SELDI-TOF-MS技术在肝癌诊断和介入治疗评价中的价值

目的:建立肝癌血清学诊断模型,探讨评估SELDI-TOF-MS技术在肝癌诊断和介入治疗评价中的价值.方法:用弱阳离子交换芯片(CM10芯片)和表面增强激光解吸电离飞行时间质谱仪(surface-enhanced laser desorption ionization time-of-flight mass spectrometry,SELDI-TOF-MS)技术,测定60例肝癌患者和60例正常对照者的血清蛋白质指纹图谱,应用BiomarkerWizard统计软件比较肝癌组和正常对照组血清蛋白质表达的差异性,采用Biomarker Pattern软件分析数据建立肝癌诊断模型,比较介入治疗前后血清蛋白质指纹图谱的差异性.结果:在质荷比(M/Z)为2000-10000范围内,和正常血清比较,肝癌的差异峰有3个(M/Z为4182Da、5710Da、6992Da;P<0.01),4182Da和5710Da下调,6992Da上调.用这3个差异蛋白峰建立肝癌诊断模型,诊断肝癌的灵敏度为93.3%(28/30),特异度为90.0%(27/30),正确率为91.7%(55/60),约登指数为0.833.差异蛋白峰(M/Z4182Da)在介入术后1mo明显上调(P<0.05).结论:应用SELDI-TOF-MS技术进行肝癌血清蛋白质指纹图谱分析,建立肝癌诊断树模型,对肝癌的诊断有一定的价值;筛选出的差异蛋白峰对肝癌的介入治疗评估有一定的应用价值.     

重症急性胰腺炎患者治疗前后血清蛋白组学的变化

目的 比较重症急性胰腺炎(SAP)患者治疗前后血清蛋白质组表达差异,寻找能够判断SAP病情好转的血清蛋白质.方法 利用表面增强激光解析离子化飞行时间质谱(surface-enhanced laser desorption/ionization time-of-flight mass spectrometry,SELDI-TOF-MS)分析20例SAP患者治疗前及治疗后7d的血清蛋白质谱,并进行APACHEⅡ评分.结果 SAP患者治疗后7d的APACHEⅡ评分为( 10.70±3.47)分,显著低于入院时的(13.00±2.21)分(t=4.272,P =0.002).治疗前后一共检测到血清中132个蛋白质峰,其中质荷比2000至40 000间有27个差异蛋白质峰(P＜0.05).分子质量47000的蛋白在SAP病情缓解后表达下调,分子质量115 000及159 000的蛋白在SAP治疗后明显上调.结论 用SELDl-TOF-MS技术能检出SAP患者治疗前后血清蛋白质的差异表达,分子质量115000和159000蛋白的上调及47000蛋白的下调可能是SAP病情好转的征兆.     

血清蛋白质组分析技术筛选肝癌自发抗体

目的采用血清蛋白质组分析技术(SERPA)筛选、鉴定肝癌自发抗体。方法双向电泳分离肝癌细胞系HCCLM3的总蛋白后将其转膜,肝癌、肝炎和正常组血清各8份与膜免疫印迹,图像分析确定不同血清免疫印迹图谱间的差异点及其与双向电泳图谱的对应关系,最后用基质辅助激光解吸飞行时间质谱进行鉴定。结果建立了高重复性HCCLM3的双向电泳图谱及其肝癌、肝炎及正常组血清的免疫印迹图谱,图谱均点数分别为603、70.75±24.25、68.50±23.44和41.38±15.05,肝癌、肝炎组图谱点数明显多于正常组,但肝癌与肝炎组差异无统计学意义。质谱鉴定确定了核蛋白、细胞骨架、代谢酶及热休克蛋白等五类肝癌自发抗体。结论SERPA是一种高通量筛选、鉴定肿瘤自发抗体的新技术,大量肝癌自发抗体的发现为肝癌进一步的免疫诊断及治疗奠定了基础。     

溃疡性结肠炎血清差异蛋白的筛选研究

目的 应用蛋白质组学寻找溃疡性结肠炎(UC)血清差异蛋白,初步探索UC可能的生物标志物.方法 收集UC患者30例和健康对照者30名的血清标本,双向凝胶电泳(2-DE)分离等量混合血清的蛋白质,运用图像分析软件进行比较和分析,识别差异表达蛋白质.应用基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)鉴定部分差异蛋白质点.结果 UC组和对照组之间年龄、体重指数、吸烟情况和饮酒量的差异均无统计学意义(P值均＞0.05).初步筛选出UC患者与健康对照者存在明显差异的39个蛋白点,选择其中9个点,经质谱分析发现触珠蛋白、热休克转录因子2、受体酪氨酸激酶、醛脱氢酶、载脂蛋白C-Ⅲ、中心粒旁物质1在UC患者中表达水平升高,角蛋白1、细丝蛋白A结合蛋白1、肌球蛋白3在UC患者中表达水平降低.结论 采用蛋白质组学2-DE和质谱技术,筛选并鉴定出与UC相关的9个血清蛋白质,为提供新的UC生物学行为研究分子标志物奠定基础.     

