乙型肝炎病毒感染的肝细胞肝癌患者血液标志物筛选
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摘要
肝细胞肝癌(hepatocellular carcinoma,HCC)是全球最常见的肿瘤之一,其死亡率位居世界第三。每年在全球肝癌发病人群中,中国患者占将近一半,在中国的癌症死亡率中排名第二。乙型肝炎病毒(hepatitis virus B,HBV)感染是肝细胞肝癌的常见病因,中国是HBV感染的高发国家。从慢性乙型肝炎发展到HCC这一漫长过程中,患者往往缺乏特异性症状,HCC被诊断时往往已经是晚期,此时治疗措施有限。改善HCC患者预后、提高生存率的最根本措施是早期诊断从而早期治疗。但是肝癌的早期诊断是个难题,如何实施慢性乙型肝炎、乙肝后肝硬化到HCC这一演变过程的监测,尤其是寻找肝癌的血液肿瘤标志物,一直以来都是中国和世界的研究目标。
目前临床上最常用的肝癌标志物是甲胎蛋白(AFP),但是有相当一部分肝癌患者的AFP不升高,也有很多慢性肝炎和肝硬化患者出现AFP的升高,因此AFP的敏感性和特异性都无法让人非常满意。
人体血液内含有大量不同分子水平的化合物,从挥发性和非挥发性的小分子如有机酸、糖类,中等分子如多肽,一直到大分子的化合物如蛋白。这些化合物涉及并涵盖了人体代谢的各个途径,可以反应机体的运行状态如生长、健康或疾病,为疾病的发生、发展和预后评估提供了很好的生物标志物库源。而对这样庞大的生物信息库的检索工具必须是允许高通量数据获取和分析的技术平台。目前,“组学”研究包括基因组学、蛋白质组学与代谢组学是生命医学领域研究的热门前沿,其中涉及的基本技术如色谱、质谱等可以满足高通量数据获取和分析的要求。
本研究的主要目的是利用“组学”的基本技术,探讨各种方法获取和分析肝癌患者血液标志物,以此作为乙肝病毒感染人群的HCC监测指标。本研究的主要内容包括:基于气相色谱-质谱联用技术的乙型肝炎病毒感染的肝癌患者血液代谢组学研究及代谢组生物标志物的筛选(顶空固相微萃取法检测全血挥发性标志物、化学衍生法检测血清非挥发性标志物);基于气相色谱-质谱联用技术的肝癌高危人群:慢性乙型肝炎患者和乙型肝炎病毒感染后肝硬化患者的血清代谢组学研究(化学衍生法);基于气相色谱-质谱联用技术的代谢组学研究方法在生物标志物定量研究中的探讨(同位素标记微波衍生法);基于基质辅助激光解析电离飞行时间质谱技术的乙型肝炎病毒感染人群血清多肽谱研究和肝癌多肽生物标志物的筛选;基于人类抗体芯片技术的乙型肝炎病毒感染人群血清细胞因子谱的研究和肝癌细胞因子生物标志物的筛选;基于同位素标记相对和绝对定量技术和液相色谱-质谱联用技术的肝癌血清蛋白质组学检测方法在蛋白质组生物标志物定量研究中的探讨。
第一部分基于气相色谱-质谱联用技术的乙型肝炎病毒感染的肝癌患者血液代谢组学研究及代谢组生物标志物的筛选
一.肝细胞肝癌全血挥发性代谢组标志物检测(顶空固相微萃取法)
目的:采用项空固相微萃取的方法分离和浓聚肝细胞肝癌患者全血标本中的挥发性生物标志物,利用气相色谱-质谱的方法对其分离和鉴定,寻找有临床诊断价值的生物标记物。
方法:优化顶空固相微萃取的条件(包括萃取的纤维、萃取时间和温度、解吸附的时间和温度),选择最佳的条件对19例HBV感染的肝癌患者和18例正常对照的全血进行挥发性代谢产物的萃取,用气相色谱/质谱联用仪对其进行检测和分析,计算峰面积,采用卡方检验比较肝癌组和正常组二组出峰的不同,筛选二组差异大的物质作为诊断标记物。
结果:最佳的萃取纤维是CAR-PDMS,最佳的萃取时间和温度是60℃和15分钟,最佳的解吸附温度和时间是250℃和30秒。用最佳条件共检测出47种挥发性小分子代谢产物,其中19种物质在二组中的差别具有统计学意义(p＜0.05,卡方检验)。我们认为以下的三种生物标志物对肝癌诊断最具有临床意义:正己醛(敏感性和特异性为97.4%和100%),1-辛烯-3-醇(敏感性和特异性为84.2%和100%),辛烷(敏感性和特异性为89.5%和100%)。
结论:项空固相微萃取联合气相色谱-质谱检测的方法是一种简单、快速、灵敏且不需要特殊溶剂的检测肝癌全血挥发性生物标志物的方法。在最佳的检测条件下,肝癌组正己醛、1-辛烯-3-醇、辛烷的阳性率显著高于正常组,且这三种物质的敏感性和特异性满足临床诊断的需要。利用顶空固相微萃取联合气相色谱-质谱法可以作为肝癌诊断的新技术,测定的肝癌患者血液中的挥发性生物标志物可以作为新型的诊断标记物,有临床应用前景。
二.肝细胞肝癌血清非挥发性代谢组标志物检测(化学衍生法)
目的:以化学衍生法作为血清样本的前处理方法,通过气相色谱-质谱仪建立肝癌患者的血清代谢物谱,寻找肝癌患者的血清小分子代谢标志物。
方法:20例经过术后证实为HBV感染的原发性肝细胞肝癌患者(男性,39～59岁)为肿瘤组,20例正常体健者(男性,38～57岁)为正常组。取二组所有病例的血清,用甲醇水溶剂萃取后进行甲氧胺吡啶和N-甲基-N-(三甲基硅烷)三氟乙酰胺(MSTFA)的二步衍生化。用气相色谱质谱(GC-MS)联用仪对衍生化产物上样检测。用逐步判别分析法筛选小分子物质,建立判别方程,鉴别二组,采用支持向量机20折交叉验证法验证。
结果:所有样本经过衍生化后进入GC-MS检测出90余个峰,鉴定出的物质包含糖类、氨基酸、脂肪酸、胆固醇、小分子羧酸、尿素等。判别分析法筛选出有判别能力的物质有丁酸、乙亚胺酸、丙三醇、L—异亮氨酸、L—缬氨酸、氨基丙二酸、D—赤鲜糖、棕榈酸、硬脂酸、9,12-十八碳二烯酸,由此建立的判别方程可以完全分开二组,误判率为零。支持向量机法20折交叉验证判别分析结果的正确率达75%。
结论:气相色谱-质谱化学衍生法可以用来进行肝癌患者的血清小分子代谢产物谱的分析;肝癌患者的血清小分子代谢产物成分有异于正常者,可以通过逐步判别分析完全区分肿瘤组和正常组,支持向量机交叉验证的结果良好。
第二部分基于气相色谱-质谱联用技术的肝癌高危人群:慢性乙型肝炎患者和乙型肝炎病毒感染后肝硬化患者的血清代谢组学研究(化学衍生法)
目的:以化学衍生法作为血清样本的前处理方法,通过气相色谱-质谱仪建立和区分肝癌的高危人群慢性乙型肝炎和肝硬化患者的血清代谢物谱。
方法:获取乙型肝炎病毒感染的20例慢性肝炎和20例肝硬化病例的血清,用甲醇水溶剂萃取后进行甲氧胺吡啶和N-甲基-N-(三甲基硅烷)三氟乙酰胺(MSTFA)的二步衍生化。用气相色谱质谱(GC-MS)联用仪对衍生化产物上样检测。用逐步判别分析法对所得的代谢组学数据进行分析。
结果:由9个代谢标志物组成的判别方程可以对慢性肝炎组做出100%的正确诊断,对肝硬化患者的诊断准确率为75%。这9个代谢物为乙酸、山梨醇、D-乳酸、己酸、1-萘胺、丁酸、磷酸、D-葡糖醇和葡萄糖。
结论:气相色谱-质谱化学衍生法可以用来进行肝癌高危人群慢性乙型肝炎和肝硬化患者的血清小分子代谢产物谱分析;慢性乙型肝炎患者的血清小分子代谢产物成分有异于肝硬化患者,逐步判别分析选择的标志物可以用来区分肝炎和肝硬化病例。
第三部分基于气相色谱-质谱联用技术的代谢组学研究方法在生物标志物定量研究中的探讨(同位素标记微波衍生法)
目的:利用稳定性同位素标记和快速微波衍生的方法对血清中的葡萄糖和氨基酸进行精确定量,应用于观察吡咯喹啉醌(PQQ)对良性肝病(酒精性肝损大鼠模型)的血清葡萄糖和异亮氨酸代谢水平的影响。
方法:将30只雄性大鼠分为对照组、酒精组和PQQ组,酒精组和PQQ组给予胃内灌注酒精4周,结束后PQQ组给予胃内灌PQQ共4周。建立模型后采集全血并分离血清,对血清用~(13)C葡萄糖和~(15)N异亮氨酸同位素进行标记、用甲醇进行蛋白沉淀、用衍生化试剂MSTFA进行微波衍生,最后用气相色谱-质谱的方法同步测定大鼠体内血糖和异亮氨酸水平,三组进行比较,结果进行t检验。
结果:通过微波衍生,葡萄糖和异亮氨酸的硅烷化能在3分钟内完成。酒精组大鼠血糖水平高于对照组(10.89±0.93mmol/L vs 8.25±1.44 mmol/L,p＜0.01)。PQQ组大鼠的血糖水平低于酒精组(9.20±0.87 mmol/L vs 10.89±0.93 mmol/L,p＜0.01)。酒精组异亮氨酸水平明显高于对照组(132.54±6.51 umol/L vs 107.83±11.72umol/L,p＜0.001),而异亮氨酸水平在酒精组和PQQ组无显著差异。
结论:通过气相色谱-质谱微波衍生和同位素标记法,可以快速、精确、定量测定浓度范围跨度很大的血清葡萄糖和异亮氨酸水平。慢性的酒精暴露可以导致大鼠血清葡萄糖和异亮氨酸的升高,PQQ能恢复酒精暴露导致的高血糖水平,异亮氨酸的代谢不受PQQ的影响。
第四部分基于基质辅助激光解析电离飞行时间质谱技术的乙型肝炎病毒感染人群血清多肽谱研究和肝癌多肽生物标志物的筛选
目的:探讨以磁性微粒富集血清多肽和基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionisation time-of-flight massspectrometry,MALDI-TOF MS)为基础的血清多肽谱检测方法是否可以区分不同的乙肝病毒感染患者,进一步筛选肝细胞肝癌的多肽生物标志物。
