表面增强激光解析电离飞行时间质谱法筛选肺癌患者血清和肺泡灌洗液中标志蛋白的研究

目的 应用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术筛选肺癌患者血清和BALF中的差异性表达蛋白,探讨是否可作为诊断肺癌的肿瘤标志物.方法 应用SELDI-TOF-MS技术通过弱阳离子交换蛋白芯片(WCX-2芯片)分别检测35例肺癌和18例肺部良性病变患者血清和BALF中的蛋白质质谱图,用Biomarker Pattern软件分析肺癌的差异蛋白并初步建立诊断模型,通过盲筛进一步验证诊断模型.结果 在肺癌患者血清中发现5个高表达的蛋白质波峰,选用其中质荷比为5639的差异蛋白波峰建立分类树模型,其诊断的敏感度为80%(28/35),特异度为78%(14/18).盲法验证的敏感度为85%(17/20),特异度为90%(9/10),粗符合率为87%(26/30),Youden指数为0.7.在肺癌患者BALF中发现8个高表达蛋白质波峰,选用其中质荷比为7976和11 809的差异蛋白波峰建立分类树模型,其诊断的敏感度为86%(30/35),特异度为72%(13/18).盲法验证的敏感度为90%(18/20),特异度为90%(9/1O),粗符合率为90%(27/30),Youden指数为0.8.平行试验结果显示两者联合应用时诊断肺癌的敏感度、准确率及特异度均为100%,具有互补作用.结论SELDI-TOF-MS技术可筛选出肺癌患者血清和BALF中差异性表达蛋白,作为一种肿瘤标志物,其诊断敏感度高,特异度好,尤其是BALF中差异性表达蛋白的测定可能具有较好的临床应用前景.

Abstract：

Objective To detect the protein markers in serum and bronchoalveolar lavage fluid (BALF) of the patients with lung cancer by surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) technology, and to explore if they can be used as markers for the diagnosis of lung cancer.Methods SELDI-TOF-MS technology and protein chips weak cation exchange (WCX-2 chip) were used to detect the protein mass spectrum in serum and BALF of 35 patients with lung cancer and 18 cases of benign pulmonary diseases.The different protein markers were analyzed by Biomarker Pattern Software and the initial diagnosis models were set up.The diagnosis models were verified further by blind screen to confirm the efficacy of diagnosis.Results Five protein peaks in the sera of the patients with lung cancer were significantly higher (P ＜ 0.05 ).The protein peak with a mass/charge ratio (M/Z)of 5639 was selected to establish the classification tree model.The sensitivity of diagnosis was 80% (28/35) and the specificity was 78% (14/18).The results verified by blind screen showed a sensitivity of 85% (17/20),a specificity of 90% (9/10), a crude accuracy (CA) of 87% ( 26/30 ) and Youden' s index (γ) of 0.7.Eight protein peaks in the BALF of the patients with lung cancer were significantly higher ( P ＜ 0.05).The different protein peaks with M/Z of 7976 and 11 809 respectively were selected to establish the classification tree model.The sensitivity of diagnosis was 86% (30/35) and the specificity was 72% (13/18).The results verified by blind screen showed a sensitivity of 90% (18/20), a specificity of 90% (9/10), a CA of 90% (27/30) and γof 0.8.There was a complementary role in combination of differential proteins in serum and BALF and the sensitivity, specificity and accuracy of diagnosis for lung cancer were 100% by parallel test.Conclusions The SELDI-TOF-MS technology can screen out the differential protein markers in serum and BALF of the patients with lung cancer, which show high sensitivity and specificity as tumor markers.The differential proteins in the BALF may be more promising for clinical application.     