肝癌发生不同病理阶段血清蛋白质组的比较

目的 比较肝癌发生不同病理阶段血清蛋白质组的表达变化情况,从中寻找特异性标志物.方法 用双向凝胶电泳分离正常人,慢性肝炎、肝硬化和肝癌患者的血清蛋白质,结合质谱技术对差异点进行鉴定; Western blot检测结合珠蛋白β链以验证蛋白质水平的表达.计量资料数据以均数±标准差(x±s)表示,采用方差分析和LSD检验进行两两比较.结果 4组血清的双向电泳图谱经软件匹配分析,并与基质辅助激光解析电离飞行时间质谱相结合,成功鉴定出结合珠蛋白,SAA1、SP40等30个差异蛋白质点,K cluster聚类分析不同的变化模式.在差异蛋白质点中,7个蛋白质点均为结合珠蛋白,呈现由正常→肝炎→肝硬化持续下调、到肝癌又上调的模式.Western blot证实结合珠蛋白β链的表达与双向电泳表达相一致.结论 在肝癌发生的动态过程中各疾病阶段的血清蛋白质差异表达有4种明显变化模式,可能为肝癌的早期诊断和预后提供依据,与肝癌发生发展相关的结合珠蛋白很可能是肝硬化发展到肝癌的一个潜在早期诊断标志物.     

肝癌及癌旁肝组织的高尔基体差异蛋白质分析

目的 筛选并鉴定肝癌及癌旁肝组织高尔基体蛋白质组中差异表达的蛋白质,从高尔基体层面解释肝癌的发生和发展机制,为早期诊断和抗癌药物的研发提供线索.方法 应用亚细胞比较蛋白质组学研究方法,比较分析肝癌及癌旁肝组织高尔基体蛋白质组.收集肝癌患者手术切除的癌组织和癌旁肝组织标本,蔗糖密度梯度离心法分离高尔基体.建立并优化两种组织高尔基体的双向电泳方法,用PD-Quest软件分析差异表达的蛋白质点,然后用质谱仪获取相应蛋白点的肽质指纹图谱,联网到Swiss-Prot蛋白质组数据库鉴定获得的差异蛋白质点,最后用Western blot验证双向电泳结果.蛋白质相对表达量的比较采用配对t检验.结果 获得了分辨率和重复性均较好的双向电泳银染图谱.与癌旁肝组织相比,肝癌高尔基体蛋白质组有包括膜联蛋白5在内的27个蛋白质位点表达上调,包括染色体修饰蛋白2b在内的20个蛋白质位点表达下调,初步鉴定出了其中17个蛋白质,并用Western blot从蛋白质水平上验证了双向凝胶电泳结果.结论 肝癌及癌旁肝组织高尔基体蛋白质组具有不同的蛋白质位点,这些差异表达的蛋白质涉及到细胞的能量代谢、肿瘤的侵袭和转移,细胞周期调控等方面,为进一步阐释高尔基体在肿瘤的发生和发展中所起的作用提供了有价值的信息.     

基于Au蛋白芯片技术的狼疮肾炎患者尿蛋白标志物研究

目的 寻找狼疮肾炎(LN)患者尿蛋白标志物,探讨基于Au蛋白芯片技术预测LN的应用价值.方法 采用表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)技术及Au蛋白芯片检测166例LN患者和对照者尿蛋白指纹图,通过t检验评价蛋白表达差异性,筛选LN患者尿标志蛋白并结合人工神经网络(ANN)技术建立诊断模型,评价其预测LN的价值.对部分筛选的差异蛋白通过比对标准蛋白质谱数据进行初步鉴定.结果 LN患者与对照者尿中差异蛋白质峰有24个(t值为-6.44～10.14,P<0.05),通过BPS软件筛选4863、9744、8762、33 832、67 403和80 806质荷比(m/z)蛋白质建立的ANN模型预测LN的灵敏度为100%(26/26),特异度为95%(38/40).其中5个11 700、22 509、33 832、67 403和80 806 m/z蛋向与标准蛋白质谱比对显示可能为β_2-微球蛋白、视黄醇结合蛋白、α_1-微球蛋白、白蛋白和转铁蛋白.结论 基于Au蛋白芯片技术的尿蛋白指纹图谱检测在LN的早期警示、判断蛋白尿类型及指导免疫抑制剂的使用中具有潜在应用价值.     