方法:用磁性微粒对94例肝细胞肝癌患者、80例慢性乙型肝炎患者和24例正常体检者的血清进行多肽的富集,用MALDI-TOF MS对富集物进行分离、鉴定。对所得的高通量数据应用遗传算法(genetic algorithm,GA),在30例肝癌、30例肝炎和24例正常者中建立肝癌的诊断模型,用64例肝癌和50例肝炎的数据对建立的肝癌模型进行验证。肝癌模型中的多肽用液相色谱-质谱质谱(liquid chromatography-massspectrometry mass spectrometry)联用技术进行进一步的身份鉴定。第二步,同样的方法额外检测乙肝病毒感染的二组人群包括乙肝病毒健康携带者(n=22)和乙肝感染后肝硬化患者(n=23)的血清多肽谱,与同一时间段额外收集的25例正常体检者、25例慢性乙型肝炎、25例肝癌患者的血清多肽谱进行比较。
结果:第一步中,三组血清多肽谱区别明显,建立二个肝癌诊断模型用于区分肝癌和正常以及肝癌和肝炎。二个模型的疾病总识别率分别为96.25%和93.33%,在验证病例中,模型对肝癌的正确诊断率分别为98.4%和76.6%。经过鉴定,肝癌的标志物包括凝血酶原前体片段、钙激活分泌蛋白-1、凋亡重复序列6抑制因子等。第二步额外检测的乙型肝炎病毒携带者和肝硬化患者的血清多肽谱明显区分于正常、慢性肝炎和肝癌患者。
结论:磁性微粒富集多肽和MALDI-TOF MS技术可以应用于乙肝感染人群的血清多肽谱研究,而且可以区分不同的乙肝感染人群;此技术适合于寻找肝癌的多肽标志物、用于乙肝病毒感染人群的肝癌监测。
第五部分基于人类抗体芯片技术的乙型肝炎病毒感染人群血清细胞因子谱的研究和肝癌细胞因子生物标志物的筛选
目的:利用抗体芯片技术检测肝细胞肝癌患者的血清细胞因子,寻找肝癌特异性细胞因子作为诊断标志物。
方法:利用Raybio~(?)生物素标记的抗体芯片Ⅰ对正常体检者(n=3)、慢性乙型肝炎患者(n=3)和肝细胞肝癌患者(n=3)进行507个人体细胞因子的差异初筛。30个组间差异最大同时组内差异最小的细胞因子被用来做扩大人群验证。验证人群是30例正常体检者,30例肝炎和40例肝细胞肝癌患者。选择one-way ANOVA和对角线判别分析来筛选肝癌的细胞因子标记物。
结果:one-way ANOVA的筛选结果示肝癌患者血清细胞因子表达有异于肝炎和正常的是IGFBP-7,MDC,MSPα链和MSPβ链。对角线判别分析筛选的肝癌细胞因子标记物MDC和MSPα链与AFP共同构建了肝癌诊断模型,其敏感性为73.2%,特异性为95%,工作受试者曲线下面积是0.89。
结论:抗体芯片是一项高敏感、高特异性的血清细胞因子检测技术,可以用来肝细胞肝癌的细胞因子标志物的筛选。
第六部分基于同位素标记相对和绝对定量技术和液相色谱-质谱联用技术的肝癌血清蛋白质组学检测方法在蛋白质组生物标志物定量研究中的探讨
目的:将同位素标记相对和绝对定量(isobaric tags for relative and absolutequantitation,iTRAQ)联合液相色谱质谱技术应用于肝细胞肝癌患者血清定量蛋白质组学研究,并初步探讨该检测方法对肝癌蛋白质组生物标志物的相对定量研究的应用前景。
方法:15例乙型肝炎病毒感染的原发性肝细胞肝癌患者和15例慢性乙型肝炎患者,收集血清分二组混合。选用高丰度蛋白分离系统(Multiple Affinity RemovalSystem,MARS)除血清高丰度蛋白、收集低丰度蛋白,用SDS-PAGE对原血清和收集的低丰度蛋白样品进行效果评估。对蛋白进行胰蛋白酶酶解后用iTRAQ试剂114和117分别标记肝癌和肝炎血样。用强阳离子交换色谱(Strong cation exchangechromatography,SCX)对标记好的多肽分层后用LC-ESI-MS/MS进行检测。选用蛋白分析软件ProteinPilot比较二组混合血清的差异蛋白。
结果:经过MARS系统去高丰度蛋白,血清样品中的低丰度蛋白得到很好的分离;经过SCX分层共40个多肽层,进入MS质谱共得到49784个多肽片段,谱库搜索的蛋白共251个,其中二组的差异度超过1.5倍的共21个蛋白。
结论:同位素标记相对和绝对定量技术和液相色谱-质谱联用技术可以用于肝癌血清蛋白质组学相对定量检测,慢性乙型肝炎和肝细胞肝癌患者的血清蛋白表达明显差异。
Liver cancer is the sixth most common cancer and the third leading cause of cancer death worldwide.Most liver cancer patients are in developing countries,among whom,Chinese account more than a half.Most liver cancers are hepatocellular carcinomas(HCC),the major risk factors for which are chronic infections with the hepatitis virus B and C.As hepatitis B virus(HBV) is more prevalent in China,we have put much attention into the disease course of HBV infection.During the long-term disease progression from chronic HBV infection to liver cirrhosis and HCC,patients are lack of characteristic symptoms.It is often late stage when HCC is diagnosed.Treatment for such patients is limited with the five year survival rate lower than 5%.In light of the high burden of HBV chronic infection,it is significant to identify reliable,reproducible,and noninvasive biomarkers or a surveillance model for HCC targeting at the HBV infected population.The most widely used serum biomarker for HCC has been alpha fetoprotein(AFP).As up to 40%of HCC patients present with normal AFP levels and elevated AFP levels are observed in patients with chronic hepatitis or cirrhosis,the low sensitivity and specificity of AFP has always been called in question.However,there has been no other serum biomarkers which could be accepted as effective as AFP.
Human blood contains a wide variety of chemically diverse molecular weight compounds ranging from volatile and nonvolatile low molecular ones such as organic acids and carbohydrates,to middle molecular ones such as polypeptides, and to large molecular ones such as proteins.These compounds differ against complex interactions between genetic factors,pathogen infection,toxicant exposure,and so on during disease process.They constructed a huge biomarker bank which could be applied for disease sceening,progression or prognosis evaluation.Techniques allowing high-flux data aquisition and analyzing are needed for researching in this huge biomarker bank.In the present,"omics" studies including genomics,proteomics and metabolomics are the newest in the biomedical field with the most rapid developed techniques such as chromatography or massspectrometry meeting the need of high-flux data aquisition and analyzing.
