食管癌自身抗体和血清小分子蛋白的鉴定
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摘要
食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)是我国常见的恶性肿瘤之一,术后5年生存率仅25%～40%,早期发现是降低死亡率的关键。肿瘤相关抗原及自身抗体的鉴定有望改善恶性肿瘤的早期诊断,发现新的有效的免疫治疗方法。目前鉴定肿瘤相关抗原的方法主要有重组cDNA表达文库的血清学筛选方法(serological analysis of recombinant cDNA expression librariesSEREX)和基于二维凝胶电泳技术的血清学蛋白质组分析方法(serologicalproteome analysis,SERPA)。血清是血液的重要成分,蕴含着丰富的疾病信息,同时样本较易获取。SELDI-TOF MS技术是广泛应用的血清蛋白多肽谱的研究方法。本研究首先应用改进的血清蛋白质组分析方法(modified serological proteomeanalysis,mSERPA)筛选和鉴定ESCC细胞系EC0156胞浆蛋白组分中存在的肿瘤相关抗原,并进一步研究了抗原蛋白的生物学特性,最后对ESCC患者血清中的自身抗体进行了验证。结果共计鉴定了8种候选的肿瘤相关抗原,其中磷酸甘油酸激酶1(phosphoglycerate kinase 1、PGK1)、105kDa热休克蛋白(105kDa heatshock protein,HSP105)、磷酸丙糖异构酶1(triosephosphate isomerase 1,TIM)和α-烯醇化酶(α-enolase,ENO1)等四种抗原在ESCC组织中的表达量均明显升高,在癌细胞中主要定位于胞浆和胞核,胞浆组分中存在不同程度的异构体或蛋白降解片段。另外研究发现TIM和ENO1抗原存在糖基化现象,ESCC血清中的自身抗体主要识别这些发生糖基化修饰的蛋白形式。PGK1、HSP105、TIM和ENO1自身抗体在ESCC患者血清中的阳性率分别为26.9%(7/26)、39.1%(9/23)、34.8%(8/23)和78.3%(18/23);在健康对照(healthy control,HC)血清中的阳性率分别为5.5%(1/18)、0(0/20)、0(0/20)、25%(5/20)。PGK1、HSP105、TIM和ENO1抗原及自身抗体有可能成为ESCC的潜在标志。另外,在前期工作的基础上应用SELDI-TOF MS技术比较分析了ESCC和健康对照血清的差异蛋白多肽谱,建立了ESCC诊断模型。应用免疫吸附的方法鉴定4097Da和(或)7774Da的差异峰是α-烯醇化酶的蛋白片段,ELISA方法验证结果表明α-烯醇化酶在ESCC血清中的表达量明显高于胰腺癌、肝细胞肝癌和健康对照血清。α-烯醇化酶可能是ESCC的潜在标志物。
肝细胞肝癌(hepatocellular carcinoma,HCC)是世界范围的恶性肿瘤,5年生存率低于10%。甲胎蛋白(alpha-fetoprotein,AFP)是临床上判断HCC的主要血清学标志,但30%左右的HCC患者血清中AFP水平并不升高(＜25ng/ml)。ClinProt技术是一种研究临床蛋白质组和蛋白多肽分子标志的新方法,适合10kDa以下的小分子蛋白和多肽。应用该方法比较研究了AFP~-HCC和肝硬化患者血清蛋白多肽谱,建立了一个由5个蛋白差异峰组成的HCC诊断模型,其诊断的敏感度和特异度分别为92.86%(26/28)和93.94%(31/33)。用未建模的HCC和肝硬化患者对诊断模型进行盲筛验证,敏感度和特异度分别为95.6%(43/45)和90.91%(20/22),其中AFP~-HCC阳性率为91.67%(22/24),AFP~+HCC阳性率为100%(21/21)。该诊断模型对辅助HCC的诊断有明显的意义。7758Da和9278Da两个差异峰共计鉴定到13种可信的蛋白质,其中粘蛋白(proteoglycan-4,PRG4)在HCC组织和血清中表达水平明显升高,PRG4蛋白有可能成为HCC的候选标志。
胰腺癌(pancreatic cancer,PC)是致死率很高的恶性肿瘤之一,5年生存率一般为1-5%。应用SELDI-TOF MS技术研究了PC和健康对照血清蛋白多肽谱的差异,建立了胰腺癌诊断模型。应用免疫吸附的方法鉴定5344Da的差异峰是血小板因子4(platelet factor 4,PF4)的蛋白片段,其在PC血清中的表达水平需要进一步验证。
Esophageal squamous cell carcinoma(ESCC) with 25-40%5-year survival rate is one of the common malignant tumors in China.Early detection is one of the most feasible means of improving outcomes.The identification of tumor associated antigens(TAAs) and autoantibody would improve the early diagnosis of cancer and develop novel immunotherapies.Serological analysis of recombinant cDNA expression libraries(SEREX) and serological proteome analysis(SERPA) based on 2-dimmensional electrophoresis(2-DE) are the main strategies for TAA identification. As one of important blood components,serum is easy to be obtained and containing abundant disease information.SELDI-TOF MS is the strategy broadly used for serum protein and peptide profiles.Firstly,TAAs in cytosolic fraction of ESCC cell line EC0156 were identified using modified serological proteome analysis and the biological characteristics of these antigens were investigated in advance. Autoantibody against these antigens in sera of ESCC patients and healthy controls was validated and evaluated.There were eight non redundant TAAs identified in this study.The four