白质或肽类物质，即食管癌血清蛋白标志物。目前可用于食管癌辅助诊断、疗效评价和术后随访的血清标志物主要包括：癌胚抗原(CEA)、鳞状上皮细胞癌相关抗原(SCC)、细胞角化素蛋白片段19(CYFRA21-1)、p53蛋白抗体(p53-Ab)等，虽具有一定的临床应用价值，但敏感性低、特异性差。因此，寻找早期诊断、无创性、高敏感度、高特异性的新的食管癌相关的特异血清标志“蛋白指纹”具有重要临床意义。The occurrence and development of tumors is a process in which normal cells undergo multi-gene and multi-step changes, gradually evolve into cancer cells with malignant proliferation characteristics, and continue to metastasize and spread. The expressions of various genes related to proliferation, apoptosis, differentiation and metastasis of tumor cells are changed, and the changes are finally manifested as changes in the expression and function of various proteins in cells, thereby affecting the life activities of cells. Proteins and small peptides that change during carcinogenesis can be metabolized, secreted and released into blood or other body fluids by tumor cells. Therefore, by studying the changes in the serum proteome of esophageal cancer, it is possible to find proteins or peptides related or specific to esophageal cancer, that is, serum protein markers of esophageal cancer. Serum markers currently available for auxiliary diagnosis, curative effect evaluation and postoperative follow-up of esophageal cancer mainly include: carcinoembryonic antigen (CEA), squamous cell carcinoma-related antigen (SCC), cytokeratin protein fragment 19 (CYFRA21-1 ), p53 protein antibody (p53-Ab), etc., although they have certain clinical application value, they have low sensitivity and poor specificity. Therefore, it is of great clinical significance to find a new specific serum marker "protein fingerprint" related to early diagnosis, non-invasive, high sensitivity and high specificity of esophageal cancer.

发明内容Contents of the invention

本发明的目的是提出一种检测食管癌血清蛋白指纹的方法，使用该方法从血清标本中寻找一组(若干个)差异表达的食管癌相关的特异蛋白质/小分子肽，并利用Biomarker Patterns Software软件分析建立最佳的树状分类模型，达到区分食管癌患者与非肿瘤健康人、区分食管癌与食管良性病变、区分食管癌与人体其它消化道肿瘤、以及早期发现食管癌的目标。The purpose of the present invention is to propose a method for detecting serum protein fingerprints of esophageal cancer, using the method to find a group (several) of differentially expressed esophageal cancer-related specific proteins/small molecular peptides from serum samples, and using Biomarker Patterns Software Software analysis establishes the best tree classification model to achieve the goals of distinguishing esophageal cancer patients from non-tumor healthy people, esophageal cancer from benign esophageal lesions, esophageal cancer from other digestive tract tumors, and early detection of esophageal cancer.

本发明的目的是这样实现的：一种检测食管癌血清蛋白指纹的新方法，其步骤是：The object of the present invention is achieved like this: a kind of novel method of detecting esophagus cancer serum albumin fingerprint, its step is:

采集食管癌组血清标本、对照组血清标本、早期食管癌组血清标本、食管良性疾病组血清标本及其它恶性肿瘤组血清标本；Collect serum samples from the esophageal cancer group, the control group, the early esophageal cancer group, the benign esophageal disease group and other malignant tumor groups;

所述血清标本在离体后2-3小时内于4℃温度下进行离心，分离血清后分装保存备用，保存温度为-80℃；The serum sample is centrifuged at 4°C within 2-3 hours after being isolated, and the serum is separated and stored in separate packages at a temperature of -80°C;

将所述血清标本置于弱阳离子亲和吸附类的蛋白质芯片上进行特异性结合与洗脱处理；placing the serum sample on a weak cationic affinity adsorption protein chip for specific binding and elution;

将所述经过处理的芯片置入蛋白质芯片阅读器(串联质谱仪)分析，获得血清标本蛋白质组图谱；Putting the processed chip into a protein chip reader (tandem mass spectrometer) for analysis to obtain a serum sample proteome map;

使用蛋白质芯片数据采集软件采集数据，对比分析食管癌组和对照组血清标本的蛋白质组图谱，结合使用蛋白质指纹树状模型分析软件获得一组食管癌组与对照组的差异蛋白质图谱即蛋白质指纹，并以该蛋白质指纹为判断标准进行盲筛试验；Using protein chip data collection software to collect data, comparative analysis of the proteome profiles of serum samples of the esophageal cancer group and the control group, combined with the use of protein fingerprint tree model analysis software to obtain a set of differential protein profiles between the esophageal cancer group and the control group, namely protein fingerprints , and use the protein fingerprint as the judgment standard to carry out blind screening test;

采用盲法对其余血清标本进行筛选试验，确定食管癌血清蛋白质指纹标准的灵敏度、特异性指标。The remaining serum samples were screened by blind method to determine the sensitivity and specificity indexes of the serum protein fingerprint standard for esophageal cancer.