The main purpose of this study is to explore new biomarkers for HCC from the HBV infected groups applying different techniques on the base of "omics" research.The contents are:the metabolomic research of HBV infected HCC patients based on Gas chromatography/Mass Spectrometry(GC/MS) and identification of HCC blood biomarkers applying the techniques of solid-phase microextraction and chemical derivatization;the serum metabolomic research in the HCC high risk groups:chronic hepatitis B patients and HBV infected cirrhosis patients based on GC/MS applying the technique of chemical derivatization;an exploration in the quantification of serum metabolomic biomarkers based on GC/MS applying the technique of microwave-assisted derivatization and isotope dilution;serum proteomic surveillance of hepatocellular carcinoma from hepatitis B virus infected groups with magnetic bead-based MALDI-TOF/MS;identification of new serum biomarkers for hepatocellular carcinoma using biotin-label-based antibody microarrays;a pilot study of identification and quantification of serum protein biomarkers in HCC based on the technique of isotope tagging for relative and absolute protein quantitation(iTRAQ).
Part One The metabolomic research of HBV infected HCC patients based on Gas chromatography/Mass Spectrometry(GC/MS) and identification of HCC blood biomarkers applying the techniques of solid-phase microextraction and chemical derivatization
Section 1.Investigation of volatile biomarkers in HCC blood using solid-phase microextraction and gas chromatography-mass spectrometry
Objective:To explore the technique of solid-phase microextraction(SPME) and gas chromatography-mass spectrometry(GC-MS) in the investigation of blood volatile biomarkers for HCC.
Methods:The fiber,duration,temperature of extraction and the duration, temperature of desorption was tested and optimized to extact the blood volatile metabolites in 19 HCC patients and 18 normal controls.GC-MS was applied in separate,detect and identificate the extracted metabolites.The peak area values were gathered and compared between groups.Chi square test was used to screen the HCC biomarkers.
Results:The best extraction fiber was CAR-PDMS.The optimal extraction duration,temperature was 15min and 60℃.The optimal extraction duration, temperature was 30s and 250℃.There were 47 volatile low molecular metabolites detected,19 of which were detected differently in the two groups(p＜0.05). Positive rates of and hexanal(sensitivity 97.4%,specificity 100%), 1-octen-3-ol(sensitivity 84.2%,specificity 100%) and octane(sensitivity 89.5%,specificity 100%)in HCC blood were found to be much higher than those in control blood.The three molecules of hexanal,1-octen-3-ol and octane were regarded as biomarkers of HCC with clinical diagnostic value.
Conclusion:SPME/GC-MS is a simple,rapid and sensitive method very suitable for investigation of volatile disease markers in human blood.
Section 2.Investigation of nonvolatile biomarkers in HCC serum using chemical derivatization and gas chromatography-mass spectrometry
Objective:To explore the technique of chemical derivatization and gas chromatography-mass spectrometry(GC-MS) in the investigation of serum nonvolatile biomarkers for HCC.
Methods:Serum metabolome was detected through chemical derivatization followed by gas chromatography/mass spectrometry(GC/MS).The serum metabolic difference was compared between hepatocellular carcinoma(HCC,n=20) male patients and normal control male subjects(n=20).The acquired GC/MS data was analyzed by stepwise discriminant analysis(SDA) and support vector machine(SVM).
Results:The metabolites including butanoic acid,ethanimidic acid, glycerol,L-isoleucine,L-valine,aminomalonic acid,D-erythrose, hexadecanoic acid,octadecanoic acid,and 9,12-octadecadienoic acid in combination with each other gave the strongest segregation between the HCC group and the control group.By applying these variables,our method provided a diagnostic model that could well discriminate between HCC and normal subjects. More important,the error count estimate for each group was 0%.The total classifying accuracy of the discriminant function tested by SVM 20-fold cross validation was 75%.