antigens phosphoglycerate kinase 1(PGK1),105kDa heat shock protein(HSP105),triosephosphate isomerase 1(TIM) andα-enolase(ENO1) were over-expressed in ESCC tissue and located in cytoplasma and nucleus in cancer cells. Different isoforms and degradated fragments of these four antigens were found in cytosolic fraction.Autoantibody against TIM or ENO1 in ESCC serum mainly reacted with its glycosylated protein.Autoantibody against PGK1,HSP105,TIM and ENO1 was detected in sera from 26.9%(7/26),39.1%(9/23),34.8%(8/23) and 78.3% (18/23) patients with ESCC,respectively.Autoantibody against PGK1 and ENO1 was detected in sera from 5.5%(1/18) and 25%(5/20) healthy controls(HC),respectively; whereas autoantibody against HSP105 or TIM was not detected in sera of healthy controls.These four antigens and autoandibodies may be the potential biomarkers of ESCC.In another research work,we compared the protein/peptide profiles of sera from ESCC with those from healthy controls based on previous study in our laboratory.The one classifier for discriminating ESCC and HC were established.The peak 4097Da and(or) 7774Da in ESCC serum was identified as fragment ofα-enolase using antibody capture method.The concentration ofα-enolase in serum of ESCC was significantly higher than that in pancreatic cancer,hepatocellular carcinoma and HC.The proteinα-enolase may be a potential biomarker for ESCC diagnosis.
Hepatocellular carcinoma(HCC) is one worldwide malignant tumor and its 5-year survival rate is less than 10%.Alpha-fetoprotein(AFP) is only an available serologic biomarker for HCC diagnosis,whereas about 30%of HCC patients with low level of serum AFP(＜25ng/ml).ClinProt technology is a novel strategy to research clinical proteome and protein/peptide biomarkers.This method was employed to uncover the different serum protein/peptide profiles of patients with AFP negative hepatocellular carcinoma(HCC) and liver cirrhosis.Five protein peaks were selected to construct the HCC classifier with 92.86%(26/28) sensitivity and 93.94%(31/33) specificity in training cohort.The pattern was able to classify the blind test cohort with 95.6% (43/45) sensitivity and 90.91%(20/22) specificity,of which the positive rate of AFP~-HCC and AFP~+ HCC is 91.67%(22/24) and 100%(21/21),respectively.The pattern may be significant for auxiliary diagnosis of HCC.Two of protein peaks 7758Da and 9278Da were purified and identified as 13 non-redundant proteins by peptide mapping.Proteoglycan-4(PRG4) was over-expressed in HCC tissue and serum,and mainly located on hepatocellular cytoplasma.PRG4 could be served as potential biomarkers of HCC.
Pancreatic cancer(PC) has the lowest survival rate for any solid cancer and its 5-year survival is only 1-5%.We compared the protein/peptide profiles of sera from PC patients with those from healthy controls.The one classifier consisting of four peaks for discriminating PC and HC were established.The peak 5344Da was identified as platelet factor 4(PF4) using antibody capture method,and its expression level in serum of PC need to be further validated.