本发明提出了一种灵敏度高、特异性强、无创性、快速、小标本量和高通量的检测方法，使用本发明的食管癌血清蛋白指纹能够对食管癌进行早期检测，其敏感性和特异性均达到80％以上，为食管癌的早期发现、早期治疗和对高危人群的预防提供了新的方法，使食管癌的早期发现、早期诊断和早期治疗成为可能。The present invention proposes a detection method with high sensitivity, strong specificity, non-invasiveness, rapidity, small sample size and high throughput. The serum protein fingerprint of esophageal cancer of the present invention can be used for early detection of esophageal cancer, and its sensitivity and The specificity reached more than 80%, which provided a new method for the early detection, early treatment and prevention of high-risk groups of esophageal cancer, and made the early detection, early diagnosis and early treatment of esophageal cancer possible.

附图说明Description of drawings

以下结合附图对本发明的实施例及积极效果作进一步说明。Embodiments and positive effects of the present invention will be further described below in conjunction with the accompanying drawings.

图1是蛋白质3975.4的胶带形图Figure 1 is a tape diagram of protein 3975.4

图2是蛋白质3975.4的峰形图Figure 2 is the peak shape diagram of protein 3975.4

图3是蛋白质2047.8的峰形图Figure 3 is the peak shape diagram of protein 2047.8

图4是线性组合分析模型ESCC-WCX2-1示意图Figure 4 is a schematic diagram of the linear combination analysis model ESCC-WCX2-1

具体实施方式Detailed ways

实施例一：Embodiment one:

一种检测食管癌血清蛋白指纹的方法，其步骤如下：A method for detecting serum protein fingerprints of esophageal cancer, the steps are as follows:

采集食管癌组血清标本、对照组血清标本、早期食管癌组血清标本、食管良性疾病组血清标本及其它恶性肿瘤组血清标本；Collect serum samples from the esophageal cancer group, the control group, the early esophageal cancer group, the benign esophageal disease group and other malignant tumor groups;

其中，in,

食管癌组为临床病理确诊为食管癌的单纯手术治疗病例，共199例；The esophageal cancer group is a total of 199 cases of esophageal cancer diagnosed by clinical pathology and surgical treatment alone;

对照组为年龄、性别匹配的非肿瘤健康志愿者，共106例；The control group consisted of age- and gender-matched non-tumor healthy volunteers, a total of 106 cases;

早期食管癌组为食管早期癌微小病变(原位癌)病例，共14例；The early esophageal cancer group included 14 cases of early esophageal minimal lesion (carcinoma in situ);

食管良性疾病组为食管炎、返流性食管炎、食管平滑肌瘤等病例，共10例；其它恶性肿瘤组为消化系统其它恶性肿瘤病例，共30例。Benign esophageal disease group included esophagitis, reflux esophagitis, and esophageal leiomyoma, a total of 10 cases; other malignant tumor group included other malignant tumors of the digestive system, a total of 30 cases.

在本实施例中，被采集者清晨空腹静脉采集全血3ml(毫升)，在离体后2-3小时内于4℃温度下进行离心，分离血清后分装保存备用，保存温度为-80℃。In this example, 3ml (milliliters) of whole blood was collected from the subject on an empty stomach in the morning, centrifuged at a temperature of 4°C within 2-3 hours after being separated from the body, and the serum was separated and stored for later use. The storage temperature was -80°C. ℃.

将所述血清标本置于弱阳离子亲和吸附类的蛋白质芯片上进行特异性结合与洗脱处理；经处理后，该芯片成为结合了特定类型蛋白质的芯片。The serum sample is placed on a weak cationic affinity adsorption protein chip for specific binding and elution treatment; after processing, the chip becomes a chip bound to a specific type of protein.