表面增强激光解析电离飞行时间质谱法筛选肺癌患者血清和肺泡灌洗液中标志蛋白的研究

Objective To detect the protein markers in serum and bronchoalveolar lavage fluid (BALF) of the patients with lung cancer by surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) technology, and to explore if they can be used as markers for the diagnosis of lung cancer.Methods SELDI-TOF-MS technology and protein chips weak cation exchange (WCX-2 chip) were used to detect the protein mass spectrum in serum and BALF of 35 patients with lung cancer and 18 cases of benign pulmonary diseases.The different protein markers were analyzed by Biomarker Pattern Software and the initial diagnosis models were set up.The diagnosis models were verified further by blind screen to confirm the efficacy of diagnosis.Results Five protein peaks in the sera of the patients with lung cancer were significantly higher (P ＜ 0.05 ).The protein peak with a mass/charge ratio (M/Z)of 5639 was selected to establish the classification tree model.The sensitivity of diagnosis was 80% (28/35) and the specificity was 78% (14/18).The results verified by blind screen showed a sensitivity of 85% (17/20),a specificity of 90% (9/10), a crude accuracy (CA) of 87% ( 26/30 ) and Youden' s index (γ) of 0.7.Eight protein peaks in the BALF of the patients with lung cancer were significantly higher ( P ＜ 0.05).The different protein peaks with M/Z of 7976 and 11 809 respectively were selected to establish the classification tree model.The sensitivity of diagnosis was 86% (30/35) and the specificity was 72% (13/18).The results verified by blind screen showed a sensitivity of 90% (18/20), a specificity of 90% (9/10), a CA of 90% (27/30) and γof 0.8.There was a complementary role in combination of differential proteins in serum and BALF and the sensitivity, specificity and accuracy of diagnosis for lung cancer were 100% by parallel test.Conclusions The SELDI-TOF-MS technology can screen out the differential protein markers in serum and BALF of the patients with lung cancer, which show high sensitivity and specificity as tumor markers.The differential proteins in the BALF may be more promising for clinical application.     

肺结核患者强化治疗前后血清差异蛋白质质谱分析

目的研究肺结核患者强化治疗前后血清蛋白质谱的变化。方法利用弱阳离子(WCX)液体蛋白质芯片和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术,对16例肺结核患者强化治疗前后的血清蛋白质谱进行比较分析。结果强化治疗前后有22种蛋白质的相对含量有明显差异,11种蛋白质在强化治疗后相对含量明显升高(P0.05),另外11种蛋白质在强化治疗后相对含量明显降低(P0.05)。结论肺结核患者强化治疗前后血清蛋白质谱存在明显差异。     

自发性高血压大鼠血清蛋白质组学分析

目的探讨自发性高血压大鼠(SHR)蛋白质组学的发病机制。方法以SHR10只为模型组,Wistar Kyoto大鼠10只为正常组,取主动脉血,低温离心,分离血清,每组随机各取5份血清样品,用双向凝胶电泳、基质辅助激光解析电离飞行时间质谱测定肽质量指纹图谱,数据库检索鉴定各肽段对应的蛋白质,获取血清差异表达蛋白质的信息。结果 2组均获得重复性好的血清蛋白质双向凝胶电泳图谱。模型组和正常组凝胶上的平均蛋白点分别为(527±34)个、(476±30)个。通过分析蛋白质点局部图谱,获得23个差异表达蛋白点。与正常组比较,模型组4个点高表达,17个点低表达,2个点缺失。23个差异点经鉴定可以确认的蛋白质点有:线粒体膜蛋白、锌指蛋白、Rho GTPase、T细胞受体、视黄醇结合蛋白、假想蛋白。结论线粒体膜蛋白、锌指蛋白、T细胞受体、假想蛋白在SHR低表达;Rho GTPase、视黄醇结合蛋白在SHR高表达,推测这些蛋白可能与高血压的发生、发展密切相关。     

应用基质辅助激光解析电离飞行时间质谱法研究原发性胆汁性肝硬化的血清多肽谱

目的应用弱阳离子交换磁珠提取多肽结合基质辅助激光解析电离飞行时间质谱系统(MALDI-TOF-MS)分析原发性胆汁性肝硬化(PBC)患者的血清多肽谱,寻找具有潜在意义的血清标志物。方法收集105例血清样本,包括55例PBC患者、25例其他肝病患者和25例正常对照者血清样本,经弱阳离子交换磁珠纯化,MALDI-TOF-MS分析及ClinProTools生物信息学方法研究其血清多肽表达谱。结果在质荷比(m/z)1000～10 000 Da的范围内3组间共检测到158个血清多肽峰,其中83个有统计学意义(P<0.001),在PBC中表达上调的多肽有31个,表达下调的多肽有52个。组合m/z 1062.47、5356.13、4268.63、846.18、1945.31和6649.53Da 6个峰建立多肽诊断指纹图谱模型,该模型盲样验证的准确率在PBC组、正常对照组和其他肝病组分别为100%、81.8%和72.7%。结论可以利用MALDI-TOF-MS技术和ClinProTools软件来筛选PBC血清标志物,其在PBC诊断方面具有一定的价值。     