Conclusion:This technique is different from traditional ones and appears to be a useful tool in the area of HCC diagnosis.
Part Two Serum metabolomic study of two groups of cases with high risk of HCC:hepatitis B virus infected chronic hepatitis and cirrhosis patients by chemical derivatization and Gas chromatography/Mass spectrometry
Objective:To investigate the serum metabolic profile of hepatitis B virus (HBV)infected chronic hepatitis and cirrhosis patients.
Methods:HBV infected non-cirrhosis male subjects(n=20) and HBV infected cirrhosis male patients(n=20) entered this experiment.Serum metabolome was detected through chemical derivatization followed by GC/MS.The high-flux metabolomic data was analyzed by stepwise discriminant analysis(SDA).
Results:From the 41 metabolites detected in serum,we selected metabolites including acetic acid,sorbitol,d-lactic acid,hexanoic acid, 1-naphthalenamine,butanoic acid,phosphoric acid,d-glucitol,glucose which in combination with each other could segregate the two groups.The error count was 0%for the non-cirrhosis group and 25%for the cirrhosis group.
Conclusion:Chemical derivatization and GC/MS technique is applicable in metabolomic profiling in patients with HCC risk.
Part Three An exploration for precise quatification of serum metabolomic biomarkers applying microwave-assisted derivatization and isotope dilution GC/MS
Objective:To precisely quatificate serum glucose and isoleucine levels applying microwave-assisted derivatization and isotope dilution GC/MS in the investigation of the effect of pyrroloquinoline quinine(PQQ) on rats with alcohol assumption.
Methods:Thirty male Sprague-Dawley rats divided into three groups.The first group was a normal control which received intragastric administration of saline for 8 weeks.Alcohol was administered intragastrically to the alcohol group and PQQ group for four weeks.PQQ was administered intragastrically to rats in the PQQ group at for another four weeks.Serum metabolites including glucose,isoleucine,the[U-~(13)C_6]-glucose and[~(15)N]-isoleucine were rapidly derivatized by N-Methyl-N-trimethylsilyltrifluoroacetamide(MSTFA) with microwave irradiation,and the trimethylsilyl derivatives were analyzed by GC/MS.T test was applied for comparison between the groups.
Results:Derivatization of was completed within 3min.Serum glucose levels were higher in alcohol group than in normal group(10.89±0.93mmol/L vs 8.25±1.44 mmol/L,p＜0.01).Serum glucose levels were lower in PQQ group than in alcohol group(9.20±0.87 mmol/L vs 10.89±0.93 mmol/L,p＜0.01).Serum isoleucine levels were higher in alcohol group than in normal group (132.54±6.51 umol/L vs 107.83±11.72 umol/L,p＜0.001).There was no difference of serum isoleucine levels between PQQ group and alcohol group
Conclusion:The combination of MAD and ID GC/MS has shown to be an accurate, rapid,simple and sensitive method for the quantification of glucose and isoleucine in serum samples.Serum glucose and isoleucine metabolism were affected by alcohol.PQQ could reverse alcohol exposure induced glucose elevation.It did not affect the metabolism of isoleucine whose level was elevated along with serum glucose.
Part Four Serum proteomic surveillance of hepatocellular carcinoma from hepatitis B virus infected groups and identification of HCc biomarkers with magnetic bead-based MALDI-TOF/MS
Objective:To determine whether serum proteomic profiling based on magnetic bead(MB) and matrix-assisted laser desorption/ionisation time-of-flight(MALDI-TOF) mass spectrometry(MS) can discriminate between hepatocellular carcinoma(HCC) and other HBV infected groups.To identify proteomic biomarkers for HCC.
Methods:One hundred ninty-eight serum samples from 80 chronic hepatitis B,94 HBV infected HCC patients and 24 normal cases were analyzed by MALDI-TOF MS after peptides enrichment by MBs.Applying genetic algorithm,diagnotic models for HCC were built between 30 HCC patients and 24 normal cases/30 hepatitis patients.Validations were done with the left cases.Markers in the models were identified through LC/MS MS.Data from 22 HBV carriers and 23 HBV-infected cirrhosis patients was laterly gathered to be separated from additional 25 chronic hepatitis B patients,25 HCC patients and 25 normal controls.
Results:The first three groups were well separated from each other.Two discrimination models were built for HCC.The overall recognition capability of the two models was 96.25%and 93.33%.The validation of them in the left patients showed misdiagnosis rates of 1.6%and 23.4%for HCC.Potential biomakers for HCC were identified as prothrombin precursor(fragment),isoform 1 of calcium-dependent activator protein for secretion,baculoviral IAP repeat-containing 6,et al.The laterly added two groups were also well separated from others.
Conclusion:MB based MALDI-TOF MS approach is applicable in identifying serum proteomic biomarkers that can be used in the surveillance of HCC from HBV infected population.
Part Five Identification of new serum biomarkers for hepatocellular carcinoma using biotin-label-based antibody microarrays
Objective:Searching for hepatocellular carcinoma(HCC) biomarkers applying antibody arrays.
Methods:In this study,serum cytokine detection using RayBio~(?) Biotin Label-based Human Antibody ArrayⅠ(507 human proteins) was applied in normal cases(n=3),chronic hepatitis B patients(n=3) and HCC patients(n=3).Thirty proteins which were the most differently detected ones between HCC patients and normal/hepatitis cases were validated in additional 30 normal,30 hepatitis and 40 HCC cases.Biomarkers for HCC were selected through one-way ANOVA and diagonal linear discriminant analysis.
Results:A diagnostic model constructed by two HCC biomarkers(MDC,MSPαchain)together with AFP had a sensitivity of 73.2%,specficity of 95%for HCC diagnosis.
Conclusion:Antibody microarrays are applicable in searching for new biomarkers for HCC.
Part Six Quantification of HCC serum proteomic biomarkers applying the methods of isobaric tags for relative and absolute quantitation(iTRAQ)and Liquid Chromatography/Mass Spectrometry
Objective:To apply the methods of isobaric tags for relative and absolute quantitation(iTRAQ)and Liquid chromatography-Mass spectrometry to hepatocellular carcinoma(HCC) patients in the preliminary study of relative quantified detection of HCC proteomic biomarker.
Methods:15 cases of hepatitis B virus infected HCC and 15 patients with chronic hepatitis B were recruited.Sera were collected and pooled within the same group.Multiple Affinity Removal System(MARS) was used for high-abundance serum proteins depletion and low-abundance proteins collection. SDS-PAGE was applied to assess the effect of MARS with the original sera and the low-abundance protein samples.After protein digestion by trypsin,iTRAQ reagents 114 and 117 were marked on peptides of HCC and hepatitis patients respectively.After being handled by Strong cation exchange chromatography (SCX),marked peptides were tested by LC-ESI-MS/MS.The software ProteinPilot was selected to make a comparison between the two different serum protein mixture.