巴马长寿人群血清长寿标记蛋白的鉴定及血脂分析

目的筛选广西巴马长寿老人的血清特异蛋白,并进行分离及鉴定,探讨相关脂蛋白与长寿的关系。方法以巴马长寿区长寿老人62例为长寿组,巴马非长寿区无长寿家族史的健康对照者41例为对照组,用表面加强激光解析电离化飞行-时间质谱检测2组人群的血清蛋白表达谱,并分析差异蛋白;一维电泳液相色谱-质谱技术分离并鉴定目的差异蛋白;再选择包括长寿组的入选者257例长寿老人为长寿老人组,包括对照组的入选者123例非长寿老人为非长寿对照组,检测2组HDL-C和TG水平。结果分子量在1000～10 000,共检测长寿组与对照组表达差异的蛋白有25个(P<0.05),在长寿组高表达的4个蛋白的相对表达量比值>2;对蛋白5336.81进行分离鉴定,其可能与血脂代谢密切相关的载脂蛋白(apo)C-Ⅱ酶解片段;长寿老人组的HDLC水平高于非长寿对照组(P<0.01),2组的TG水平比较,差异无统计学意义(P>0.05)。结论巴马长寿老人与巴马非长寿区对照人群的血清蛋白图谱存在差异;apoC-Ⅱ蛋白在巴马长寿老人中的适度高表达,可能是其出现动脉粥样硬化、冠心病、脑血栓等心脑血管疾病发生较少的原因之一。     

用飞行质谱技术筛选肝棘球蚴病患者血清蛋白质组学标记物

目的 筛选肝棘球蚴病患者血清蛋白质组学标记物,建立血清蛋白质指纹图诊断模型,评价其应用价值.方法 肝棘球蚴病患者68例,对照组73例(其他肝病患者33例,健康人40例),将全部样品分为训练组(37例)和测试组(67例).用弱阳离子交换蛋白芯片结合表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术检测肝棘球蚴病患者和对照者的血清蛋白质谱.分别比较训练组中37例肝棘球蚴患者和37例对照者、5例肝囊型棘球蚴病(HCE)患者和5例肝泡球蚴病(HAE)患者、8例肝棘球蚴病患者手术前后两组血清蛋白质峰的差异.利用ZJU-PDAS软件进行数据处理,通过支持向量机(SVM)运算建立蛋白质指纹图诊断模型,并用测试组标本采用盲法对模型的灵敏度和特异度进行验证.结果 肝棘球蚴病组和对照组共筛选出9个差异蛋白峰,其中相对分子质量为1044、1047、1073、1075、1338、6453、6649、8714 m/z的8个蛋白质峰在病例组中下调,相对分子质量为5651 m/z的蛋白峰在病例组中上调(P<0.05),盲法验证表明所建模型对肝棘球蚴病诊断的灵敏度为77.4%(24/31),特异度为66.7%(24/36);阳性预测值为66.7%(24/36),阴性预测值为77.4%(24/31).HCE和HAE患者之间筛选出2个差异表达蛋白峰,质荷比分别为8716和2751 m/z(P<0.05);8例肝棘球蚴病患者手术前后两组共筛选出6个差异表达蛋白质峰,相对分子质量分别为1297、1505、1525、1534、5921,5941 m/z(P<0.05).结论 SELDI-TOF-MS技术结合生物信息学方法能筛选出肝棘球蚴病患者血清蛋白质组学标记物,对肝棘球蚴病诊断及预后判断具有潜在的应用价值.     