质谱仪在真菌鉴定中的应用与评价

目的探讨质谱仪在真菌感染中的应用,评价其鉴定真菌的能力。方法选取分离自我院住院患者的真菌菌株327株,用质谱仪进行快速鉴定,并与VITEK-2(酵母样真菌)和显微镜检查(丝状真菌)的鉴定结果进行比对,差异结果用分子生物学方法确认鉴定。结果依照质谱仪评分标准,227株酵母样真菌在种水平(2.0)的鉴定率为90.31%,在属水平(1.7)为98.68%;丝状真菌在种水平的鉴定率为74.00%,在属水平为94.00%。对临床常见的曲霉菌鉴定正确率可达96.74%。结论质谱仪在鉴定真菌的种属水平上都达到了令人满意的结果,尤其鉴定酵母菌和曲霉菌的能力更为突出,可作为临床实验室的常规检测方法。     

奈达铂耐药食管鳞癌患者血清双向电泳联合基质辅助激光解吸电离飞行时间质谱技术的蛋白筛选

目的探讨奈达铂耐药食管鳞癌患者及奈达铂敏感患者的血清蛋白表达谱的差异性及食管鳞癌奈达铂耐药的相关血清蛋白。方法采用固相p H梯度双向凝胶电泳分离奈达铂敏感及奈达铂耐药患者的血清蛋白样本共40例,建立双向凝胶电泳(2-DE)图谱,再应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术及数据库鉴定部分差异蛋白质。结果采用2-DE技术成功获得分辨率高和重复性好的蛋白表达图谱,凝胶图像软件分析后获得差异≥1.5倍以上的蛋白质共68个,经质谱分析鉴定出8种蛋白,其中表达增高的有谷胱甘肽转硫酶(GST)P1、乳酸脱氢酶(LDH)-B、Sox-2、胰岛素样生长因子结合蛋白(IGFBP)-1和基质金属蛋白酶(MMP)-7,表达降低的有E-钙黏蛋白(E-cadherin)、激活素结合蛋白(FS)和磷酸甘油酸激酶(PGK)1。结论应用2-DE技术分离奈达铂耐药食管鳞癌患者及奈达铂敏感患者的血清蛋白表达谱,并通过MALDITOF-MS技术鉴定出与食管鳞癌奈达铂耐药相关的蛋白,为食管鳞癌耐药相关标志物的筛选提供了新的技术方法及候选分子。     

Bruker Biotyper和Vitek MS系统质谱分析鉴定革兰阴性菌临床分离株的比较研究

目的比较并评价两种基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization time of flight mass spectrometry,MALDI-TOF-MS)系统——Bruker MALDI Biotyper系统(以下简称Bruker Biotyper系统)和MALDITOF Vitek MS系统(以下简称Vitek MS系统)在革兰阴性菌临床分离株鉴定中的应用。方法收集沈阳军区总医院2012年3月—2013年1月分离自血液、尿液、脑脊液、分泌物、伤口拭子和痰液临床标本的革兰阴性菌共120株。应用Vitek 2 Compact生化鉴定系统将每株细菌鉴定到种,而后采用Bruker Biotyper和Vitek MS系统对其进行鉴定,三者鉴定结果存在差异的菌株经16S rDNA测序最终确认菌种。结果对于120株革兰阴性菌临床分离株,Bruker Biotyper和Vitek MS系统在属的水平上正确鉴定率分别为95.0%和92.5%,在种的水平上正确鉴定率分别为89.2%和86.7%,差异均无统计学意义。结论 Bruker Biotyper和Vitek MS系统对革兰阴性菌临床分离株的正确鉴定率不存在差异,两种系统的鉴定结果与各自的图谱数据库有密切关系。     