将所述经过处理的芯片置入蛋白质芯片阅读器(串联质谱仪)分析，获得血清标本蛋白质组图谱。Put the processed chip into a protein chip reader (tandem mass spectrometer) for analysis to obtain the proteome map of the serum sample.

使用蛋白质芯片数据采集软件采集数据，对比分析食管癌组和对照组血清标本的蛋白质组图谱，结合使用蛋白质指纹树状模型分析软件获得一组食管癌组与对照组的差异蛋白质图谱即蛋白质指纹，并以该蛋白质指纹为判断标准进行盲筛试验。Using protein chip data acquisition software to collect data, comparative analysis of the proteome profiles of the serum samples of the esophageal cancer group and the control group, combined with the use of protein fingerprint tree model analysis software to obtain a set of differential protein profiles between the esophageal cancer group and the control group, namely protein fingerprints , and use the protein fingerprint as the criterion to carry out the blind screening test.

采用盲法对其余血清标本进行筛选试验，确定食管癌血清蛋白质指纹标准的灵敏度、特异性指标。The remaining serum samples were screened by blind method to determine the sensitivity and specificity indexes of the serum protein fingerprint standard for esophageal cancer.

实施例二：Embodiment two:

一种检测食管癌血清蛋白指纹的新方法，其步骤如下：A new method for detecting the serum protein fingerprint of esophageal cancer, the steps are as follows:

采集食管癌组血清标本、对照组血清标本、早期食管癌组血清标本、食管良性疾病组血清标本及其它恶性肿瘤组血清标本。Serum samples of esophageal cancer group, control group, early esophageal cancer group, benign esophageal disease group and other malignant tumors were collected.

其中，in,

食管癌组为临床病理确诊为食管癌的单纯手术治疗病例，共199例；The esophageal cancer group is a total of 199 cases of esophageal cancer diagnosed by clinical pathology and surgical treatment alone;

对照组为年龄、性别匹配的非肿瘤健康志愿者，共106例；The control group consisted of age- and gender-matched non-tumor healthy volunteers, a total of 106 cases;

早期食管癌组为食管早期癌微小病变病例，共14例；The early esophageal cancer group included 14 early esophageal cancer minimal lesion cases;

食管良性疾病组为食管炎、返流性食管炎、食管平滑肌瘤等病例，共10例；其它恶性肿瘤组为消化系统其它恶性肿瘤病例，共30例。Benign esophageal disease group included esophagitis, reflux esophagitis, and esophageal leiomyoma, a total of 10 cases; other malignant tumor group included other malignant tumors of the digestive system, a total of 30 cases.

在本实施例中，被采集者清晨空腹静脉采集全血3ml，在离体后2-3小时内于4℃温度下进行离心，分离血清后分装保存备用，保存温度为-80℃。In this example, 3ml of whole blood was collected intravenously from the subject on an empty stomach in the morning, and centrifuged at 4°C within 2-3 hours after being separated from the body. The serum was separated and stored in separate packages at -80°C.

将所述血清标本置于弱阳离子亲和吸附类的蛋白质芯片上进行特异性结合与洗脱处理；所用蛋白质芯片为美国赛弗吉公司(Ciphergen Biosystems，Inc.出品。The serum sample was placed on a weak cationic affinity adsorption protein chip for specific binding and elution; the protein chip used was produced by Ciphergen Biosystems, Inc., USA.

在本实施例中，所述芯片装入特定的加样器(Bioprocessor)中，每个加样点上加200μl(微升)结合缓冲液(50mM醋酸钠，pH 4.0，Sigma公司产品)，摇床孵育5分钟后弃去结合缓冲液。重复此过程一次。弃去孔中的液体，备用。In this embodiment, the chip is loaded into a specific sampler (Bioprocessor), and 200 μl (microliter) of binding buffer (50 mM sodium acetate, pH 4.0, product of Sigma Company) is added to each sample point, shake Binding buffer was discarded after 5 min of bed incubation. Repeat this process once. Discard the liquid in the wells and set aside.

所述血清标本需经稀释处理，该稀释处理的步骤是：血清标本先经蛋白浓度定量，(Micro BCA Protein Assay Kit，PIERCE公司产品)。The serum sample needs to be diluted, and the dilution process is as follows: the serum sample is first quantified by protein concentration (Micro BCA Protein Assay Kit, product of PIERCE company).