早期肺鳞癌相关蛋白的筛选及其表达验证

目的应用蛋白质组学技术筛选早期肺癌癌变相关蛋白。方法利用双向凝胶电泳方法分离人早期肺鳞癌组织和相应癌旁正常组织总蛋白,选择差异蛋白质点进行质谱分析。免疫组织化学法验证部分差异蛋白质的表达差异。结果用DeCyder软件DIA模块分析显示,每块2-DE胶分离近1470左右个蛋白质点,再经过BVA模块分析,找出在9张图像中均出现,差异有统计学意义(P0.05)、两组间蛋白含量比值在1.5倍以上的差异性蛋白质点24个,成功鉴定14个。选择在癌组织中高表达的差异蛋白点进行质谱分析,最终鉴定为AnnexinⅡ、Cathcpsin D等蛋白质。免疫组织化学检测结果显示,AnnexinⅡ、Cathcpsin D蛋白在肺鳞癌组织中的阳性表达率均显著增高(P0.05)。结论蛋白质组学方法是一种应用于初步筛选早期肺癌相关蛋白的有效方法 ,所鉴定的蛋白为进一步筛选用于肺癌早期诊断及其治疗的分子标志物奠定了前期基础。     

Screening serum hepatocellular carcinoma-associated proteins by SELDI-based protein spectrum analysis

AIM： To find out potential serum hepatocellular carcinoma （HCC）-associated proteins with low molecular weight and low abundance 13y SELDI-based serum protein spectra analysis, that will have much application in the diagnosis or differentiated diagnosis of HCC, as well as giving a better understanding of the mechanism of hepato-card nogenesis.
METHODS： Total serum samples were collected with informed consent from 81 HCC patients with HBV（＋）/ cirrhosis（＋）, 36 cirrhosis patients and 43 chronic hepatitis B patients. Serum protein fingerprint profiles were first generated by selected WCX2 protein chip capture integrating with SELDI-TOF-MS, then normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard..Comparative analysis of the intensity of corresponding protein fingerprint peaks in normalized protein spectra, some protein peaks with significant difference between H.CC and cirrhosis or chronic hepatitis B were found.
RESULTS： One hundred and twenty-eight serum protein peaks betweeri.2000 and 30000Da were identified under the condition of signal-to-noise 〉 5 and minimum threshold for cluster 〉 20%. Eighty-seven of these proteins were showed significant differences in intensity between HCC and cirrhosis （P 〈 0.05）. Of the above differential proteins, 45 proteins had changes greater than two-fold, including 15 upregulated proteins and 30 downregulated proteins i.n HCC serum. Between HCC and chronic hepatitis B, 9 of 52 differential proteins （P 〈 0.05） had intensities of more than two-fold, including 2 upregulated proteins and 7 downregulated proteins in HCC serum. Between cirrhosis and chronic hepatitis B, 28 of 79 significant differential proteins （P 〈 0.05） changes greater than two-fold in intensity, including 17 upregulated proteins and 11 downregulated proteins in cirrhosis serum. For the analysis of these leading differential proteins in subtraction difference mode among three diseases, the five common downregulated proteins in HCC serum （M/Z 287     

肝细胞癌门静脉癌栓相关血清小分子量蛋白质标志物的筛选

目的筛选肝细胞癌门静脉癌栓相关的血清小分子量蛋白质标志物。方法收集健康志愿者、肝细胞癌无癌栓患者和肝细胞癌门静脉癌栓患者3组血清各l2份，用16％SDS-PAGE进行双向电泳，得到3组血清小分子量蛋白质表达图谱；经Image Master软件比对分析后，用基质辅助激光解析飞行时间质谱对差异点进行鉴定。结果在16％SDS-PAGE凝胶中，3×10^3～20×10^3区域内蛋白质条带间距较12．5％SDS-PAGE明显增宽，条带数目明显增多，且3组血清小分子量蛋白质表达图谱中均可清晰显示〈20×10^3的小分子量蛋白质斑点。组间比较显示，3组血清共有15个小分子量差异蛋白质点，经鉴定为5种蛋白质。与健康组比较，无癌栓组中载脂蛋白A-I、脂蛋白CⅢ、转甲状腺素蛋白和DNA拓扑异构酶Ⅱ表达降低，而结合珠蛋白-2表达增高；门静脉癌栓组5种蛋白质表达均较无癌栓组降低。结论在肝细胞癌的发生和发展过程中，血清小分子量蛋白质表达谱发生明显变化，差异表达的小分子量蛋白质有可能为肝细胞癌和门静脉癌栓早期预测及治疗监测提供参考。     

Surface enhanced laser desorption/ionization profiling: New diagnostic method of HBV-related hepatocellular carcinoma

