喉癌及下咽癌血浆肿瘤标志物的蛋白质组学筛选及验证
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摘要
目的和意义
喉癌及下咽癌是严重危害人们生命和健康的常见头颈部恶性肿瘤,病理类型多为鳞状细胞癌。近年来,由于环境等多种不同致癌因素的共同作用,喉癌及下咽癌的发病率呈逐渐上升的趋势。尽管以外科手术为主的综合治疗措施取得较大进展,但是喉癌患者综合治疗后的5年生存率仍无明显的提高。尽管如此,临床大量数据表明,治疗的早晚对患者预后产生重要的影响。据统计,早期喉癌、下咽癌治疗效果相对较好,喉功能多可以保留；晚期喉癌、下咽癌由局部浸润和处淋巴结转移,治疗效果较差,5年生存率下降。由此,提高喉癌、下咽癌疗效的关键在于的早期诊断、早期治疗。然而,喉癌及下咽癌早期诊断困难,目前尚没有可靠的用于早期诊断的特异性指标,因此,临床上亟待寻找喉癌及下咽癌相关的特异性肿瘤标志物以辅助早期诊断。
随着分子生物学技术的快速发展及对肿瘤研究深入展开,人们对肿瘤的病因及发病机制认识逐步加深,一些与肿瘤发生、发展及预后有关的癌基因和抑癌基因相继被发现。基因的功能是通过其编码的蛋白质来实现的,蛋白质才是生命活动的真正执行者,蛋白质在肿瘤形成中的作用越来越受到重视。蛋白质组学技术的发展,为从整体水平研究蛋白质组在肿瘤发生、发展及演变过程的作用提供了新的技术平台,同时为发现新的特异性肿瘤标记物奠定了技术基础。
蛋白质组学(proteomics)是研究细胞、组织或生物体全体蛋白质的科学,旨在阐明生物体全部蛋白质的表达及功能,包括蛋白质的表达、结构、功能和相互作用等。通过检测蛋白质在不同环境、不同时期、药物干预、各种疾病及病理生理过程中的变化,来阐述这些外在因素对细胞代谢的影响,分析改变的蛋白质在各种疾病过程中发挥的作用,解释各种病理生理过程的分子机理。在肿瘤研究中,通过蛋白质组学的方法可以对肿瘤组织蛋白质进行全面和深入研究,找到各种异常表达的蛋白质,作为肿瘤诊断的标志物和筛选肿瘤治疗药物的靶点,也是肿瘤蛋白组学研究的重点。近年来,蛋白质组学技术在肺癌、肝癌、胃癌、肾癌、宫颈癌等恶性肿瘤的早期诊断方面的研究取得初步的进展,发现了一系列肿瘤相关蛋白和潜在的肿瘤标记物,为肿瘤相关研究提供了丰富资源。国、内外学者已应用蛋白组学技术初步观察到头颈癌及癌旁组织、原发癌与淋巴结转移细胞系、头颈癌患者的唾液及血浆等蛋白质表达谱,证实了组织之间的蛋白质表达谱确实存在差异,并筛选到了一些与头颈癌相关的肿瘤相关蛋白。然而,既往研究的这些成果仍然尚处于初级阶段,尚没有可以应用于头颈肿瘤临床诊断的肿瘤标记物。既往国外研究是将头颈癌作为一个整体进行研究,未对每种头颈癌进行分类研究,研究指标缺乏特异性。国、内外大多研究未进行肿瘤病理分型,也未进行分级和分期,未对治疗前后疗效判断的对比研究,尚缺乏对预后判断的指标。目前为止,国内外对喉癌及下咽癌的血浆蛋白质组学研究报道较少,尚没有可供用喉癌及下咽癌早期诊断、治疗及预后监测的特异性肿瘤标志物应用于临床。
基于血浆蛋白质组学在喉癌、下咽癌肿瘤标志物的研究现状及其意义,本课题研究的目的是应用蛋白组学技术筛选血浆中差异表达的蛋白质组,寻找潜在的与喉癌、下咽癌患者密切相关的血浆肿瘤分子标志物,为喉癌及下咽癌的早期诊断提供可靠的实验依据。
材料和方法
1.临床标本搜集
20例喉癌患者及20例下咽癌患者血浆标本来自2010年1月～2012年6月南方医院耳鼻咽喉头颈外科门诊及住院患者,均为治疗前患者血浆。喉癌组男性16例,女性4例,年龄53-60岁,平均年龄56.3岁。其中声门型10例,声门上型6例,声门下型3例,贯声门型1例。TNM分期为T1-4N1-3MO,其中Ⅰ期2例,Ⅱ期3例,Ⅲ期12例,Ⅳ期3例。病理证实为喉鳞状细胞癌。下咽癌组男性18例,女性2例,年龄51-63岁,平均年龄55.4岁。其中梨状窝区癌15例,下咽后壁癌4例,环后区癌1例。TNM分期为T1-4N1-3Mo,其中Ⅰ期1例,Ⅱ期2例,Ⅲ期13例,Ⅳ期4例。病理证实为下咽鳞状细胞癌。20例正常健康组血浆标本来自于2012年6月南方医院体检科健康体检者,男性15例,女性5例,年龄50-60岁,平均年龄54.6岁。标本保存于-80℃冰箱中备用。
2.血浆样品去除高丰度蛋白处理
应用SIGMA ProteoPrep Blue Albumin and IgG Depletion Kit试剂盒,除去albumin及IgG等高丰度蛋白,加强低丰度蛋白的显影。
3.血浆蛋白样品的纯化
用2-D Clean-up Kit试剂盒除去盐、脂肪、多糖等干扰双向电泳的物质。
4.蛋白定量
用EttanTN2-D Quant Kit试剂盒测定样本中蛋白浓度。
5. CyDye染料标记
内标由样品蛋白各取25μg混合而成,每块胶内标总量为50μg,用Cy2染料进行标记。使用三种特殊的荧光染料Cy2、Cy3、Cy5以50μg蛋白:400pmol荧光染料的比例进行标记反应。
6.双向凝胶电泳
标记好的样品蛋白上样到pH3-10NL的24cm非线性IPG胶条进行水化反应,避光过夜。置入EttanTM PGPhor进行第一向电泳,接着用EttanTM DALT Six电泳仪进行第二向电泳。
7.分析胶的成像
用Typhoon Imager9400成像仪对电泳后胶板图像扫描,Cy2、Cy3以及Cy5分别以488/520nm、532/580nm、633/670nm波长激光进行扫描,设定的PMT值以整块胶或感兴趣的区域内的最大灰度值在60,000-90,000范围内为准。通过ImageQuant TL软件可以看至Cy2、Cy3以及Cy5的图像分别为蓝色、绿色及红色。
8.制备胶的制作
将含有同等量各组样品蛋白混合后的样品共600-800μg加样到制备胶进行一向和二向电泳,具体步骤和参数与分析胶的制备要求相同。用胶体考染法进行后染色,所得凝胶在DeCyder2D差异分析软件中可与DIGE分析凝胶组相匹配后进行点切取。
9.差异点的软件分析及挖胶
用DeCyder V6.5分析软件先进行胶内差异分析(differential in-gel analysis,DIA),再进行胶间差异分析(biological variation analysis,BVA),找到感兴趣的差异蛋白点,然后对分析到的差异点在制备胶上匹配到相对应的蛋白点,最后利用EttanTM Spot picker全自动斑点切取系统进行挖胶。
10.考染胶粒的胶内酶解
取出冻存的胶粒,加入胰蛋白酶后,37℃,恒温水浴中酶解14-16hr；抽提后的酶解肽段用真空冷冻干燥仪冻干至10μl左右。使用ZipTip(?)18纯化浓缩样品,最后用2μl含5mg/ml CHCA基质和洗脱蛋白混合,并直接点到清洁干燥的MALDI样品靶上。
11.质谱鉴定
将样品靶输入ABI4800MALDI-TOF/TOF质谱仪中,内标校正,获取肽质量指纹图谱(peptide mass fingerprinting,PMF),自动选择最强的10个母离子(precursor)进行MS/MS二级质谱,进行氨基酸测序,得到肽序列标签(peptide sequence tag,PST)。使用Mascot软件对质谱的PMF及PST进行数据库检索比对,采用MS和MS/MS联合模式搜索数据库NCBInr。取蛋白评分大于95%的鉴定结果为高度可信。取可信度高的结果做进一步的生物信息学分析。
12. Western blot验证
随机选取4例喉癌患者血浆、8例下咽癌患者血浆及8例健康人血浆,进行Western-blot验证试验。
13. ELISA验证
分别取20例喉癌患者、20例下咽癌患者血浆及20例健康人血浆,进行ELISA验证试验。
14.统计学方法
使用统计软件为SPSS17.0,统计方法为两独立样本均数的t检验,方差齐性检验用Levene检验,取α=0.05为检验标准。所有计量资料用均数±标准误(x±SE)来反映样本均数的离散趋势。
结果
1.蛋白定量结果
采用2-D Quant kit的定量方法的同时,结合SDS-PAGE的染色图片直观显示来核实定量的准确性。每组取50μg上样做SDS-PAGE电泳,结果显示各组间蛋白条带辉度基本一致,说明定量准确。
2.分析胶荧光图像扫描
经过荧光差异双向凝胶电泳及Typhoon Imager9400成像仪对胶进行荧光扫描,1号胶和2号胶均得到了清晰的蛋白质表达谱,利用ImageQuant TL分析软件观察,不同染料标记的样本呈现不同颜色的图谱。
3. DeCyder2D软件分析蛋白差异点
使用DeCyder2D差异分析软件对DIGE结果进行分析,经过胶内差异分析和胶间差异分析,喉癌组血浆总蛋白和健康对照组相比较,共找到了28个差异蛋白点,其中上调的有9个,下调的有19个。下咽癌患者与健康人血浆相比较,筛选出表达量差异2倍以上的差异蛋白点共36个,其中表达上调的有16个,表达下调的有20个,
4.差异蛋白质的质谱鉴定