Results:With the depletion of high-abundance proteins by MARS system, low-abundance proteins have been well collected.There were 40 fractions after SCX handling,resulting in a total of more than 49,784 peptide fragments detected in the MS-MS system.Two hundred and fifty one proteins were identified through online spectra library search,twenty one of them presented quitely differently(more than 1.5 times the difference) in the two different serum pools.
Conclusion:ITRAQ and liquid chromatography-mass spectrometry can be used for relative quantified detection of HCC proteomic biomarkers. There are significant difference of serum protein levels between patients with chronic hepatitis B and HCC.
目前临床上最常用的肝癌标志物是甲胎蛋白(AFP),但是有相当一部分肝癌患者的AFP不升高,也有很多慢性肝炎和肝硬化患者出现AFP的升高,因此AFP的敏感性和特异性都无法让人非常满意。
人体血液内含有大量不同分子水平的化合物,从挥发性和非挥发性的小分子如有机酸、糖类,中等分子如多肽,一直到大分子的化合物如蛋白。这些化合物涉及并涵盖了人体代谢的各个途径,可以反应机体的运行状态如生长、健康或疾病,为疾病的发生、发展和预后评估提供了很好的生物标志物库源。而对这样庞大的生物信息库的检索工具必须是允许高通量数据获取和分析的技术平台。目前,“组学”研究包括基因组学、蛋白质组学与代谢组学是生命医学领域研究的热门前沿,其中涉及的基本技术如色谱、质谱等可以满足高通量数据获取和分析的要求。
本研究的主要目的是利用“组学”的基本技术,探讨各种方法获取和分析肝癌患者血液标志物,以此作为乙肝病毒感染人群的HCC监测指标。本研究的主要内容包括:基于气相色谱-质谱联用技术的乙型肝炎病毒感染的肝癌患者血液代谢组学研究及代谢组生物标志物的筛选(顶空固相微萃取法检测全血挥发性标志物、化学衍生法检测血清非挥发性标志物);基于气相色谱-质谱联用技术的肝癌高危人群:慢性乙型肝炎患者和乙型肝炎病毒感染后肝硬化患者的血清代谢组学研究(化学衍生法);基于气相色谱-质谱联用技术的代谢组学研究方法在生物标志物定量研究中的探讨(同位素标记微波衍生法);基于基质辅助激光解析电离飞行时间质谱技术的乙型肝炎病毒感染人群血清多肽谱研究和肝癌多肽生物标志物的筛选;基于人类抗体芯片技术的乙型肝炎病毒感染人群血清细胞因子谱的研究和肝癌细胞因子生物标志物的筛选;基于同位素标记相对和绝对定量技术和液相色谱-质谱联用技术的肝癌血清蛋白质组学检测方法在蛋白质组生物标志物定量研究中的探讨。
第一部分基于气相色谱-质谱联用技术的乙型肝炎病毒感染的肝癌患者血液代谢组学研究及代谢组生物标志物的筛选
一.肝细胞肝癌全血挥发性代谢组标志物检测(顶空固相微萃取法)
目的:采用项空固相微萃取的方法分离和浓聚肝细胞肝癌患者全血标本中的挥发性生物标志物,利用气相色谱-质谱的方法对其分离和鉴定,寻找有临床诊断价值的生物标记物。
方法:优化顶空固相微萃取的条件(包括萃取的纤维、萃取时间和温度、解吸附的时间和温度),选择最佳的条件对19例HBV感染的肝癌患者和18例正常对照的全血进行挥发性代谢产物的萃取,用气相色谱/质谱联用仪对其进行检测和分析,计算峰面积,采用卡方检验比较肝癌组和正常组二组出峰的不同,筛选二组差异大的物质作为诊断标记物。
结果:最佳的萃取纤维是CAR-PDMS,最佳的萃取时间和温度是60℃和15分钟,最佳的解吸附温度和时间是250℃和30秒。用最佳条件共检测出47种挥发性小分子代谢产物,其中19种物质在二组中的差别具有统计学意义(p＜0.05,卡方检验)。我们认为以下的三种生物标志物对肝癌诊断最具有临床意义:正己醛(敏感性和特异性为97.4%和100%),1-辛烯-3-醇(敏感性和特异性为84.2%和100%),辛烷(敏感性和特异性为89.5%和100%)。
结论:项空固相微萃取联合气相色谱-质谱检测的方法是一种简单、快速、灵敏且不需要特殊溶剂的检测肝癌全血挥发性生物标志物的方法。在最佳的检测条件下,肝癌组正己醛、1-辛烯-3-醇、辛烷的阳性率显著高于正常组,且这三种物质的敏感性和特异性满足临床诊断的需要。利用顶空固相微萃取联合气相色谱-质谱法可以作为肝癌诊断的新技术,测定的肝癌患者血液中的挥发性生物标志物可以作为新型的诊断标记物,有临床应用前景。
二.肝细胞肝癌血清非挥发性代谢组标志物检测(化学衍生法)
目的:以化学衍生法作为血清样本的前处理方法,通过气相色谱-质谱仪建立肝癌患者的血清代谢物谱,寻找肝癌患者的血清小分子代谢标志物。
方法:20例经过术后证实为HBV感染的原发性肝细胞肝癌患者(男性,39～59岁)为肿瘤组,20例正常体健者(男性,38～57岁)为正常组。取二组所有病例的血清,用甲醇水溶剂萃取后进行甲氧胺吡啶和N-甲基-N-(三甲基硅烷)三氟乙酰胺(MSTFA)的二步衍生化。用气相色谱质谱(GC-MS)联用仪对衍生化产物上样检测。用逐步判别分析法筛选小分子物质,建立判别方程,鉴别二组,采用支持向量机20折交叉验证法验证。
结果:所有样本经过衍生化后进入GC-MS检测出90余个峰,鉴定出的物质包含糖类、氨基酸、脂肪酸、胆固醇、小分子羧酸、尿素等。判别分析法筛选出有判别能力的物质有丁酸、乙亚胺酸、丙三醇、L—异亮氨酸、L—缬氨酸、氨基丙二酸、D—赤鲜糖、棕榈酸、硬脂酸、9,12-十八碳二烯酸,由此建立的判别方程可以完全分开二组,误判率为零。支持向量机法20折交叉验证判别分析结果的正确率达75%。
结论:气相色谱-质谱化学衍生法可以用来进行肝癌患者的血清小分子代谢产物谱的分析;肝癌患者的血清小分子代谢产物成分有异于正常者,可以通过逐步判别分析完全区分肿瘤组和正常组,支持向量机交叉验证的结果良好。
第二部分基于气相色谱-质谱联用技术的肝癌高危人群:慢性乙型肝炎患者和乙型肝炎病毒感染后肝硬化患者的血清代谢组学研究(化学衍生法)
目的:以化学衍生法作为血清样本的前处理方法,通过气相色谱-质谱仪建立和区分肝癌的高危人群慢性乙型肝炎和肝硬化患者的血清代谢物谱。
方法:获取乙型肝炎病毒感染的20例慢性肝炎和20例肝硬化病例的血清,用甲醇水溶剂萃取后进行甲氧胺吡啶和N-甲基-N-(三甲基硅烷)三氟乙酰胺(MSTFA)的二步衍生化。用气相色谱质谱(GC-MS)联用仪对衍生化产物上样检测。用逐步判别分析法对所得的代谢组学数据进行分析。
结果:由9个代谢标志物组成的判别方程可以对慢性肝炎组做出100%的正确诊断,对肝硬化患者的诊断准确率为75%。这9个代谢物为乙酸、山梨醇、D-乳酸、己酸、1-萘胺、丁酸、磷酸、D-葡糖醇和葡萄糖。
结论:气相色谱-质谱化学衍生法可以用来进行肝癌高危人群慢性乙型肝炎和肝硬化患者的血清小分子代谢产物谱分析;慢性乙型肝炎患者的血清小分子代谢产物成分有异于肝硬化患者,逐步判别分析选择的标志物可以用来区分肝炎和肝硬化病例。
第三部分基于气相色谱-质谱联用技术的代谢组学研究方法在生物标志物定量研究中的探讨(同位素标记微波衍生法)
目的:利用稳定性同位素标记和快速微波衍生的方法对血清中的葡萄糖和氨基酸进行精确定量,应用于观察吡咯喹啉醌(PQQ)对良性肝病(酒精性肝损大鼠模型)的血清葡萄糖和异亮氨酸代谢水平的影响。
方法:将30只雄性大鼠分为对照组、酒精组和PQQ组,酒精组和PQQ组给予胃内灌注酒精4周,结束后PQQ组给予胃内灌PQQ共4周。建立模型后采集全血并分离血清,对血清用~(13)C葡萄糖和~(15)N异亮氨酸同位素进行标记、用甲醇进行蛋白沉淀、用衍生化试剂MSTFA进行微波衍生,最后用气相色谱-质谱的方法同步测定大鼠体内血糖和异亮氨酸水平,三组进行比较,结果进行t检验。
结果:通过微波衍生,葡萄糖和异亮氨酸的硅烷化能在3分钟内完成。酒精组大鼠血糖水平高于对照组(10.89±0.93mmol/L vs 8.25±1.44 mmol/L,p＜0.01)。PQQ组大鼠的血糖水平低于酒精组(9.20±0.87 mmol/L vs 10.89±0.93 mmol/L,p＜0.01)。酒精组异亮氨酸水平明显高于对照组(132.54±6.51 umol/L vs 107.83±11.72umol/L,p＜0.001),而异亮氨酸水平在酒精组和PQQ组无显著差异。
结论:通过气相色谱-质谱微波衍生和同位素标记法,可以快速、精确、定量测定浓度范围跨度很大的血清葡萄糖和异亮氨酸水平。慢性的酒精暴露可以导致大鼠血清葡萄糖和异亮氨酸的升高,PQQ能恢复酒精暴露导致的高血糖水平,异亮氨酸的代谢不受PQQ的影响。
第四部分基于基质辅助激光解析电离飞行时间质谱技术的乙型肝炎病毒感染人群血清多肽谱研究和肝癌多肽生物标志物的筛选
目的:探讨以磁性微粒富集血清多肽和基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionisation time-of-flight massspectrometry,MALDI-TOF MS)为基础的血清多肽谱检测方法是否可以区分不同的乙肝病毒感染患者,进一步筛选肝细胞肝癌的多肽生物标志物。
