【作者】 杨洋；

【导师】 王建军；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 第一部分：非小细胞肺癌血清蛋白质标记物的检测目的检测非小细胞肺癌血清蛋白质组,筛选特异性的蛋白质标记物,构建可用于非小细胞肺癌早期检测和诊断的判别模型。方法应用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术检测112例非小细胞肺癌和123例对照血清样本的蛋白质质谱,并结合支持向量机(SVM)对数据进行分析。结果经检测及生物信息学分析处理,发现P<0.01的差异性m/z峰22个。从差异性显著的蛋白质峰的任意组合中,运用SVM筛选出预测值Youden指数最高的组合模型,获得m/z峰值位于6628、9191和11412的蛋白质标记物3个。与正常健康组相比,m/z峰值位于6628的蛋白质在非小细胞肺癌中明显低表达,而峰值位于9191和11412的蛋白质呈明显高表达,且表达强度与分期也有一定的相关性。盲法测试结果显示,联合3种潜在蛋白质标记物,该判别模型区分非小细胞肺癌和对照组的敏感性为96.56%,特异性为94.79%,阳性预测值为95.0%。结论m/z峰值位于6628、9191和11412的蛋白质可以作为非小细胞肺癌潜在的生物学标记物。SELDI-TOF-MS蛋白质芯片技术为非小细胞肺癌血清蛋白质标记物的检测提供了有效的手段。第二部分：非小细胞肺癌血清蛋白质标记物的鉴定目的结合蛋白组学技术对m/z峰值位于6628、9191、11412的蛋白质标记物进行结构鉴定。方法运用HPLC对3种蛋白质标记物进行分离纯化,随后采用MALDI-TOF-MS质谱技术对分离后的不同时间点的蛋白质纯化液进行检测,以跟踪目标蛋白质所在的样本。最后,对纯化后的蛋白质标记物进行酶解,并应用nano-LC-ESI-MS/MS进行测序。结果m/z峰值位于6628的蛋白质经鉴定为载脂蛋白C-Ⅰ；m/z峰值位于9191、11412的蛋白质分别为触珠蛋白α1链、S100A4蛋白。结论联合运用HPLC、MALDI-TOF-MS和nano-LC-ESI-MS/MS等蛋白组学技术,可有效地对肿瘤蛋白质标记物进行结构鉴定。m/z峰值位于6628、9191和11412的蛋白质分别被鉴定为载脂蛋白C-Ⅰ,触珠蛋白α1链和S100A4蛋白。第三部分：非小细胞肺癌血清蛋白质标记物的验证目的运用抗体芯片技术对鉴定出的载脂蛋白C-Ⅰ、触珠蛋白α1链和S100A4蛋白质进行免疫学检测,以验证鉴定结果的可靠性。方法分别运用螯合有抗人载脂蛋白C-Ⅰ抗体、抗人触珠蛋白α抗体和抗人S100A4抗体的蛋白质芯片对非小细胞肺癌患者和对照血清样本进行检测。结果m/z峰值位于6628的蛋白质可被偶联有抗载脂蛋白C-I抗体的芯片捕获,而m/z峰值位于9191和11412的蛋白质可分别与偶联有抗触珠蛋白α抗体和抗S100A4抗体的芯片相结合。结论3种蛋白质均可被抗体芯片特异性捕获,证明了前期鉴定结果的可靠性。载脂蛋白C-Ⅰ、触珠蛋白α1链和S100A4蛋白在非小细胞肺癌患者和对照组血清中存在显著的差异性表达,三者联合应用对非小细胞肺癌的早期检测和诊断具有良好的应用价值和广阔的发展前景。更多还原

【Abstract】 Part I Detection of Serum protein Biomarkers of Non-small Cell Lung CancerObjective To detect the specific serum protein biomarkers and establish the discriminant model for early screening and diagnosis of non-small cell lung cancer (NSCLC).Methods Serum proteomic profiles of 112 NSCLC and 123 controls were analyzed using surfaced enhanced laser desorption/ionization time of flight mass spectroscopy (SELDI-TOF-MS), combing a support vector machine (SVM) classifier to deal with the data.Results After detecting and analyzing by bioinformation,22 peaks with p value <0.01 were obtained. From the random combination of protein peaks with remakable variation, SVM screened out the combined model with maximum Youden index of the predicted value, identifying 3 markers positioned at 6628,9191 and 11412. In the NSCLC group, the 6628 Da protein was remarkably decreased while 9191 and 11412 Da proteins were significantly elevated. In addition, the levels of these proteins were associated with the clinical stages. Combining these 3 potential markers, the sensitivity, specificity and positive predictive value of the model were 96.56%,94.79% and 95.0% respectively in the blind testing set.Conclusions These proteins with m/z of 6628,9191 and 11412 could be as potential biomarks of NSCLC. SELDI-TOF-MS has been proved an effective method for serum protein biomarkers detection of NSCLC. PartⅡIdentification of Serum protein Biomarkers of Non-small Cell Lung CancerObjective To identify the candidate protein biomarkers with m/z of 6628,9191 and 11412 using proteomics technologies.Methods The HPLC was used to purify the 3 candidate protein biomarkers, and then the MALDI-TOF-MS technology was applied to detect each fraction to trace the candidate protein biomarkers. After digestion with modified trypsin, the peptide mixture was analyzed by nano-LC-MS/MS.Results The candidate biomarker with m/z of 6628 was identified as apolipoprotein C-I, while another two biomarkers were identified as haptoglobin alpha-1 chain (9191 Da) and S100A4 (11412 Da).Conclusions An efficient strategy, including HPLC purification, MALDI-TOF-MS trace and LC-MS/MS identification, has been proved a powerful tool to identify the target proteins. The candidate biomarker with m/z of 6628,9191 and 11412 were identified as apolipoprotein C-I, haptoglobin alpha-1 chain and S100A4. PartⅢConfirmation of Serum protein Biomarkers of Non-small Cell Lung CancerObjective To confirm the identity of apolipoprotein C-I, haptoglobin alpha-1 chain and S100A4 with the ProteinChip-array-based immunoassay.Methods Serum samples with NSCLC and controls were analyzed using specific antibody arrays which were prepared by covalently coupling the appropriate antibodies to preactivated ProteinChip arrays.Results The anti-apolipoprotein C-I array captured apolipoprotein C-I (6628 Da), the anti-haptoglobin alpha-chain antibody specifically captured the haptoglobin alpha-chain (9191 Da) protein, and the S100A4 antibody specifically captured the S100A4 protein (11412 Da).Conclusions These 3 biomarkers could be specifically captured by the corresponding antibody arrays, which proved the reliability of results with identification. The expression of apolipoprotein C-I, haptoglobin alpha-1 chain and S100A4 in patients with NSCLC are significantly different from nomal controls, which have a good value and broad application prospects for early screening and diagnosis of non-small cell lung cancer.更多还原