使用ABI4800MALDI-TOF/TOF质谱仪对差异蛋白点进行鉴定。通过mascot软件检索Swiss-Prot数据库鉴定蛋白,去除蛋白名称相同的蛋白,喉癌血浆中共鉴定出16种蛋白质,分别为碱性磷酸酶、a-1抗胰蛋白酶、免疫球蛋白γ-3链C结构域、纤维蛋白原γ链、α1酸性糖蛋白1、载脂蛋白L1、结合珠蛋白、结合珠蛋白相关蛋白、免疫球蛋白λ-1链C结构域、免疫球蛋白κ链C结构域、载脂蛋白A1、C反应蛋白、血浆铜蓝蛋白、转甲状腺素蛋白、血红蛋白α亚基、载脂蛋白C3。下咽癌血浆中共鉴定出18种差异蛋白,分别为血清白蛋白、碱性磷酸酶、α1抗糜蛋白酶、α2-HS-糖蛋白、血管紧张素原、免疫球蛋白γ-3链C结构域、纤维蛋白原γ链、补体C4-B、载脂蛋白L1、结合珠蛋白、胶原蛋白a1链、免疫球蛋白λ-1链C结构域、免疫球蛋白K链C结构域、载脂蛋白A1、血浆铜蓝蛋白、转甲状腺素蛋白、血红蛋白α亚基、载脂蛋白C3。
5. Western blot验证结果
Western blot实验结果发现,AGP1在喉癌患者血浆中浓度和健康对照组相比,呈现升高趋势；AHSG在下咽癌患者血浆中浓度和健康对照组相比,呈现升高趋势,均与蛋白质组学结果一致。
6. ELISA验证结果
ELISA实验结果发现,AGP1在喉癌患者(n=20)血浆中浓度和健康对照组(n=20)相比,呈现升高趋势,与蛋白质组学结果一致,见图1-11。AGP1在健康对照组的浓度为965.53±49.24μg/ml(x±SE),在喉癌患者血浆中浓度为2109.13±±99.94μg/ml (x±SE)。Levene方差齐性检验结果显示两组数据方差齐性,用两独立样本的t检验,t=10.282,P<0.05(0.000),喉癌组和健康对照组血浆中AGP1浓度差异具有统计学意义。AHSG在下咽癌患者(n=20)血浆中浓度和健康对照组(n=20)相比,呈现升高趋势,与蛋白质组学结果一致。AHSG在健康对照组的浓度为129.36±4.04μg/ml(x±SE),在下咽癌患者血浆中浓度为261.61±8.39μg/ml (x±E)。 Levene方差齐性检验结果显示两组数据方差齐性,用两独立样本的t检验,t=-14.197,P<0.05(0.000),下咽癌组和健康对照组血浆中AHSG浓度差异具有统计学意义。
结论
本研究应用基于DIGE-MALDI-TOF/TOF质谱技术-生物信息学分析的差异蛋白质组学技术平台,成功分离并鉴定出喉癌及下咽癌患者血浆的差异蛋白质组,并对两个感兴趣的差异蛋白进行了初步的功能验证,验证结果与实验结果相一致。本研究可以得到以下4点结论：
1.成功建立了喉癌组及正常对照组血浆的DIGE差异图谱,分析后得到28个差异蛋白质点,并利用自动切胶仪成功挖取出28个蛋白胶粒。利用质谱仪成功鉴定出28个差异蛋白,除去蛋白名称相同的蛋白,共鉴定出16种差异蛋白质。
2.成功建立了下咽癌组及正常对照组血浆的DIGE差异图谱,分析后得到36个差异蛋白质点,并利用自动切胶仪成功挖取出36个蛋白胶粒。利用质谱仪成功鉴定出36个差异蛋白,除去蛋白名称相同的蛋白,共鉴定出18种差异蛋白质
3.利用Western-blot及ELISA对候选蛋白AHSG及AGP1进行功能验证,AHSG及AGP1的血浆表达与蛋白质组学结果一致。
4.实验表明：AGP1及AHSG在喉癌及下咽癌中高表达,经统计学检验,差异有统计学意义,可为喉癌及下咽癌早期诊断的特异性候选肿瘤标志物。
基于上述结果,血浆蛋白在喉癌和下咽癌发生、发展过程中发挥重要作用,血浆蛋白在喉癌、下咽癌和正常人血浆的差异表达表明,这些差异蛋白与喉癌及下咽癌密切相关,可成为肿瘤患者的候选特异性血浆肿瘤标志物。蛋白质组学技术为寻找血液中肿瘤相关分子标志物、为头颈肿瘤的诊断提供了新的途径。
Objectives and Significance
Laryngeal and hypopharyngeal cancers were common malignant tumors of head and neck which threaten people's lives and healths seriously, and the most common pathological type was squamous cell carcinoma. In recent years, due to the interaction of different carcinogenic factors such as environment, laryngeal and hypopharyngeal cancer incidences have been on a gradual upward trend. While the progresses of combination treatment which based on surgical measures mainly have been made, the5-year survival rate of laryngeal carcinoma patients was still no noticeable improvement after multimodality treatment. However, clinical data suggested that different stages had significant influences on prognosis of patients with treatment. According to statistics data, the results of treatments on the early-stage of laryngeal and hypopharyngeal cancers were better, and the laryngeal functions were better reservated commonly; however, because of the local infiltration and metastasis of lymph nodes for advanced laryngeal and hypopharyngeal carcinomas, the treatment results were disappointed and the5-year survival rate was decreased. Thus, to improve the consequences of laryngeal and hypopharyngeal carcinoma, early diagnosis and early treatment was important. However, since there were no reliable and specific indicators for early diagnosis of the tumors currently, early diagnosis of laryngeal and hypopharyngeal carcinoma were difficult, therefore, it was urgently to look for specific tumor markers of laryngeal and hypopharyngeal cancer for early clinical diagnosis.
With the development of techniques of molecular biology and the further research on cancers, the understanding of etiology and pathogenesis of cancers were gradually deepened, and some relevant oncogenes and tumor suppressor genes about tumorigenesis, development and prognostic have been found. Functions of gene were performed by proteins which encoded, and proteins were the true performers for lives. The development of proteomics technology have provided a new technology platform for overall study on tumor development and malignant transformation process, as well as found technical basis of looking for the new specific tumor markers.