将3μl血清和6μl样本稀释液U9(9M Urea、2％CHAPS、50mM Tris-HCl、pH9.0和1％DTT)混合，置于冰上30分钟，每隔5分钟轻微混匀一次。在上述稀释血清中加入108μl结合缓冲液，混匀后置于冰上。Mix 3 μl serum and 6 μl sample diluent U9 (9M Urea, 2% CHAPS, 50mM Tris-HCl, pH9.0 and 1% DTT), place on ice for 30 minutes, and mix gently every 5 minutes. Add 108 μl of binding buffer to the above diluted serum, mix well and place on ice.

所述特异性结合与洗脱处理的步骤是：The steps of the specific binding and elution treatment are:

在所述蛋白质芯片的每个加样孔中加入100μl稀释的血清，盖上锡纸，室温下在摇床上孵育1小时。Add 100 μl of diluted serum to each sample well of the protein chip, cover with tin foil, and incubate on a shaker at room temperature for 1 hour.

弃去血清样品，每个加样孔中加入200μl结合缓冲液，摇床上洗5分钟；弃去结合缓冲液，重复此步骤两次；弃去结合缓冲液，每个加样孔中加入200μl HPLC级纯净水(Millipore公司产品)，轻摇后立即弃去水，从加样器上卸下所述芯片，该芯片在室温下自然干燥。Discard the serum sample, add 200 μl binding buffer to each well, and wash on a shaker for 5 minutes; discard the binding buffer, repeat this step twice; discard the binding buffer, add 200 μl HPLC to each well Grade pure water (Millipore company product), discard the water immediately after gently shaking, unload the chip from the sampler, and let the chip dry naturally at room temperature.

加能量吸收分子(Energy Absorbing Molecules，EAM)：Add Energy Absorbing Molecules (EAM):

取出所述芯片待微干后，在每个加样孔上加0.5μl饱和SPA(50％乙腈(V/V)、0.5％三氟乙酸(V/V)，Sigma公司产品)，SPA(Sinapinic acid，EAM的一种，Ciphergen公司产品)。After taking out the chip and waiting for micro-drying, add 0.5 μ l saturated SPA (50% acetonitrile (V/V), 0.5% trifluoroacetic acid (V/V), Sigma company product), SPA (Sinapinic acid, a kind of EAM, product of Ciphergen Company).

将芯片放入芯片阅读器(PBS II-C)，可以检测到结合到芯片上的所有蛋白质，通过蛋白质芯片数据采集软件(ProteinChip@Software)读取数据，得到每一个加样点上的所有的蛋白质图谱。仪器的主要参数设置如下：检测分子量范围0-50,000道尔顿；激光强度175；检测敏感度8；检测点24-84区(即，对芯片每点上结合蛋白恒定的激光轰击范围，两相邻激光激发位置间隔5个区，覆盖每点中央60％的最佳蛋白结合区域)。Put the chip into the chip reader (PBS II-C), and all the proteins bound to the chip can be detected, and the data can be read through the protein chip data acquisition software (ProteinChip@Software), and all the proteins on each sample loading point can be obtained. Protein Atlas. The main parameters of the instrument are set as follows: the detection molecular weight range is 0-50,000 Daltons; the laser intensity is 175; the detection sensitivity is 8; The adjacent laser excitation position is separated by 5 regions, covering the central 60% of the optimal protein binding region of each point).

血清蛋白质组图谱的初步分析：Preliminary analysis of the serum proteome profile:

利用蛋白质芯片系统专用软件Ciphergen Proteinchip Software(Ciphergen公司)对已经整合的所有数据进行初步处理，选择其中的一些参数，如调整基线(baseline)，并将所有的蛋白质图谱标准化到相同的总的粒子流(totalioncurrent)，再用Biomarker Wizard软件检测数据对已经得到的蛋白质图谱进行分析(选择信噪比S/N参数为5：最小峰参数为20％)，系统共探测到一百多个蛋白质峰，经过T检测得到49个P＜0.05的蛋白质峰。Use Ciphergen Proteinchip Software (Ciphergen Company), a special software for protein chip system, to conduct preliminary processing on all the integrated data, select some of the parameters, such as adjusting the baseline (baseline), and normalize all protein maps to the same total particle flow (totalioncurrent), and then use the Biomarker Wizard software to detect the data and analyze the obtained protein map (select the signal-to-noise ratio S/N parameter to be 5: the minimum peak parameter is 20%), and the system has detected more than one hundred protein peaks. 49 protein peaks with P<0.05 were obtained by T detection.