肝细胞肝癌(hepatocellular carcinoma,HCC)是世界范围的恶性肿瘤,5年生存率低于10%。甲胎蛋白(alpha-fetoprotein,AFP)是临床上判断HCC的主要血清学标志,但30%左右的HCC患者血清中AFP水平并不升高(＜25ng/ml)。ClinProt技术是一种研究临床蛋白质组和蛋白多肽分子标志的新方法,适合10kDa以下的小分子蛋白和多肽。应用该方法比较研究了AFP~-HCC和肝硬化患者血清蛋白多肽谱,建立了一个由5个蛋白差异峰组成的HCC诊断模型,其诊断的敏感度和特异度分别为92.86%(26/28)和93.94%(31/33)。用未建模的HCC和肝硬化患者对诊断模型进行盲筛验证,敏感度和特异度分别为95.6%(43/45)和90.91%(20/22),其中AFP~-HCC阳性率为91.67%(22/24),AFP~+HCC阳性率为100%(21/21)。该诊断模型对辅助HCC的诊断有明显的意义。7758Da和9278Da两个差异峰共计鉴定到13种可信的蛋白质,其中粘蛋白(proteoglycan-4,PRG4)在HCC组织和血清中表达水平明显升高,PRG4蛋白有可能成为HCC的候选标志。
胰腺癌(pancreatic cancer,PC)是致死率很高的恶性肿瘤之一,5年生存率一般为1-5%。应用SELDI-TOF MS技术研究了PC和健康对照血清蛋白多肽谱的差异,建立了胰腺癌诊断模型。应用免疫吸附的方法鉴定5344Da的差异峰是血小板因子4(platelet factor 4,PF4)的蛋白片段,其在PC血清中的表达水平需要进一步验证。
Esophageal squamous cell carcinoma(ESCC) with 25-40%5-year survival rate is one of the common malignant tumors in China.Early detection is one of the most feasible means of improving outcomes.The identification of tumor associated antigens(TAAs) and autoantibody would improve the early diagnosis of cancer and develop novel immunotherapies.Serological analysis of recombinant cDNA expression libraries(SEREX) and serological proteome analysis(SERPA) based on 2-dimmensional electrophoresis(2-DE) are the main strategies for TAA identification. As one of important blood components,serum is easy to be obtained and containing abundant disease information.SELDI-TOF MS is the strategy broadly used for serum protein and peptide profiles.Firstly,TAAs in cytosolic fraction of ESCC cell line EC0156 were identified using modified serological proteome analysis and the biological characteristics of these antigens were investigated in advance. Autoantibody against these antigens in sera of ESCC patients and healthy controls was validated and evaluated.There were eight non redundant TAAs identified in this study.The four antigens phosphoglycerate kinase 1(PGK1),105kDa heat shock protein(HSP105),triosephosphate isomerase 1(TIM) andα-enolase(ENO1) were over-expressed in ESCC tissue and located in cytoplasma and nucleus in cancer cells. Different isoforms and degradated fragments of these four antigens were found in cytosolic fraction.Autoantibody against TIM or ENO1 in ESCC serum mainly reacted with its glycosylated protein.Autoantibody against PGK1,HSP105,TIM and ENO1 was detected in sera from 26.9%(7/26),39.1%(9/23),34.8%(8/23) and 78.3% (18/23) patients with ESCC,respectively.Autoantibody against PGK1 and ENO1 was detected in sera from 5.5%(1/18) and 25%(5/20) healthy controls(HC),respectively; whereas autoantibody against HSP105 or TIM was not detected in sera of healthy controls.These four antigens and autoandibodies may be the potential biomarkers of ESCC.In another research work,we compared the protein/peptide profiles of sera from ESCC with those from healthy controls based on previous study in our laboratory.The one classifier for discriminating ESCC and HC were established.The peak 4097Da and(or) 7774Da in ESCC serum was identified as fragment ofα-enolase using antibody capture method.The concentration ofα-enolase in serum of ESCC was significantly higher than that in pancreatic cancer,hepatocellular carcinoma and HC.The proteinα-enolase may be a potential biomarker for ESCC diagnosis.
Hepatocellular carcinoma(HCC) is one worldwide malignant tumor and its 5-year survival rate is less than 10%.Alpha-fetoprotein(AFP) is only an available serologic biomarker for HCC diagnosis,whereas about 30%of HCC patients with low level of serum AFP(＜25ng/ml).ClinProt technology is a novel strategy to research clinical proteome and protein/peptide biomarkers.This method was employed to uncover the different serum protein/peptide profiles of patients with AFP negative hepatocellular carcinoma(HCC) and liver cirrhosis.Five protein peaks were selected to construct the HCC classifier with 92.86%(26/28) sensitivity and 93.94%(31/33) specificity in training cohort.The pattern was able to classify the blind test cohort with 95.6% (43/45) sensitivity and 90.91%(20/22) specificity,of which the positive rate of AFP~-HCC and AFP~+ HCC is 91.67%(22/24) and 100%(21/21),respectively.The pattern may be significant for auxiliary diagnosis of HCC.Two of protein peaks 7758Da and 9278Da were purified and identified as 13 non-redundant proteins by peptide mapping.Proteoglycan-4(PRG4) was over-expressed in HCC tissue and serum,and mainly located on hepatocellular cytoplasma.PRG4 could be served as potential biomarkers of HCC.
Pancreatic cancer(PC) has the lowest survival rate for any solid cancer and its 5-year survival is only 1-5%.We compared the protein/peptide profiles of sera from PC patients with those from healthy controls.The one classifier consisting of four peaks for discriminating PC and HC were established.The peak 5344Da was identified as platelet factor 4(PF4) using antibody capture method,and its expression level in serum of PC need to be further validated.
引文
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