Proteomics is a scientific study of proteom compositions and their variation rules of cells, tissues or organism, and the aims of which is to clarify the mode of protein expression patterns and functional patterns of all living organisms, including proteins expression, existences, structures, functions and interactions and so on. We can understand the effects of external factors to cell metabolism, the functions of changed proteins in various disease, and molecular mechanisms of all the pathophysiological processes by detecting the level of proteins in different environments, different time, drugs treatment, diseases and changes in physiological processes. In cancer research, advanced studies have been performed by proteomic approach, which were mainly on protein quantities, structures, characters, relationships and biological functions of tumor tissues, to further clarify the mechanism of tumor development, to look for all kinds of abnormal expression of proteins which can be used as markers for cancer diagnosis and screening of drugs target for cancer treatment. In recent years, the proteomics techniques were well progressed in early diagnosis of lung cancer, liver cancer, stomach cancer, renal cell carcinoma, and cervical cancer, and a series of potential cancer-related proteins and tumor markers were discovered, which may provide wealthy resources for cancer-related research. Both domestic and overseas studies have observed that there were differential expressioned proteins between head and neck cancers and adjacent normal, primary cancers and lymph node cells, and in salivary proteins, additionly, the results were confirmed by experimental data. At present, there were few plasma proteomics reports on laryngeal and hypopharyngeal carcinoma, and there were no available tumor markers for early diagnosis, treatment and prognosis for clinical monitoring.
According to the study situation of plasma proteomics and its significance of tumor markers for laryngeal carcinoma and hypopharyngeal carcinoma, the purpose of this study is to utilize proteomics technology to screen differential expressed plasma proteome to look for potential serum tumor markers for patients with laryngeal and hypopharyngeal cancers, therefore, our study will provide reliable experimental data for early diagnosis of laryngeal and hypopharyngeal carcinoma.
Materials and methods
1. Clinical samples collection
The plasma samples from20cases of laryngeal cancer patients and20cases of hypopharyngeal cancer patients were collected in Otolaryngology Head and Neck Surgery of Nanfang Hospital from January2010to June2012, and all the samples were collected before treatment. There were16male and4female patients in laryngeal cancer group, with53-60years old, T1-4N1-3M0TNM stage, and laryngeal squamous cell carcinoma (LSCC) of pathologically type; there were18male and2female patients in hypopharyngeal cancer group, with50-60years old, T1-4N1-3M0TNM stage, and hypopharyngeal squamous cell carcinoma (HSCC) of pathologically type.20cases of normal plasma samples were collected from healthy physical examination department of Nanfang Hospital in June2012, with15male and5female donors, and50-60years old. The samples were deposited in-80℃refrigerator.
2. High abundance proteins removement of plasma samples
To remove high abundance proteins such as albumin and IgG to strengthen the development of low abundance proteins, a SIGMA ProteoPrep Blue Albumin and IgG Depletion Kit was used.
3. Purification of plasma protein samples
2-D Clean-up Kit was used to remove salt, fat, polysaccharides and other material which interfere with two-dimensional electrophoresis.
4. Protein concentration determination
The protein concentration of the samples were determined by EttanTM2-D Quant Kit.
5. CyDye labeling
Internal standard was protein mixture of all the samples with25μg each, and50μg total protein for internal standard in one gel, which was marked with Cy2dye. Three special fluorescent dyes named Cy2, Cy3and Cy5were labeled with50μg protein:400pmol dyes.