食管癌患者血清差异表达蛋白质峰的获得：Obtaining peaks of differentially expressed proteins in serum of patients with esophageal cancer:

使用Biomarker Patterns Software高级树状模型分析软件处理已经初步得到的蛋白质的数据，选择特定的参数配置，通过对59例食管癌和61例正常对照的分析，寻找出最佳的分析模型(命名为线性组合分析模型ESCC-WCX2-1)。食管癌血清蛋白指纹由12个不同质荷比(M/Z)的蛋白质组成。在这种分析模型中得到12个在食管癌组和对照组中具有差异表达的蛋白质峰，并联合应用这12种蛋白质的差异可以有效地区分食管癌和非肿瘤正常对照组。这12种蛋白质的线性组合系数和分子量分别为+0.278(1028.35Da)；-0.268(1098.97Da)；-0.239(1301.71Da)；-0.235(2047.86Da)；+0.0693(2742.74Da)；-0.328(4130.07Da)；+0.308(3975.46Da)；+0.0912(4283.96Da)；-0.409(4301.17Da)；+0.153(5635.14Da)；+0.293(6203.80Da)；和+0.496(13749.1Da)。在对这两组共计120例标本的分析中，检测的灵敏度为91.53％，特异性为86.89％。Use the Biomarker Patterns Software advanced tree model analysis software to process the protein data that has been initially obtained, select a specific parameter configuration, and find the best analysis model (named linear) by analyzing 59 cases of esophageal cancer and 61 cases of normal controls Combined analysis model ESCC-WCX2-1). The serum protein fingerprint of esophageal cancer consists of 12 proteins with different mass-to-charge ratios (M/Z). In this analysis model, 12 protein peaks with differential expression between the esophageal cancer group and the control group were obtained, and the combined application of the differences of these 12 proteins can effectively distinguish esophageal cancer from the non-tumor normal control group. The linear combination coefficients and molecular weights of these 12 proteins were +0.278 (1028.35Da); -0.268 (1098.97Da); -0.239 (1301.71Da); -0.235 (2047.86Da); +0.0693 (2742.74Da); +0.308 (3975.46 Da); +0.0912 (4283.96 Da); -0.409 (4301.17 Da); +0.153 (5635.14 Da); +0.293 (6203.80 Da); and +0.496 (13749.1 Da). In the analysis of a total of 120 samples from these two groups, the detection sensitivity was 91.53%, and the specificity was 86.89%.

线性组合分析模型ESCC-WCX2-1的盲筛应用：Blind screening application of linear combination analysis model ESCC-WCX2-1:

进一步选用了3组共195例血清标本，其中包括食管癌血清140例、非肿瘤正常对照血清45例和食管良性病变血清10例。A total of 195 serum samples were selected from 3 groups, including 140 cases of esophageal cancer serum, 45 cases of non-tumor normal control serum and 10 cases of benign esophageal lesions.

采用相同的实验操作条件，相同的软件处理参数，得到的蛋白质图谱应用Biomarker Patterns Software高级线性组合模型分析软件的盲筛功能进行筛选，观察运用该线性组合分析模型ESCC-WCX2-1判断的灵敏度、特异性等指标。实验者在检测时不知道这些标本的临床诊断信息。Using the same experimental operating conditions and the same software processing parameters, the obtained protein maps were screened using the blind screening function of Biomarker Patterns Software advanced linear combination model analysis software, and the sensitivity, indicators of specificity. The experimenters were blinded to the clinical diagnosis of these specimens at the time of testing.

检测结果参见图1、图2、图3、图4，其中，The test results are shown in Figure 1, Figure 2, Figure 3, and Figure 4, in which,

图1显示了蛋白质3975.4的在蛋白质图谱中的表达，对照中的表达要高于肿瘤的表达。标记的为该蛋白质的分子量的大小。Figure 1 shows the expression of protein 3975.4 in the protein map, the expression in the control is higher than the expression in the tumor. Marked is the size of the molecular weight of the protein.

图2显示了蛋白质3975.4的在蛋白质图谱中的另一种表达，对照中的表达要高于肿瘤的表达。标记的为该蛋白质的分子量的大小。Figure 2 shows another expression of protein 3975.4 in the protein map, the expression in the control is higher than the expression in the tumor. Marked is the size of the molecular weight of the protein.