Background and Aim:  To screen for serum biomarkers of HBV-related hepatocellular carcinoma (HCC) and HBV-related liver cirrhosis (LC) in an attempt to seek a new method for differential diagnosis of HCC and LC using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) techniques.
Methods:  Using SELDI-TOF-MS, serum proteins/peptide profiles on the immobilized metal ion affinity capture (IMAC) protein chips were obtained from 29 HCC patients and 30 LC patients. Discriminant analysis was carried out to establish new diagnostic methods using protein/peptide peaks with or without α-fetoprotein (AFP).
Results:  Forty-five protein/peptide peaks changed much more in the HCC group than they did in the LC group. Discriminant analysis using the Wilcoxon rank-sum test showed high sensitivity and specificity in distinguishing HCC from LC. The most significantly differentiating peak, 3892, offered 69.0% sensitivity, 83.3% specificity and 80% positive predictive value in distinguishing HCC and LC. Interestingly, six HCC patients with negative serum AFP were confirmed by peak 3892. The combination of multi-protein peaks (m/z = 9297, 29 941) with AFP offered an 82.8% sensitivity, 93.3% specificity and 92.3% positive predictive value, which was much better than AFP alone ( P  = 0.013).
Conclusions:  Special proteins/peptides of serum may differentiate HBV-related HCC and HBV-related LC, indicating that SELDI-TOF-MS may be useful to distinguish HCC from LC with the proper discriminant analytical method. SELDI peak 3892 may be a complementary diagnostic marker to positive AFP for HCC and a potential marker for the diagnosis of AFP-negative HCC as well.     

Screening and detection of portal vein tumor thrombi-associated serum low molecular weight protein biomarkers in human hepatocellular carcinoma

Purpose Serum low molecular weight protein biomarkers might be important in relation to portal vein tumor thrombi (PVTT) in hepatocellular carcinoma (HCC). This study aimed to screen and to detect these biomarkers. Methods We selected sera of 3 groups from 12 healthy volunteers, 12 HCC patients without PVTT and 12 HCC patients with PVTT, respectively. By using two-dimensional gel electrophoresis (2-DE) in which the first dimension was 16% SDS-PAGE, serum protein images of 3 groups were analyzed by Image Master Software. The differential protein spots were further identified by MALDI-TOF MS/MS. Results Compared with 12.5% SDS-PAGE gel, there were more protein bands between 3 and 20 kDa in 16% SDS-PAGE gel and low molecular weight (MW) protein spots (<20 kDa) were clearly shown. Fifteen differential protein spots representing five proteins were found in the three groups by inter-class comparison and were then identified. Compared with the healthy group, apolipoprotein A-I, lipoprotein CIII, transthyretin and DNA topoisomerase II were down regulated in HCC groups while haptoglobin-2 was over expressed. All the five proteins were less in PVTT group than in non-PVTT group. Conclusion The expression of low MW serum protein changes obviously in the beginning and progressive stage of HCC, and differentially expressed low MW proteins might be the potential biomarkers in early prognostication and surveillance of treatment for HCC and PVTT.     

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.     

Identification of two portal vein tumor thrombosis associated proteins in hepatocellular carcinoma: protein disulfide-isomerase A6 and apolipoprotein A-I

Background and Aim: Portal vein tumor thrombus (PVTT) is one of the factors that can affect prognosis and survival of hepatocellular carcinoma (HCC). In the present study, we aimed to find out some biomarkers associated with vascular invasion features of HCC with the method of comparative proteomic analysis. Methods: The proteins were extracted from a pair of HCC tissues with PVTT and without PVTT, and then separated by two‐dimensional polyacrylamide gel electrophoresis. Differentially expressed protein spots were identified by matrix assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Further analysis of two proteins were completed using real‐time fluorescence quantitative polymerase chain reaction and western‐blot in 40 HCC tissues with PVTT (n = 20) and without PVTT (n = 20). Results: Among 465 protein spots displayed on the gels, 33 unique proteins (> twofold change, P < 0.01) were identified, including 24 upregulated in HCC tissue without PVTT and nine upregulated in HCC tissue with PVTT. The real‐time fluorescence quantitative PCR showed no statistically significant difference between HCC tissues with PVTT and without PVTT for mRNA expressions of protein disulfide‐isomerase, A6 (PDI A6) (P = 0.137) and apolipoprotein A‐I (Apo A‐I) (P = 0.718). However, compared with HCC tissues without PVTT, protein expression of PDI A6 was higher in HCC tissues with PVTT (P < 0.001), while protein expression of Apo A‐I was lower in HCC tissues with PVTT (P = 0.012). Conclusions: PDI A6 and Apo A‐I are closely related to vascular invasion feature of HCC.     