6. Two dimentional electrophoresis
The rehydration process was performed with immobilized non-linear pH gradient (IPG) strips (pH3-10NL,24cm) which were later rehydrated by CyDye-labeled samples in the dark at room temperature overnight. Isoelectric focusing was then performed using a IPGphor apparatus. The gels were then run in an Ettan DALT Six gel tank.
7. Image scanning of analytic gel
The gels after electrophoresis were scanned with Typhoon Imager Scanner9400, and the channels of Cy2, Cy3and Cy5were respectively scanned by488/520nm,532/580nm and633/670nm, and the PMT value was set as the biggest grey value within the range60000-90000in the whole gel or the interested region. The blue, green, and red images which were labeled with Cy2, Cy3and Cy5dyes were observed respectively.
8. Production of preparative gel
A preparative gel, containing600-800μg of unlabeled internal standard mixture proteins, was prepared for2DE, the steps and parameters were the same as the analytic gels. After post-stainning with colloid staining dyes, the preparative gel was matched with analytic gels to pick the differential spots analyzed with DeCyder 2D software.
9. Differential spots analysis with software and spot picking
Differential in-gel analysis and biological variation analysis were analyzed with DeCyder V6.5software to look for the differential expressed protein spots, and then the varied spots were matched on the preparative gel, finally, they were picked by EttanTM Spot picker automatically.
10. In-gel digestion of differential expressed spots
The picked spots, which were added trypsin, were placed in37℃constant temperature water bath for14-16hr; After the extraction of digested peptides were concentrated to about10μl by vacuum freeze drying apparatus. ZipTip (?) C18were used to purify and concentrate samples, and finally,2μl CHCA matrix (5mg/ml) was used to elute the protein mixture, and then placed to the clean and dry MALDI sample plate directly.
11. Mass spectrometry identification
Sample target was input to ABI4800MALDI-TOF/TOF mass spectrometer, after internal standard calibration, the peptide mass fingerprint (PMF) were aquired automatically, and then the strongest tenth precursor ions were selected for MS/MS mass spectrometry, and finally, the peptide sequence tags (PST) were got after amino acid sequencing. PMF and PST data were used to search in database with Mascot software, and MS combined MS/MS pattern was matched to NCBInr database, protein score which was greater than95%was highly credible. Bioinformatics analysis were performed for those highly credible proteins.
12. Western blot
4plasma samples of laryngeal cancer,8of hypopharyngeal cancer and8of healthy persons were selected randomly to perform Western blot verification.
13. Enzyme-linked immuno sorbent assay
20plasma samples of laryngeal cancer, hypopharyngeal cancer and healthy persons were respectively selected randomly to perform ELISA verification.
14. Statistical methods
For ELISA analysis, independent samples Student's t-test was used to determine mean differences between two groups, and Levene test was used in homogeneity test of variances, and a P<0.05was used to assess significance of differences using SPSS17.0software. All measurement data was shown with x±SE to reflect the discrete tendency of sample mean.
Results
1. Results of protein concentration assay
2-D Quant kit was used to assay protein concentration firstly, and then a stained pictures of SDS-PAGE was to verify the accuracy of protein concentration. The results showed that each protein bands were equal.
2. Image scanning of analytic gel
Clear protein expression spectrums were got after two-dimensional gel electrophoresis and Typhoon Imager9400image scanning. Samples labeled with different dyes showed different color maps by ImageQuant TL software.
3. Differential expressed spots analyzed by Decyder2D software
According to the DeCyder software analysis,28protein spots had significant differences occurring between the LSCC patients and the healthy honors, among those spots,9were up-regulated and19down-regulated. Addtionally,36protein spots had significant differences occurring between the HSCC patients and the healthy honors, among those spots,16were up-regulated and20down-regulated.
4. MS identification of picked spots
The differential expressed protein spots were analyzed by ABI4800MALD-TOF/TOF mass spectrometry. Mascot software was used to search the proteins in Swiss-Prot database, except proteins with the same name,16proteins were identified in LSCC and18proteins in HSCC.
5. Verification results of Western blot
According to Western blot results, AGP1was significantly increased in the plasma of LSCC patients, and AHSG was significantly increased in the plasma of HSCC patients. These results were consistent with the data from the proteomics experiments.
6. Verification results of ELISA
According to ELISA results, AGP1was increased between LSCC patients (n=20) and healthy donors (n=20), which was consistent with the data from the proteomics experiments. The plasma AGP1concentration of control group is965.53±49.24μg/ml (x±SE), and LSCC group is2109.13±99.94μg/ml (x±SE). Levene test results showed that the variances was equal, and after independent samples t test, t=10.282, P<0.05(0.000) was observed, therefore, the results suggested that AGP1concentration had a significant difference between LSCC and healthy controls. AHSG was increased between HSCC patients (n=20) and healthy donors (n=20), which was consistent with the data from the proteomics experiments. The plasma AHSG concentration of control group is129.36+4.04μg/ml (x±SE), and HSCC group is261.61±8.39μg/ml (x±SE). Levene test results showed that the variances was equal, and after independent samples t test, t=-14.197, P<0.05(0.000) was observed, therefore, the results suggested that AHSG concentration had a significant difference between HSCC and healthy controls.
Conclusions
This study identified differentially-expressed plasma proteomes of LSCC and HSCC by DIGE and MALDI-TOF/TOF platform, and two of the interested proteins were consistent with the results of functional experiments. Through this study,4conclusions could be drawn as follows:
1. The differentially expressed DIGE maps of plasma were established successfully in LSCC patients and healthy controls, and28varied spots were picked by an spot picker automaticly. These28protein were identified by mass spectrometer successfully, except for proteins with the same name,16kinds of proteins were got at last.
2. The differentially expressed DIGE maps of plasma were established successfully in HSCC patients and healthy controls, and36varied spots were picked by an spot picker automaticly. These36protein were identified by mass spectrometer successfully, except for proteins with the same name,18kinds of proteins were got at last.
3. Among the candidates proteins, AGP1and AHSG were verificated by Western blot and ELISA, and the results were consistent with the data from the proteomics experiments.
4. Both AGP1and AHSG were upregulated in plasma of LSCC and HSCC patients, the differences were significant by statistical tests, therefore, they were expected to be specific candidate serum tumor biomarkers for the early diagnosis of LSCC and HSCC.
According to the results above, the candidate differential proteins should play an important role in the process of development of LSCC and HSCC, and these proteins should be as the specific serum tumor markers for these cancers. Proteomics technology could provide a new way for look for tumor related biomarkers in blood to help diagnosis of head and neck cancers.