图3显示了蛋白质2047.8在蛋白质图谱中的表达，肿瘤中的表达要高于对照的表达。标记的为该蛋白质的分子量的大小。Figure 3 shows the expression of protein 2047.8 in the protein map, the expression in the tumor is higher than that in the control. Marked is the size of the molecular weight of the protein.

使用该线性组合分析模型ESCC-WCX2-1检测这195例标本得到的灵敏度为85％，特异性可达到84.44％。Using the linear combination analysis model ESCC-WCX2-1 to detect these 195 samples obtained a sensitivity of 85%, and a specificity of 84.44%.

与食管其它良性肿瘤的关系：Relationship with other benign tumors of the esophagus:

选择10例食管其它良性疾病对照，线性组合分析模型ESCC-WCX2-1可以将之与食管癌区分开来。Select 10 cases of other benign esophageal disease controls, linear combination analysis model ESCC-WCX2-1 can distinguish it from esophageal cancer.

与早期食管癌(原位癌)的关系：Relationship with early esophageal cancer (carcinoma in situ):

选择14例早期食管癌(原位癌)微小病变血清，线性组合分析模型ESCC-WCX2-1可以将12例区分为食管癌，准确率为85.71％(12/14)。Select 14 cases of early esophageal carcinoma (carcinoma in situ) minimal lesion serum, linear combination analysis model ESCC-WCX2-1 can distinguish 12 cases as esophageal cancer, the accuracy rate is 85.71% (12/14).

与消化系统其它恶性肿瘤的关系：Relationship with other malignant tumors of the digestive system:

选择22例消化系统其它恶性肿瘤血清，线性组合分析模型ESCC-WCX2-1可以将1例区分为食管癌，准确率为95.45％(1/22)。Select 22 cases of other malignant tumors of the digestive system, the linear combination analysis model ESCC-WCX2-1 can distinguish 1 case as esophageal cancer, the accuracy rate is 95.45% (1/22).

Claims (2)

1. A new method for detecting serum protein fingerprint of esophageal cancer is characterized in that:

collecting esophageal cancer group serum samples, control group serum samples, early esophageal cancer group serum samples, esophageal benign disease group serum samples and other malignant tumor group serum samples;

centrifuging the serum specimen at 4 ℃ within 2-3 hours after separation, separating the serum, packaging and storing for later use, wherein the storage temperature is-80 ℃;

placing the serum sample on a weak cation affinity adsorption protein chip for specific binding and elution treatment;

putting the processed chip into a protein chip reader (tandem mass spectrometer) for analysis to obtain a proteome map of the serum sample;

acquiring data by using protein chip data acquisition software, comparing and analyzing proteome maps of serum samples of an esophageal cancer group and a control group, obtaining a group of differential protein maps of the esophageal cancer group and the control group, namely protein fingerprints, by combining protein fingerprint tree-like model analysis software, and performing blind screening test by taking the protein fingerprints as a judgment standard;

and (4) screening and testing the rest serum samples by adopting a blind method to determine the sensitivity and specificity indexes of the esophageal cancer serum protein fingerprint standard.

2. The method for detecting serum protein fingerprint of esophageal cancer according to claim 1, comprising the following steps: the method is characterized in that:

the serum sample is morning fasting venous serum;

the serum sample needs to be subjected to dilution treatment, and the dilution treatment comprises the following steps:

the serum specimen is firstly subjected to protein concentration quantification,

mu.l serum and 6. mu.l sample dilution U9 were mixed and placed on ice for 30 minutes and gently mixed once every 5 minutes. Adding 108 μ l of binding buffer solution into the diluted serum, mixing, and placing on ice;

the specific binding and elution process steps are:

adding 100 μ l of diluted serum into each well of the protein chip, covering with tinfoil, and incubating on a shaker for 1 hour at room temperature;

discarding the serum sample, adding 200 μ l of binding buffer solution into each sample adding hole, and washing for 5 minutes on a shaking table; discarding the binding buffer and repeating this step twice; discarding the binding buffer, adding 200 μ l of purified water into each sample well, shaking gently, immediately discarding water, removing the chip from the sample applicator, and naturally drying the chip at room temperature;

after the chip is taken out and dried slightly, 0.5 mul of saturated SPA is added to each sample adding hole, and the step is repeated once after the chip is naturally dried.

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