帕金森病脑脊液蛋白质组学研究

目的寻找头痛和帕金森病(PD)患者用药前、后脑脊液差异蛋白质点,确定PD疾病特异性蛋白(DSPs)和药物治疗对蛋白质表达谱的影响。方法收集2004—2006年重庆医科大学附属第一医院神经内科收治的24例PD患者及12例对照组头痛患者脑脊液。用双向凝胶电泳(2D-PAGE)分离蛋白质,银染染色,分析图像寻找有意义的差异蛋白点;进行质谱(MS)鉴定。结果脑脊液蛋白质点主要集中在pH 4~7,头痛组与帕金森用药前后脑脊液电泳图谱蛋白质点分布基本一致,符合临床脑脊液生化常规检查,甲状腺激素结合蛋白、载脂蛋白E在PD患者用药前后相对于对照组都消失,血清转铁蛋白在PD患者用药前后相对于对照组都明显上调。结论用药前后PD患者脑脊液蛋白质点与头痛组脑脊液蛋白质点存在差异,药物治疗对PD患者脑脊液蛋白质表达谱影响不明显。     

溃疡性结肠炎相关性结肠癌差异表达的miRNA的筛选研究

[目的]分析结肠癌与正常结肠黏膜、结肠炎肠黏膜的微小RNA（miRNA）表达谱差异.[方法]通过建立溃疡性结肠炎（ulcerative colitis,UC）及UC相关性结肠癌（ulcerative colitis related colorectal cancer,UCRCC）小鼠模型,抽提、纯化RNA,加入荧光标记后与miRNA寡核甘酸基因芯片（Affymetrix公司）杂交,应用SAM软件进行数据分析,对有显著差异的miRNA进行实时荧光定量PCR验证,采用靶基因分析软件分析miRNA功能.[结果]has-miR-194、has-miR-215、has-miR-93、has-miR-192、has-miR-92a、has-miR-29b、has-miR-20a等7个miRNA在肿瘤组织中显著上调[中位假基因验出率（FDR）＜5％],has-miR-1231、has-miR-195、has-miR-143、has-miR-145等4个miRNA显著下调（FDR＜5％）.[结论]UCRCC与正常结肠黏膜、UC肠黏膜之间存在明显的miRNA差异表达,这些miRNA的差异性表达可能与UCRCC的发病有关.     

炎症性肠病患者血浆差异蛋白质的筛选

目的 应用表面增强激光解析电离飞行质谱技术(SELDI-TOF-MS)寻找溃疡性结肠炎(UC)和克罗恩病(CD)患者的血浆差异表达蛋白,建立筛选模型.方法 采用CM10芯片对24例UC、25例CD和25例正常对照者血浆进行分析,从蛋白表达图谱中发现差异蛋白,建立区分CD和正常对照、UC和正常对照、UC和CD以及炎症性肠病(IBD)和正常对照的决策树分析模型,并进行盲法筛选.结果 在质荷比(m/z)2000～30 000范围内,CD和正常对照之间、UC和正常对照之间、IBD和正常对照之间具有2倍以上差异的蛋白峰数分别为9个、5个和11个(P＜0.05),软件自动选取m/z为8208、8837的蛋白质建立区分CD和正常对照的决策树模型,m/z为6985的蛋白质建立区分UC和正常对照的决策树模型,m/z为8208、1752、28 840和1702的4个蛋白质建立IBD和正常对照的决策树模型,3个决策树模型的灵敏度分别为96%、82%、91%,特异度分别为100%、85%、100%.结论 m/z 8208对于CD筛选价值高,特异度好,值得进一步探索.     

应用蛋白质组学技术筛检肝癌血清差异蛋白质并分离鉴定阿朴脂蛋白AⅡ

目的 应用蛋白质组学技术筛选并鉴定肝癌患者血清中的差异蛋白质.方法 采用表面增强激光解析及电离飞行时间质谱技术,对33例肝癌患者和33例正常对照的血清蛋白质进行了检测分析,筛选肝癌的差异蛋白;用等电点沉淀法、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及高效液相色谱串联质谱等一系列方法分离鉴定差异蛋白质.肝癌组与正常组蛋白质峰差异比较采用两样本t检验. 结果 肝癌组与正常人组血清蛋白质图谱在相对分子质量为2 000～10 000范围内,有65个蛋白质峰差异有统计学意义(P<0.05),由此组建的诊断模型灵敏度为100%,特异度为96.97%其中包括8706.5 M/Z和8579.2M/Z(t值分别为2.562和2.783,P值均<0.05),经分离鉴定其为阿朴脂蛋白AⅡ. 结论 阿朴脂蛋白AⅡ是肝癌的差异蛋白之一,可能与肝癌的发生有关.     