喉癌及下咽癌是严重危害人们生命和健康的常见头颈部恶性肿瘤,病理类型多为鳞状细胞癌。近年来,由于环境等多种不同致癌因素的共同作用,喉癌及下咽癌的发病率呈逐渐上升的趋势。尽管以外科手术为主的综合治疗措施取得较大进展,但是喉癌患者综合治疗后的5年生存率仍无明显的提高。尽管如此,临床大量数据表明,治疗的早晚对患者预后产生重要的影响。据统计,早期喉癌、下咽癌治疗效果相对较好,喉功能多可以保留；晚期喉癌、下咽癌由局部浸润和处淋巴结转移,治疗效果较差,5年生存率下降。由此,提高喉癌、下咽癌疗效的关键在于的早期诊断、早期治疗。然而,喉癌及下咽癌早期诊断困难,目前尚没有可靠的用于早期诊断的特异性指标,因此,临床上亟待寻找喉癌及下咽癌相关的特异性肿瘤标志物以辅助早期诊断。
随着分子生物学技术的快速发展及对肿瘤研究深入展开,人们对肿瘤的病因及发病机制认识逐步加深,一些与肿瘤发生、发展及预后有关的癌基因和抑癌基因相继被发现。基因的功能是通过其编码的蛋白质来实现的,蛋白质才是生命活动的真正执行者,蛋白质在肿瘤形成中的作用越来越受到重视。蛋白质组学技术的发展,为从整体水平研究蛋白质组在肿瘤发生、发展及演变过程的作用提供了新的技术平台,同时为发现新的特异性肿瘤标记物奠定了技术基础。
蛋白质组学(proteomics)是研究细胞、组织或生物体全体蛋白质的科学,旨在阐明生物体全部蛋白质的表达及功能,包括蛋白质的表达、结构、功能和相互作用等。通过检测蛋白质在不同环境、不同时期、药物干预、各种疾病及病理生理过程中的变化,来阐述这些外在因素对细胞代谢的影响,分析改变的蛋白质在各种疾病过程中发挥的作用,解释各种病理生理过程的分子机理。在肿瘤研究中,通过蛋白质组学的方法可以对肿瘤组织蛋白质进行全面和深入研究,找到各种异常表达的蛋白质,作为肿瘤诊断的标志物和筛选肿瘤治疗药物的靶点,也是肿瘤蛋白组学研究的重点。近年来,蛋白质组学技术在肺癌、肝癌、胃癌、肾癌、宫颈癌等恶性肿瘤的早期诊断方面的研究取得初步的进展,发现了一系列肿瘤相关蛋白和潜在的肿瘤标记物,为肿瘤相关研究提供了丰富资源。国、内外学者已应用蛋白组学技术初步观察到头颈癌及癌旁组织、原发癌与淋巴结转移细胞系、头颈癌患者的唾液及血浆等蛋白质表达谱,证实了组织之间的蛋白质表达谱确实存在差异,并筛选到了一些与头颈癌相关的肿瘤相关蛋白。然而,既往研究的这些成果仍然尚处于初级阶段,尚没有可以应用于头颈肿瘤临床诊断的肿瘤标记物。既往国外研究是将头颈癌作为一个整体进行研究,未对每种头颈癌进行分类研究,研究指标缺乏特异性。国、内外大多研究未进行肿瘤病理分型,也未进行分级和分期,未对治疗前后疗效判断的对比研究,尚缺乏对预后判断的指标。目前为止,国内外对喉癌及下咽癌的血浆蛋白质组学研究报道较少,尚没有可供用喉癌及下咽癌早期诊断、治疗及预后监测的特异性肿瘤标志物应用于临床。
基于血浆蛋白质组学在喉癌、下咽癌肿瘤标志物的研究现状及其意义,本课题研究的目的是应用蛋白组学技术筛选血浆中差异表达的蛋白质组,寻找潜在的与喉癌、下咽癌患者密切相关的血浆肿瘤分子标志物,为喉癌及下咽癌的早期诊断提供可靠的实验依据。
材料和方法
1.临床标本搜集
20例喉癌患者及20例下咽癌患者血浆标本来自2010年1月～2012年6月南方医院耳鼻咽喉头颈外科门诊及住院患者,均为治疗前患者血浆。喉癌组男性16例,女性4例,年龄53-60岁,平均年龄56.3岁。其中声门型10例,声门上型6例,声门下型3例,贯声门型1例。TNM分期为T1-4N1-3MO,其中Ⅰ期2例,Ⅱ期3例,Ⅲ期12例,Ⅳ期3例。病理证实为喉鳞状细胞癌。下咽癌组男性18例,女性2例,年龄51-63岁,平均年龄55.4岁。其中梨状窝区癌15例,下咽后壁癌4例,环后区癌1例。TNM分期为T1-4N1-3Mo,其中Ⅰ期1例,Ⅱ期2例,Ⅲ期13例,Ⅳ期4例。病理证实为下咽鳞状细胞癌。20例正常健康组血浆标本来自于2012年6月南方医院体检科健康体检者,男性15例,女性5例,年龄50-60岁,平均年龄54.6岁。标本保存于-80℃冰箱中备用。
2.血浆样品去除高丰度蛋白处理
应用SIGMA ProteoPrep Blue Albumin and IgG Depletion Kit试剂盒,除去albumin及IgG等高丰度蛋白,加强低丰度蛋白的显影。
3.血浆蛋白样品的纯化
用2-D Clean-up Kit试剂盒除去盐、脂肪、多糖等干扰双向电泳的物质。
4.蛋白定量
用EttanTN2-D Quant Kit试剂盒测定样本中蛋白浓度。
5. CyDye染料标记
内标由样品蛋白各取25μg混合而成,每块胶内标总量为50μg,用Cy2染料进行标记。使用三种特殊的荧光染料Cy2、Cy3、Cy5以50μg蛋白:400pmol荧光染料的比例进行标记反应。
6.双向凝胶电泳
标记好的样品蛋白上样到pH3-10NL的24cm非线性IPG胶条进行水化反应,避光过夜。置入EttanTM PGPhor进行第一向电泳,接着用EttanTM DALT Six电泳仪进行第二向电泳。
7.分析胶的成像
用Typhoon Imager9400成像仪对电泳后胶板图像扫描,Cy2、Cy3以及Cy5分别以488/520nm、532/580nm、633/670nm波长激光进行扫描,设定的PMT值以整块胶或感兴趣的区域内的最大灰度值在60,000-90,000范围内为准。通过ImageQuant TL软件可以看至Cy2、Cy3以及Cy5的图像分别为蓝色、绿色及红色。
8.制备胶的制作
将含有同等量各组样品蛋白混合后的样品共600-800μg加样到制备胶进行一向和二向电泳,具体步骤和参数与分析胶的制备要求相同。用胶体考染法进行后染色,所得凝胶在DeCyder2D差异分析软件中可与DIGE分析凝胶组相匹配后进行点切取。
9.差异点的软件分析及挖胶
用DeCyder V6.5分析软件先进行胶内差异分析(differential in-gel analysis,DIA),再进行胶间差异分析(biological variation analysis,BVA),找到感兴趣的差异蛋白点,然后对分析到的差异点在制备胶上匹配到相对应的蛋白点,最后利用EttanTM Spot picker全自动斑点切取系统进行挖胶。
10.考染胶粒的胶内酶解
取出冻存的胶粒,加入胰蛋白酶后,37℃,恒温水浴中酶解14-16hr；抽提后的酶解肽段用真空冷冻干燥仪冻干至10μl左右。使用ZipTip(?)18纯化浓缩样品,最后用2μl含5mg/ml CHCA基质和洗脱蛋白混合,并直接点到清洁干燥的MALDI样品靶上。
11.质谱鉴定
将样品靶输入ABI4800MALDI-TOF/TOF质谱仪中,内标校正,获取肽质量指纹图谱(peptide mass fingerprinting,PMF),自动选择最强的10个母离子(precursor)进行MS/MS二级质谱,进行氨基酸测序,得到肽序列标签(peptide sequence tag,PST)。使用Mascot软件对质谱的PMF及PST进行数据库检索比对,采用MS和MS/MS联合模式搜索数据库NCBInr。取蛋白评分大于95%的鉴定结果为高度可信。取可信度高的结果做进一步的生物信息学分析。
12. Western blot验证
随机选取4例喉癌患者血浆、8例下咽癌患者血浆及8例健康人血浆,进行Western-blot验证试验。
13. ELISA验证
分别取20例喉癌患者、20例下咽癌患者血浆及20例健康人血浆,进行ELISA验证试验。
14.统计学方法
使用统计软件为SPSS17.0,统计方法为两独立样本均数的t检验,方差齐性检验用Levene检验,取α=0.05为检验标准。所有计量资料用均数±标准误(x±SE)来反映样本均数的离散趋势。
结果
1.蛋白定量结果
采用2-D Quant kit的定量方法的同时,结合SDS-PAGE的染色图片直观显示来核实定量的准确性。每组取50μg上样做SDS-PAGE电泳,结果显示各组间蛋白条带辉度基本一致,说明定量准确。
2.分析胶荧光图像扫描
经过荧光差异双向凝胶电泳及Typhoon Imager9400成像仪对胶进行荧光扫描,1号胶和2号胶均得到了清晰的蛋白质表达谱,利用ImageQuant TL分析软件观察,不同染料标记的样本呈现不同颜色的图谱。
3. DeCyder2D软件分析蛋白差异点
使用DeCyder2D差异分析软件对DIGE结果进行分析,经过胶内差异分析和胶间差异分析,喉癌组血浆总蛋白和健康对照组相比较,共找到了28个差异蛋白点,其中上调的有9个,下调的有19个。下咽癌患者与健康人血浆相比较,筛选出表达量差异2倍以上的差异蛋白点共36个,其中表达上调的有16个,表达下调的有20个,
4.差异蛋白质的质谱鉴定