方法:用磁性微粒对94例肝细胞肝癌患者、80例慢性乙型肝炎患者和24例正常体检者的血清进行多肽的富集,用MALDI-TOF MS对富集物进行分离、鉴定。对所得的高通量数据应用遗传算法(genetic algorithm,GA),在30例肝癌、30例肝炎和24例正常者中建立肝癌的诊断模型,用64例肝癌和50例肝炎的数据对建立的肝癌模型进行验证。肝癌模型中的多肽用液相色谱-质谱质谱(liquid chromatography-massspectrometry mass spectrometry)联用技术进行进一步的身份鉴定。第二步,同样的方法额外检测乙肝病毒感染的二组人群包括乙肝病毒健康携带者(n=22)和乙肝感染后肝硬化患者(n=23)的血清多肽谱,与同一时间段额外收集的25例正常体检者、25例慢性乙型肝炎、25例肝癌患者的血清多肽谱进行比较。
结果:第一步中,三组血清多肽谱区别明显,建立二个肝癌诊断模型用于区分肝癌和正常以及肝癌和肝炎。二个模型的疾病总识别率分别为96.25%和93.33%,在验证病例中,模型对肝癌的正确诊断率分别为98.4%和76.6%。经过鉴定,肝癌的标志物包括凝血酶原前体片段、钙激活分泌蛋白-1、凋亡重复序列6抑制因子等。第二步额外检测的乙型肝炎病毒携带者和肝硬化患者的血清多肽谱明显区分于正常、慢性肝炎和肝癌患者。
结论:磁性微粒富集多肽和MALDI-TOF MS技术可以应用于乙肝感染人群的血清多肽谱研究,而且可以区分不同的乙肝感染人群;此技术适合于寻找肝癌的多肽标志物、用于乙肝病毒感染人群的肝癌监测。
第五部分基于人类抗体芯片技术的乙型肝炎病毒感染人群血清细胞因子谱的研究和肝癌细胞因子生物标志物的筛选
目的:利用抗体芯片技术检测肝细胞肝癌患者的血清细胞因子,寻找肝癌特异性细胞因子作为诊断标志物。
方法:利用Raybio~(?)生物素标记的抗体芯片Ⅰ对正常体检者(n=3)、慢性乙型肝炎患者(n=3)和肝细胞肝癌患者(n=3)进行507个人体细胞因子的差异初筛。30个组间差异最大同时组内差异最小的细胞因子被用来做扩大人群验证。验证人群是30例正常体检者,30例肝炎和40例肝细胞肝癌患者。选择one-way ANOVA和对角线判别分析来筛选肝癌的细胞因子标记物。
结果:one-way ANOVA的筛选结果示肝癌患者血清细胞因子表达有异于肝炎和正常的是IGFBP-7,MDC,MSPα链和MSPβ链。对角线判别分析筛选的肝癌细胞因子标记物MDC和MSPα链与AFP共同构建了肝癌诊断模型,其敏感性为73.2%,特异性为95%,工作受试者曲线下面积是0.89。
结论:抗体芯片是一项高敏感、高特异性的血清细胞因子检测技术,可以用来肝细胞肝癌的细胞因子标志物的筛选。
第六部分基于同位素标记相对和绝对定量技术和液相色谱-质谱联用技术的肝癌血清蛋白质组学检测方法在蛋白质组生物标志物定量研究中的探讨
目的:将同位素标记相对和绝对定量(isobaric tags for relative and absolutequantitation,iTRAQ)联合液相色谱质谱技术应用于肝细胞肝癌患者血清定量蛋白质组学研究,并初步探讨该检测方法对肝癌蛋白质组生物标志物的相对定量研究的应用前景。
方法:15例乙型肝炎病毒感染的原发性肝细胞肝癌患者和15例慢性乙型肝炎患者,收集血清分二组混合。选用高丰度蛋白分离系统(Multiple Affinity RemovalSystem,MARS)除血清高丰度蛋白、收集低丰度蛋白,用SDS-PAGE对原血清和收集的低丰度蛋白样品进行效果评估。对蛋白进行胰蛋白酶酶解后用iTRAQ试剂114和117分别标记肝癌和肝炎血样。用强阳离子交换色谱(Strong cation exchangechromatography,SCX)对标记好的多肽分层后用LC-ESI-MS/MS进行检测。选用蛋白分析软件ProteinPilot比较二组混合血清的差异蛋白。
结果:经过MARS系统去高丰度蛋白,血清样品中的低丰度蛋白得到很好的分离;经过SCX分层共40个多肽层,进入MS质谱共得到49784个多肽片段,谱库搜索的蛋白共251个,其中二组的差异度超过1.5倍的共21个蛋白。
结论:同位素标记相对和绝对定量技术和液相色谱-质谱联用技术可以用于肝癌血清蛋白质组学相对定量检测,慢性乙型肝炎和肝细胞肝癌患者的血清蛋白表达明显差异。
Liver cancer is the sixth most common cancer and the third leading cause of cancer death worldwide.Most liver cancer patients are in developing countries,among whom,Chinese account more than a half.Most liver cancers are hepatocellular carcinomas(HCC),the major risk factors for which are chronic infections with the hepatitis virus B and C.As hepatitis B virus(HBV) is more prevalent in China,we have put much attention into the disease course of HBV infection.During the long-term disease progression from chronic HBV infection to liver cirrhosis and HCC,patients are lack of characteristic symptoms.It is often late stage when HCC is diagnosed.Treatment for such patients is limited with the five year survival rate lower than 5%.In light of the high burden of HBV chronic infection,it is significant to identify reliable,reproducible,and noninvasive biomarkers or a surveillance model for HCC targeting at the HBV infected population.The most widely used serum biomarker for HCC has been alpha fetoprotein(AFP).As up to 40%of HCC patients present with normal AFP levels and elevated AFP levels are observed in patients with chronic hepatitis or cirrhosis,the low sensitivity and specificity of AFP has always been called in question.However,there has been no other serum biomarkers which could be accepted as effective as AFP.
Human blood contains a wide variety of chemically diverse molecular weight compounds ranging from volatile and nonvolatile low molecular ones such as organic acids and carbohydrates,to middle molecular ones such as polypeptides, and to large molecular ones such as proteins.These compounds differ against complex interactions between genetic factors,pathogen infection,toxicant exposure,and so on during disease process.They constructed a huge biomarker bank which could be applied for disease sceening,progression or prognosis evaluation.Techniques allowing high-flux data aquisition and analyzing are needed for researching in this huge biomarker bank.In the present,"omics" studies including genomics,proteomics and metabolomics are the newest in the biomedical field with the most rapid developed techniques such as chromatography or massspectrometry meeting the need of high-flux data aquisition and analyzing.