血清多标志物蛋白质谱在原发性肝细胞癌诊断中的应用

HCC早期诊断和早期治疗是提高患者生存率的关键。表面增强激光解吸电离飞行时间质谱（SELDI-TOF-MS）技术是一项近年来新兴的临床蛋白组学实用技术,已被广泛地用于肿瘤标记物的筛查等研究。本研究比较了HCC患者和健康对照者血清蛋白质质谱,通过决策树分类技术筛选出一组特异的标志蛋白,建立HCC诊断模型,为HCC的早期预警和早期诊断提供更敏感可靠的指标。[第一段]     

高发区食管癌及癌前病变的血清蛋白质谱分析

目的 检测高发区食管癌及癌前病变患者血清蛋白质谱,建立蛋白指纹图谱模型并探究其筛查价值.方法 收集38名健康对照者、63例食管鳞状上皮不典型增生患者(I级26例,Ⅱ级26例,Ⅲ级11例)和36例进展期食管癌患者的内镜活检和血清标本,用CM10蛋白芯片及表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术检测标本的蛋白表达谱.支持向量机算法分别建立食管癌及癌前病变诊断模型,并经留一法交叉验证.结果 ①区分进展期食管癌和健康对照的诊断模型特异性为89.47%,敏感性为83.33%.②区分进展期食管癌和Ⅰ、Ⅱ、Ⅲ级不典型增生的诊断模型的特异性分别为92.31%、80.77%、90.91%,敏感性分别为80.56%、83.33%、94.44%.③在上述诊断模型中,质荷比(m/z)峰值在相对分子质量4291、5644、5664、8775处重复出现.结论 SELDI-TOF-MS技术和支持向量机算法的应用,为高发区高危人群中食管癌及癌前病变的早期筛查和诊断提供了一条新途径.4291、5644、5664、8775 4个质荷比峰对食管各级病变有相似的分类作用,可能是与食管癌变过程中相关的生物学标志物.     

Interleukin-17 SNPs and serum levels increase ulcerative colitis risk: A meta-analysis

AIM: To investigate the associations of interleukin-17 (IL-17) genetic polymorphisms and serum levels with ulcerative colitis (UC) risk.METHODS: Relevant articles were identified through a search of the following electronic databases, excluding language restriction: (1) the Cochrane Library Database (Issue 12, 2013); (2) Web of Science (1945-2013); (3) PubMed (1966-2013); (4) CINAHL (1982-2013); (5) EMBASE (1980-2013); and (6) the Chinese Biomedical Database (1982-2013). Meta-analysis was conducted using STATA 12.0 software. Crude odds ratios and standardized mean differences (SMDs) with corresponding 95% confidence intervals (CIs) were calculated. All of the included studies met all of the following five criteria: (1) the study design must be a clinical cohort or a case-control study; (2) the study must relate to the relationship between IL-17A/F genetic polymorphisms or serum IL-17 levels and the risk of UC; (3) all patients must meet the diagnostic criteria for UC; (4) the study must provide sufficient information about single nucleotide polymorphism frequencies or serum IL-17 levels; and (5) the genotype distribution of healthy controls must conform to the Hardy-Weinberg equilibrium (HWE). The Newcastle-Ottawa Scale (NOS) criteria were used to assess the methodological quality of the studies. The NOS criteria included three aspects: (1) subject selection: 0-4; (2) comparability of subjects: 0-2; and (3) clinical outcome: 0-3. NOS scores ranged from 0 to 9, with a score ≥ 7 indicating good quality.RESULTS: Of the initial 177 articles, only 16 case-control studies met all of the inclusion criteria. A total of 1614 UC patients and 2863 healthy controls were included in this study. Fourteen studies were performed on Asian populations, and two studies on Caucasian populations. Results of the meta-analysis revealed that IL-17A and IL-17F genetic polymorphisms potentially increased UC risk under both allele and dominant models (P < 0.001 for all). The results also showed that UC patients had higher serum IL-17 levels than healthy controls (SMD = 5.95, 95%CI: 4.25-7.65, P < 0.001). Furthermore, serum IL-17 levels significantly correlated with the severity of UC (moderate vs mild: SMD = 2.59, 95%CI: 0.03-5.16, P < 0.05; severe vs mild: SMD = 7.09, 95%CI: 3.96-10.23, P < 0.001; severe vs moderate: SMD = 5.84, 95%CI: 5.09-6.59, P < 0.001). The NOS score was ≥ 5 for all of the included studies. Based on the sensitivity analysis, no single study influenced the overall pooled estimates. Neither the Begger’s funnel plots nor Egger’s test displayed strong statistical evidence for publication bias (IL-17A/F genetic polymorphisms: t = -2.60, P = 0.019; serum IL-17 levels: t = -1.54, P = 0.141).CONCLUSION: The findings strongly suggest that IL-17A/F genetic polymorphisms and serum IL-17 levels contribute to the development and progression of UC.     