使用ABI4800MALDI-TOF/TOF质谱仪对差异蛋白点进行鉴定。通过mascot软件检索Swiss-Prot数据库鉴定蛋白,去除蛋白名称相同的蛋白,喉癌血浆中共鉴定出16种蛋白质,分别为碱性磷酸酶、a-1抗胰蛋白酶、免疫球蛋白γ-3链C结构域、纤维蛋白原γ链、α1酸性糖蛋白1、载脂蛋白L1、结合珠蛋白、结合珠蛋白相关蛋白、免疫球蛋白λ-1链C结构域、免疫球蛋白κ链C结构域、载脂蛋白A1、C反应蛋白、血浆铜蓝蛋白、转甲状腺素蛋白、血红蛋白α亚基、载脂蛋白C3。下咽癌血浆中共鉴定出18种差异蛋白,分别为血清白蛋白、碱性磷酸酶、α1抗糜蛋白酶、α2-HS-糖蛋白、血管紧张素原、免疫球蛋白γ-3链C结构域、纤维蛋白原γ链、补体C4-B、载脂蛋白L1、结合珠蛋白、胶原蛋白a1链、免疫球蛋白λ-1链C结构域、免疫球蛋白K链C结构域、载脂蛋白A1、血浆铜蓝蛋白、转甲状腺素蛋白、血红蛋白α亚基、载脂蛋白C3。
5. Western blot验证结果
Western blot实验结果发现,AGP1在喉癌患者血浆中浓度和健康对照组相比,呈现升高趋势；AHSG在下咽癌患者血浆中浓度和健康对照组相比,呈现升高趋势,均与蛋白质组学结果一致。
6. ELISA验证结果
ELISA实验结果发现,AGP1在喉癌患者(n=20)血浆中浓度和健康对照组(n=20)相比,呈现升高趋势,与蛋白质组学结果一致,见图1-11。AGP1在健康对照组的浓度为965.53±49.24μg/ml(x±SE),在喉癌患者血浆中浓度为2109.13±±99.94μg/ml (x±SE)。Levene方差齐性检验结果显示两组数据方差齐性,用两独立样本的t检验,t=10.282,P<0.05(0.000),喉癌组和健康对照组血浆中AGP1浓度差异具有统计学意义。AHSG在下咽癌患者(n=20)血浆中浓度和健康对照组(n=20)相比,呈现升高趋势,与蛋白质组学结果一致。AHSG在健康对照组的浓度为129.36±4.04μg/ml(x±SE),在下咽癌患者血浆中浓度为261.61±8.39μg/ml (x±E)。 Levene方差齐性检验结果显示两组数据方差齐性,用两独立样本的t检验,t=-14.197,P<0.05(0.000),下咽癌组和健康对照组血浆中AHSG浓度差异具有统计学意义。
结论
本研究应用基于DIGE-MALDI-TOF/TOF质谱技术-生物信息学分析的差异蛋白质组学技术平台,成功分离并鉴定出喉癌及下咽癌患者血浆的差异蛋白质组,并对两个感兴趣的差异蛋白进行了初步的功能验证,验证结果与实验结果相一致。本研究可以得到以下4点结论：
1.成功建立了喉癌组及正常对照组血浆的DIGE差异图谱,分析后得到28个差异蛋白质点,并利用自动切胶仪成功挖取出28个蛋白胶粒。利用质谱仪成功鉴定出28个差异蛋白,除去蛋白名称相同的蛋白,共鉴定出16种差异蛋白质。
2.成功建立了下咽癌组及正常对照组血浆的DIGE差异图谱,分析后得到36个差异蛋白质点,并利用自动切胶仪成功挖取出36个蛋白胶粒。利用质谱仪成功鉴定出36个差异蛋白,除去蛋白名称相同的蛋白,共鉴定出18种差异蛋白质
3.利用Western-blot及ELISA对候选蛋白AHSG及AGP1进行功能验证,AHSG及AGP1的血浆表达与蛋白质组学结果一致。
4.实验表明：AGP1及AHSG在喉癌及下咽癌中高表达,经统计学检验,差异有统计学意义,可为喉癌及下咽癌早期诊断的特异性候选肿瘤标志物。
基于上述结果,血浆蛋白在喉癌和下咽癌发生、发展过程中发挥重要作用,血浆蛋白在喉癌、下咽癌和正常人血浆的差异表达表明,这些差异蛋白与喉癌及下咽癌密切相关,可成为肿瘤患者的候选特异性血浆肿瘤标志物。蛋白质组学技术为寻找血液中肿瘤相关分子标志物、为头颈肿瘤的诊断提供了新的途径。
Objectives and Significance
Laryngeal and hypopharyngeal cancers were common malignant tumors of head and neck which threaten people's lives and healths seriously, and the most common pathological type was squamous cell carcinoma. In recent years, due to the interaction of different carcinogenic factors such as environment, laryngeal and hypopharyngeal cancer incidences have been on a gradual upward trend. While the progresses of combination treatment which based on surgical measures mainly have been made, the5-year survival rate of laryngeal carcinoma patients was still no noticeable improvement after multimodality treatment. However, clinical data suggested that different stages had significant influences on prognosis of patients with treatment. According to statistics data, the results of treatments on the early-stage of laryngeal and hypopharyngeal cancers were better, and the laryngeal functions were better reservated commonly; however, because of the local infiltration and metastasis of lymph nodes for advanced laryngeal and hypopharyngeal carcinomas, the treatment results were disappointed and the5-year survival rate was decreased. Thus, to improve the consequences of laryngeal and hypopharyngeal carcinoma, early diagnosis and early treatment was important. However, since there were no reliable and specific indicators for early diagnosis of the tumors currently, early diagnosis of laryngeal and hypopharyngeal carcinoma were difficult, therefore, it was urgently to look for specific tumor markers of laryngeal and hypopharyngeal cancer for early clinical diagnosis.
With the development of techniques of molecular biology and the further research on cancers, the understanding of etiology and pathogenesis of cancers were gradually deepened, and some relevant oncogenes and tumor suppressor genes about tumorigenesis, development and prognostic have been found. Functions of gene were performed by proteins which encoded, and proteins were the true performers for lives. The development of proteomics technology have provided a new technology platform for overall study on tumor development and malignant transformation process, as well as found technical basis of looking for the new specific tumor markers.