The main purpose of this study is to explore new biomarkers for HCC from the HBV infected groups applying different techniques on the base of "omics" research.The contents are:the metabolomic research of HBV infected HCC patients based on Gas chromatography/Mass Spectrometry(GC/MS) and identification of HCC blood biomarkers applying the techniques of solid-phase microextraction and chemical derivatization;the serum metabolomic research in the HCC high risk groups:chronic hepatitis B patients and HBV infected cirrhosis patients based on GC/MS applying the technique of chemical derivatization;an exploration in the quantification of serum metabolomic biomarkers based on GC/MS applying the technique of microwave-assisted derivatization and isotope dilution;serum proteomic surveillance of hepatocellular carcinoma from hepatitis B virus infected groups with magnetic bead-based MALDI-TOF/MS;identification of new serum biomarkers for hepatocellular carcinoma using biotin-label-based antibody microarrays;a pilot study of identification and quantification of serum protein biomarkers in HCC based on the technique of isotope tagging for relative and absolute protein quantitation(iTRAQ).
Part One The metabolomic research of HBV infected HCC patients based on Gas chromatography/Mass Spectrometry(GC/MS) and identification of HCC blood biomarkers applying the techniques of solid-phase microextraction and chemical derivatization
Section 1.Investigation of volatile biomarkers in HCC blood using solid-phase microextraction and gas chromatography-mass spectrometry
Objective:To explore the technique of solid-phase microextraction(SPME) and gas chromatography-mass spectrometry(GC-MS) in the investigation of blood volatile biomarkers for HCC.
Methods:The fiber,duration,temperature of extraction and the duration, temperature of desorption was tested and optimized to extact the blood volatile metabolites in 19 HCC patients and 18 normal controls.GC-MS was applied in separate,detect and identificate the extracted metabolites.The peak area values were gathered and compared between groups.Chi square test was used to screen the HCC biomarkers.
Results:The best extraction fiber was CAR-PDMS.The optimal extraction duration,temperature was 15min and 60℃.The optimal extraction duration, temperature was 30s and 250℃.There were 47 volatile low molecular metabolites detected,19 of which were detected differently in the two groups(p＜0.05). Positive rates of and hexanal(sensitivity 97.4%,specificity 100%), 1-octen-3-ol(sensitivity 84.2%,specificity 100%) and octane(sensitivity 89.5%,specificity 100%)in HCC blood were found to be much higher than those in control blood.The three molecules of hexanal,1-octen-3-ol and octane were regarded as biomarkers of HCC with clinical diagnostic value.
Conclusion:SPME/GC-MS is a simple,rapid and sensitive method very suitable for investigation of volatile disease markers in human blood.
Section 2.Investigation of nonvolatile biomarkers in HCC serum using chemical derivatization and gas chromatography-mass spectrometry
Objective:To explore the technique of chemical derivatization and gas chromatography-mass spectrometry(GC-MS) in the investigation of serum nonvolatile biomarkers for HCC.
Methods:Serum metabolome was detected through chemical derivatization followed by gas chromatography/mass spectrometry(GC/MS).The serum metabolic difference was compared between hepatocellular carcinoma(HCC,n=20) male patients and normal control male subjects(n=20).The acquired GC/MS data was analyzed by stepwise discriminant analysis(SDA) and support vector machine(SVM).
Results:The metabolites including butanoic acid,ethanimidic acid, glycerol,L-isoleucine,L-valine,aminomalonic acid,D-erythrose, hexadecanoic acid,octadecanoic acid,and 9,12-octadecadienoic acid in combination with each other gave the strongest segregation between the HCC group and the control group.By applying these variables,our method provided a diagnostic model that could well discriminate between HCC and normal subjects. More important,the error count estimate for each group was 0%.The total classifying accuracy of the discriminant function tested by SVM 20-fold cross validation was 75%.
Conclusion:This technique is different from traditional ones and appears to be a useful tool in the area of HCC diagnosis.
Part Two Serum metabolomic study of two groups of cases with high risk of HCC:hepatitis B virus infected chronic hepatitis and cirrhosis patients by chemical derivatization and Gas chromatography/Mass spectrometry
Objective:To investigate the serum metabolic profile of hepatitis B virus (HBV)infected chronic hepatitis and cirrhosis patients.