WCX磁珠联合MALDI-TOF-MS技术在结直肠癌诊断中的应用

目的:比较结直肠癌与正常人血清蛋白质谱的变化,筛选特异性蛋白标志物,建立结直肠癌诊断分类树模型.方法:收集血清样本133例(其中结直肠癌67例,正常人66例),随机分为建模组和验证组.运用弱阳离子纳米磁珠(magnetic bead-weak cation exchange,MB-WCX)联合基质辅助激光解吸离子飞行质谱(matrix-assistedlaser desorption/ionization time-of-flight massspectrometry,MALDI-TOF-MS),建立结直肠癌与正常人血清蛋白质谱.用Flex Analysis2.4软件收集数据,应用ClinproTools2.2软件对建模组34例结直肠癌和33例正常人血清差异蛋白质谱进行定量分析,应用Genetic Algorithm算法建立结直肠癌诊断模型,应用所获取的诊断模型对验证组样本(33例结直肠癌和33例正常人)进行分类诊断,以评价诊断模型的诊断价值.结果:通过比较分析结直肠癌与正常人血清蛋白质谱,发现共有33个差异蛋白峰(P<0.05),其中在结直肠癌中表达上调25个,表达下调8个.利用其中5个差异峰(Mr分别为759,3316,4645,4248,2645Dr)建立诊断模型,获得了94.12%(32/34)敏感性和96.97%(32/33)的特异性,经独立样本双盲验证,其灵敏度为93.94%(31/33),特异度为96.97%(32/33).结论:基于磁珠分离和MALDI-TOF-MS技术能直接检测出结直肠癌患者血清差异表达蛋白,建立的诊断模型具有较高的敏感性和特异性,对提高结直肠癌的诊断具有一定的临床意义.     

Identification of a new marker of hepatocellular carcinoma by serum protein profiling of patients with chronic liver diseases

Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a proteomic technique that enables the profiling of proteins present in any biological material studied. We used this approach to identify new biomarkers of hepatocellular carcinoma (HCC) in the sera of patients with cirrhosis. Sera from 82 patients with cirrhosis, either without (n = 38) or with (n = 44) HCC, were analyzed by SELDI-TOF MS, and the results of the two groups were compared. The most efficient protein peaks leading to discrimination of patients with HCC were selected (receiver operative characteristic curves). The highest-scoring peak combination was established in a first group of serum samples (multinomial regression) and was tested in an independent group. The protein corresponding to the highest discrimination was purified and characterized further. The intensity of 30 protein peaks significantly differed between cirrhotic patients with and without HCC. An algorithm including the six highest-scoring peaks allowed correct classification (presence or absence of HCC) of 92.5% of patients in the test sample set and 90% in the validation sample set. The highest discriminating peak (8900 Da) was purified further and was characterized as the C-terminal part of the V10 fragment of vitronectin. An in vitro study suggested that the increase of the 8900-Da fragment in the serum of patients with HCC may proceed from the cleavage of native vitronectin with metalloproteases, a family of enzymes whose activity is enhanced in HCC. In conclusion, global protein profiling is an efficient approach that enabled us to identify a catalytic fragment ofvitronectin as a new serum marker of HCC in patients with chronic liver diseases.     

Early diagnostic potential for hepatocellular carcinoma using the SELDI ProteinChip system

Early detection of HCC increases the potential for curative treatment and improves survival. To facilitate early detection of HCC, this study sought to identify novel diagnostic markers of HCC using surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF/MS) ProteinChip technology. Serum samples were obtained from 153 patients with or without HCC, all of whom had been diagnosed with HCV-associated chronic liver disease. To identify proteins associated with HCC, serum samples were analyzed using SELDI-TOF/MS. We constructed an initial decision tree for the correct diagnosis of HCC using serum samples from patients with (n = 35) and without (n = 44) HCC. Six protein peaks were selected to construct a decision tree using this first group. The efficacy of the decision tree was then assessed using a second group of patients with (n = 29) and without (n = 33) HCC. The sensitivity and specificity of this decision tree for the diagnosis of HCC were 83% and 76%, respectively. For a third group, we analyzed sera from seven patients with HCC obtained before the diagnosis of HCC by ultrasonography (US) and from five patients free of HCC for the past 3 years. Use of these diagnostic markers predicted the diagnosis of HCC in six of these seven patients before HCC was clinically apparent without any false positives. CONCLUSION: Serum profiling using the SELDI ProteinChip system is useful for the early detection and prediction of HCC in patients with chronic HCV infection.