Proteomics is a scientific study of proteom compositions and their variation rules of cells, tissues or organism, and the aims of which is to clarify the mode of protein expression patterns and functional patterns of all living organisms, including proteins expression, existences, structures, functions and interactions and so on. We can understand the effects of external factors to cell metabolism, the functions of changed proteins in various disease, and molecular mechanisms of all the pathophysiological processes by detecting the level of proteins in different environments, different time, drugs treatment, diseases and changes in physiological processes. In cancer research, advanced studies have been performed by proteomic approach, which were mainly on protein quantities, structures, characters, relationships and biological functions of tumor tissues, to further clarify the mechanism of tumor development, to look for all kinds of abnormal expression of proteins which can be used as markers for cancer diagnosis and screening of drugs target for cancer treatment. In recent years, the proteomics techniques were well progressed in early diagnosis of lung cancer, liver cancer, stomach cancer, renal cell carcinoma, and cervical cancer, and a series of potential cancer-related proteins and tumor markers were discovered, which may provide wealthy resources for cancer-related research. Both domestic and overseas studies have observed that there were differential expressioned proteins between head and neck cancers and adjacent normal, primary cancers and lymph node cells, and in salivary proteins, additionly, the results were confirmed by experimental data. At present, there were few plasma proteomics reports on laryngeal and hypopharyngeal carcinoma, and there were no available tumor markers for early diagnosis, treatment and prognosis for clinical monitoring.
According to the study situation of plasma proteomics and its significance of tumor markers for laryngeal carcinoma and hypopharyngeal carcinoma, the purpose of this study is to utilize proteomics technology to screen differential expressed plasma proteome to look for potential serum tumor markers for patients with laryngeal and hypopharyngeal cancers, therefore, our study will provide reliable experimental data for early diagnosis of laryngeal and hypopharyngeal carcinoma.
Materials and methods
1. Clinical samples collection
The plasma samples from20cases of laryngeal cancer patients and20cases of hypopharyngeal cancer patients were collected in Otolaryngology Head and Neck Surgery of Nanfang Hospital from January2010to June2012, and all the samples were collected before treatment. There were16male and4female patients in laryngeal cancer group, with53-60years old, T1-4N1-3M0TNM stage, and laryngeal squamous cell carcinoma (LSCC) of pathologically type; there were18male and2female patients in hypopharyngeal cancer group, with50-60years old, T1-4N1-3M0TNM stage, and hypopharyngeal squamous cell carcinoma (HSCC) of pathologically type.20cases of normal plasma samples were collected from healthy physical examination department of Nanfang Hospital in June2012, with15male and5female donors, and50-60years old. The samples were deposited in-80℃refrigerator.
2. High abundance proteins removement of plasma samples
To remove high abundance proteins such as albumin and IgG to strengthen the development of low abundance proteins, a SIGMA ProteoPrep Blue Albumin and IgG Depletion Kit was used.
3. Purification of plasma protein samples
2-D Clean-up Kit was used to remove salt, fat, polysaccharides and other material which interfere with two-dimensional electrophoresis.
4. Protein concentration determination
The protein concentration of the samples were determined by EttanTM2-D Quant Kit.
5. CyDye labeling
Internal standard was protein mixture of all the samples with25μg each, and50μg total protein for internal standard in one gel, which was marked with Cy2dye. Three special fluorescent dyes named Cy2, Cy3and Cy5were labeled with50μg protein:400pmol dyes.