Methods:HBV infected non-cirrhosis male subjects(n=20) and HBV infected cirrhosis male patients(n=20) entered this experiment.Serum metabolome was detected through chemical derivatization followed by GC/MS.The high-flux metabolomic data was analyzed by stepwise discriminant analysis(SDA).
Results:From the 41 metabolites detected in serum,we selected metabolites including acetic acid,sorbitol,d-lactic acid,hexanoic acid, 1-naphthalenamine,butanoic acid,phosphoric acid,d-glucitol,glucose which in combination with each other could segregate the two groups.The error count was 0%for the non-cirrhosis group and 25%for the cirrhosis group.
Conclusion:Chemical derivatization and GC/MS technique is applicable in metabolomic profiling in patients with HCC risk.
Part Three An exploration for precise quatification of serum metabolomic biomarkers applying microwave-assisted derivatization and isotope dilution GC/MS
Objective:To precisely quatificate serum glucose and isoleucine levels applying microwave-assisted derivatization and isotope dilution GC/MS in the investigation of the effect of pyrroloquinoline quinine(PQQ) on rats with alcohol assumption.
Methods:Thirty male Sprague-Dawley rats divided into three groups.The first group was a normal control which received intragastric administration of saline for 8 weeks.Alcohol was administered intragastrically to the alcohol group and PQQ group for four weeks.PQQ was administered intragastrically to rats in the PQQ group at for another four weeks.Serum metabolites including glucose,isoleucine,the[U-~(13)C_6]-glucose and[~(15)N]-isoleucine were rapidly derivatized by N-Methyl-N-trimethylsilyltrifluoroacetamide(MSTFA) with microwave irradiation,and the trimethylsilyl derivatives were analyzed by GC/MS.T test was applied for comparison between the groups.
Results:Derivatization of was completed within 3min.Serum glucose levels were higher in alcohol group than in normal group(10.89±0.93mmol/L vs 8.25±1.44 mmol/L,p＜0.01).Serum glucose levels were lower in PQQ group than in alcohol group(9.20±0.87 mmol/L vs 10.89±0.93 mmol/L,p＜0.01).Serum isoleucine levels were higher in alcohol group than in normal group (132.54±6.51 umol/L vs 107.83±11.72 umol/L,p＜0.001).There was no difference of serum isoleucine levels between PQQ group and alcohol group
Conclusion:The combination of MAD and ID GC/MS has shown to be an accurate, rapid,simple and sensitive method for the quantification of glucose and isoleucine in serum samples.Serum glucose and isoleucine metabolism were affected by alcohol.PQQ could reverse alcohol exposure induced glucose elevation.It did not affect the metabolism of isoleucine whose level was elevated along with serum glucose.
Part Four Serum proteomic surveillance of hepatocellular carcinoma from hepatitis B virus infected groups and identification of HCc biomarkers with magnetic bead-based MALDI-TOF/MS
Objective:To determine whether serum proteomic profiling based on magnetic bead(MB) and matrix-assisted laser desorption/ionisation time-of-flight(MALDI-TOF) mass spectrometry(MS) can discriminate between hepatocellular carcinoma(HCC) and other HBV infected groups.To identify proteomic biomarkers for HCC.
Methods:One hundred ninty-eight serum samples from 80 chronic hepatitis B,94 HBV infected HCC patients and 24 normal cases were analyzed by MALDI-TOF MS after peptides enrichment by MBs.Applying genetic algorithm,diagnotic models for HCC were built between 30 HCC patients and 24 normal cases/30 hepatitis patients.Validations were done with the left cases.Markers in the models were identified through LC/MS MS.Data from 22 HBV carriers and 23 HBV-infected cirrhosis patients was laterly gathered to be separated from additional 25 chronic hepatitis B patients,25 HCC patients and 25 normal controls.
Results:The first three groups were well separated from each other.Two discrimination models were built for HCC.The overall recognition capability of the two models was 96.25%and 93.33%.The validation of them in the left patients showed misdiagnosis rates of 1.6%and 23.4%for HCC.Potential biomakers for HCC were identified as prothrombin precursor(fragment),isoform 1 of calcium-dependent activator protein for secretion,baculoviral IAP repeat-containing 6,et al.The laterly added two groups were also well separated from others.
Conclusion:MB based MALDI-TOF MS approach is applicable in identifying serum proteomic biomarkers that can be used in the surveillance of HCC from HBV infected population.
Part Five Identification of new serum biomarkers for hepatocellular carcinoma using biotin-label-based antibody microarrays
Objective:Searching for hepatocellular carcinoma(HCC) biomarkers applying antibody arrays.
Methods:In this study,serum cytokine detection using RayBio~(?) Biotin Label-based Human Antibody ArrayⅠ(507 human proteins) was applied in normal cases(n=3),chronic hepatitis B patients(n=3) and HCC patients(n=3).Thirty proteins which were the most differently detected ones between HCC patients and normal/hepatitis cases were validated in additional 30 normal,30 hepatitis and 40 HCC cases.Biomarkers for HCC were selected through one-way ANOVA and diagonal linear discriminant analysis.
Results:A diagnostic model constructed by two HCC biomarkers(MDC,MSPαchain)together with AFP had a sensitivity of 73.2%,specficity of 95%for HCC diagnosis.
Conclusion:Antibody microarrays are applicable in searching for new biomarkers for HCC.
Part Six Quantification of HCC serum proteomic biomarkers applying the methods of isobaric tags for relative and absolute quantitation(iTRAQ)and Liquid Chromatography/Mass Spectrometry
Objective:To apply the methods of isobaric tags for relative and absolute quantitation(iTRAQ)and Liquid chromatography-Mass spectrometry to hepatocellular carcinoma(HCC) patients in the preliminary study of relative quantified detection of HCC proteomic biomarker.
Methods:15 cases of hepatitis B virus infected HCC and 15 patients with chronic hepatitis B were recruited.Sera were collected and pooled within the same group.Multiple Affinity Removal System(MARS) was used for high-abundance serum proteins depletion and low-abundance proteins collection. SDS-PAGE was applied to assess the effect of MARS with the original sera and the low-abundance protein samples.After protein digestion by trypsin,iTRAQ reagents 114 and 117 were marked on peptides of HCC and hepatitis patients respectively.After being handled by Strong cation exchange chromatography (SCX),marked peptides were tested by LC-ESI-MS/MS.The software ProteinPilot was selected to make a comparison between the two different serum protein mixture.
Results:With the depletion of high-abundance proteins by MARS system, low-abundance proteins have been well collected.There were 40 fractions after SCX handling,resulting in a total of more than 49,784 peptide fragments detected in the MS-MS system.Two hundred and fifty one proteins were identified through online spectra library search,twenty one of them presented quitely differently(more than 1.5 times the difference) in the two different serum pools.
Conclusion:ITRAQ and liquid chromatography-mass spectrometry can be used for relative quantified detection of HCC proteomic biomarkers. There are significant difference of serum protein levels between patients with chronic hepatitis B and HCC.
引文
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