小鼠肝癌细胞H22膜蛋白组的双向电泳

目的 运用双向电泳及生物信息学技术,对小鼠肝癌细胞H22与正常肝细胞的膜蛋白进行比较和鉴定,以期为探索膜蛋白在肝癌发生,发展及侵袭转移过程中的作用提供实验依据.方法 运用顺序抽提法结合与双向电泳兼容的膜蛋白抽提试剂盒提取小鼠肝癌细胞H22和正常小鼠肝细胞膜蛋白,用考马斯亮蓝法定量,经双向凝胶电泳分离、银染,Image Master 2D Platinum软件分析,切取差异蛋白点,采用基质辅助激光解吸附飞行时间质谱技术检测,Aldente软件检索Swiss-Prot数据库进行匹配鉴定. 结果与正常小鼠肝细胞膜蛋白双向电泳图谱对比分析鉴定了8个在小鼠肝癌细胞H22中表达明显增强的膜蛋白,分别为硫酸酯酶修饰因子2,蛋白激酶C和肌酸激酶Ⅱ底物蛋白3,序列装配结构组件50同系物,巨噬细胞清道夫受体Ⅰ/Ⅱ,非特异蛋白C9或f135同系物,紧密结合蛋白ZO-2,3-羟基-3-甲基戊二酰辅酶A还原酶,空泡蛋白序列相关蛋白52同系物.结论 已鉴定的8个膜蛋白的生理功能涉及细胞代谢、细胞增殖、细胞信号转导、细胞骨架等,且在肝癌细胞H22中表达明显增强,提示小鼠肝癌细胞H22发生发展及侵袭转移可能涉及相关膜蛋白生理功能的改变.     

基质辅助激光解吸电离飞行时间质谱在结核病领域的应用进展

基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)具有高通量、高速度、自动化等优点,成为生物大分子研究的重要工具。其在临床研究领域取得显著进展,在结核病研究领域的作用也日益凸显。作者主要介绍了MALDI-TOF MS在分枝杆菌菌种鉴定、结核病耐药性研究、结核病血清标志物的筛选、结核病易感基因多态性检测和抗结核药物代谢研究方面的应用进展,以加强对MALDI-TOF MS在结核病研究领域的认识。     

白细胞介素在溃疡性结肠炎中的作用

溃疡性结肠炎(ulcerative colitis,UC)是一种以慢性肠道炎症为特征的炎症性肠病。UC的发病机制还未完全明确,其发生与免疫、遗传、环境及感染因素有关,细胞因子在UC等炎症性肠病中起到重要作用。白细胞介素(interleukin,IL)是细胞因子中最主要的具有多种生物活性的一组淋巴因子,并在免疫细胞的发育、分化、免疫应答等过程中有重要调节作用。目前研究发现很多IL在UC的发病中起重要作用。本文对在UC中起重要作用的IL作一概述。