6. Two dimentional electrophoresis
The rehydration process was performed with immobilized non-linear pH gradient (IPG) strips (pH3-10NL,24cm) which were later rehydrated by CyDye-labeled samples in the dark at room temperature overnight. Isoelectric focusing was then performed using a IPGphor apparatus. The gels were then run in an Ettan DALT Six gel tank.
7. Image scanning of analytic gel
The gels after electrophoresis were scanned with Typhoon Imager Scanner9400, and the channels of Cy2, Cy3and Cy5were respectively scanned by488/520nm,532/580nm and633/670nm, and the PMT value was set as the biggest grey value within the range60000-90000in the whole gel or the interested region. The blue, green, and red images which were labeled with Cy2, Cy3and Cy5dyes were observed respectively.
8. Production of preparative gel
A preparative gel, containing600-800μg of unlabeled internal standard mixture proteins, was prepared for2DE, the steps and parameters were the same as the analytic gels. After post-stainning with colloid staining dyes, the preparative gel was matched with analytic gels to pick the differential spots analyzed with DeCyder 2D software.
9. Differential spots analysis with software and spot picking
Differential in-gel analysis and biological variation analysis were analyzed with DeCyder V6.5software to look for the differential expressed protein spots, and then the varied spots were matched on the preparative gel, finally, they were picked by EttanTM Spot picker automatically.
10. In-gel digestion of differential expressed spots
The picked spots, which were added trypsin, were placed in37℃constant temperature water bath for14-16hr; After the extraction of digested peptides were concentrated to about10μl by vacuum freeze drying apparatus. ZipTip (?) C18were used to purify and concentrate samples, and finally,2μl CHCA matrix (5mg/ml) was used to elute the protein mixture, and then placed to the clean and dry MALDI sample plate directly.
11. Mass spectrometry identification
Sample target was input to ABI4800MALDI-TOF/TOF mass spectrometer, after internal standard calibration, the peptide mass fingerprint (PMF) were aquired automatically, and then the strongest tenth precursor ions were selected for MS/MS mass spectrometry, and finally, the peptide sequence tags (PST) were got after amino acid sequencing. PMF and PST data were used to search in database with Mascot software, and MS combined MS/MS pattern was matched to NCBInr database, protein score which was greater than95%was highly credible. Bioinformatics analysis were performed for those highly credible proteins.
12. Western blot
4plasma samples of laryngeal cancer,8of hypopharyngeal cancer and8of healthy persons were selected randomly to perform Western blot verification.
13. Enzyme-linked immuno sorbent assay
20plasma samples of laryngeal cancer, hypopharyngeal cancer and healthy persons were respectively selected randomly to perform ELISA verification.
14. Statistical methods
For ELISA analysis, independent samples Student's t-test was used to determine mean differences between two groups, and Levene test was used in homogeneity test of variances, and a P<0.05was used to assess significance of differences using SPSS17.0software. All measurement data was shown with x±SE to reflect the discrete tendency of sample mean.
Results
1. Results of protein concentration assay
2-D Quant kit was used to assay protein concentration firstly, and then a stained pictures of SDS-PAGE was to verify the accuracy of protein concentration. The results showed that each protein bands were equal.
2. Image scanning of analytic gel
Clear protein expression spectrums were got after two-dimensional gel electrophoresis and Typhoon Imager9400image scanning. Samples labeled with different dyes showed different color maps by ImageQuant TL software.
3. Differential expressed spots analyzed by Decyder2D software
According to the DeCyder software analysis,28protein spots had significant differences occurring between the LSCC patients and the healthy honors, among those spots,9were up-regulated and19down-regulated. Addtionally,36protein spots had significant differences occurring between the HSCC patients and the healthy honors, among those spots,16were up-regulated and20down-regulated.
4. MS identification of picked spots
The differential expressed protein spots were analyzed by ABI4800MALD-TOF/TOF mass spectrometry. Mascot software was used to search the proteins in Swiss-Prot database, except proteins with the same name,16proteins were identified in LSCC and18proteins in HSCC.
5. Verification results of Western blot
According to Western blot results, AGP1was significantly increased in the plasma of LSCC patients, and AHSG was significantly increased in the plasma of HSCC patients. These results were consistent with the data from the proteomics experiments.
6. Verification results of ELISA
According to ELISA results, AGP1was increased between LSCC patients (n=20) and healthy donors (n=20), which was consistent with the data from the proteomics experiments. The plasma AGP1concentration of control group is965.53±49.24μg/ml (x±SE), and LSCC group is2109.13±99.94μg/ml (x±SE). Levene test results showed that the variances was equal, and after independent samples t test, t=10.282, P<0.05(0.000) was observed, therefore, the results suggested that AGP1concentration had a significant difference between LSCC and healthy controls. AHSG was increased between HSCC patients (n=20) and healthy donors (n=20), which was consistent with the data from the proteomics experiments. The plasma AHSG concentration of control group is129.36+4.04μg/ml (x±SE), and HSCC group is261.61±8.39μg/ml (x±SE). Levene test results showed that the variances was equal, and after independent samples t test, t=-14.197, P<0.05(0.000) was observed, therefore, the results suggested that AHSG concentration had a significant difference between HSCC and healthy controls.
Conclusions
This study identified differentially-expressed plasma proteomes of LSCC and HSCC by DIGE and MALDI-TOF/TOF platform, and two of the interested proteins were consistent with the results of functional experiments. Through this study,4conclusions could be drawn as follows:
1. The differentially expressed DIGE maps of plasma were established successfully in LSCC patients and healthy controls, and28varied spots were picked by an spot picker automaticly. These28protein were identified by mass spectrometer successfully, except for proteins with the same name,16kinds of proteins were got at last.
2. The differentially expressed DIGE maps of plasma were established successfully in HSCC patients and healthy controls, and36varied spots were picked by an spot picker automaticly. These36protein were identified by mass spectrometer successfully, except for proteins with the same name,18kinds of proteins were got at last.
3. Among the candidates proteins, AGP1and AHSG were verificated by Western blot and ELISA, and the results were consistent with the data from the proteomics experiments.
4. Both AGP1and AHSG were upregulated in plasma of LSCC and HSCC patients, the differences were significant by statistical tests, therefore, they were expected to be specific candidate serum tumor biomarkers for the early diagnosis of LSCC and HSCC.
According to the results above, the candidate differential proteins should play an important role in the process of development of LSCC and HSCC, and these proteins should be as the specific serum tumor markers for these cancers. Proteomics technology could provide a new way for look for tumor related biomarkers in blood to help diagnosis of head and neck cancers.
引文
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