【作者】 华丛笑；

【导师】 李一石；

【作者基本信息】 中国协和医科大学 ， 内科学， 2008， 博士

【摘要】 研究背景蛋白质是多种致病因子作用于机体的靶分子，也是绝大多数药物的靶点。药物对机体功能的调整有可能是作用于多个靶点，即直接或间接通过影响多个基因的表达而实现。蛋白质组学拓宽了药物靶点研究，能深入研究疾病和药物引起的多种细胞内、外蛋白质及代谢功能的变化，具有多靶点研究的优势。比较给药前后蛋白质谱的变化，能深入了解药物作用和不良反应的分子机制。药物疗效或不良反应的生物标记物被确定后，可用于药物疗效或不良反应的临床评价。蛋白质组学技术可以全面监测、分析体内各种组织及血液中蛋白质的表达，已经成功用于肿瘤、糖尿病等多种疾病相关标记物的检测，成为疾病诊断和监测的有力工具。因此，比较药物治疗前后的血浆或组织蛋白质表达谱，探索药物治疗相关的生物标记物的方法是可行的。而差异凝胶电泳技术是蛋白质组学技术中常用的双向电泳的新发展，它引入内标以消除凝胶之间的差异，排除了由电泳的重复性问题导致的误差，提高了筛选差异表达蛋白质的准确性。国内外前瞻性研究已经证实，血清总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）增高、高密度脂蛋白胆固醇（HDL-C）降低是冠心病和缺血性脑卒中的独立危险因素。近十年来，我国高脂血症发病率呈增长态势，根据2002年调查，我国成人的血脂异常总患病率为18．6％，目前血脂异常患者达2亿人。因此，研究血脂异常患者的调脂治疗有重要的现实意义。近年来研究认为，调脂药物具有降脂和降脂外作用，并可能影响血脂相关的多种血浆标记物。他汀类是目前应用最为广泛的调脂药物，是肝细胞内胆固醇合成早期阶段的限速酶即三羟基三甲基戊二酰辅酸A（HMG-CoA）还原酶的抑制剂，能抑制内源性胆固醇的合成，并能增加肝细胞表面LDL-C受体的数量和活性，促进胆固醇清除。匹伐他汀是第三代他汀类调脂药物，具有肝细胞选择性高和半衰期长的特点。洛伐他汀也是HMG-CoA还原酶抑制剂，能增加肝脏的胆固醇合成，促进血液中的胆固醇清除，它对降低LDL具有显著效果，但增加HDL的作用较弱；烟酸能抑制肝脏合成TG、VLDL，促进VLDL分解，并抑制apoAl的降解，具有升高HDL的作用，但降低LDL作用较弱。两药合用能够作用于脂质合成代谢的不同环节，作用互补，加强调脂作用。目前临床应用的生物标记物尚不能满足调脂药物疗效评价的需要。血浆中含有大量分泌蛋白质，包括急性期反应蛋白、炎症介质和细胞因子等，血浆蛋白质表达谱的改变可能反映不同调脂药物的疗效。因此，探索调脂相关的生物标记物评价调脂药物的疗效和药物作用的机制是需要的。但是，目前国内外应用血浆蛋白质组学技术进行高脂血症患者调脂治疗标记物研究的资料还很少。研究目的1．比较高脂血症患者与健康对照人群的血浆蛋白质表达谱，筛选高脂血症相关的血浆蛋白质。2．比较高脂血症患者服用调脂药物前后的血浆蛋白质表达谱，筛选出调脂药物治疗相关的血浆蛋白质。研究对象1．高脂血症患者：选取2005年5月～2006年12月阜外医院门诊高脂血症患者51例。其中15例，服匹伐他汀2mg，每天一次，晚间睡前口服；36例患者，服洛伐他汀／烟酸缓释片10mg／500mg，每天一次，晚间睡前口服。两药均治疗8周。高脂血症患者的入选标准：18～70岁的原发性高脂血症患者，停服调血脂药并饮食控制4周，LDL-C≥140～160mg／dl（3．6～4．13mmol／dL），并且TG≤400mg／dl（4．52mmol／dL）。患者均签署书面知情同意书。2．健康对照者：同期在阜外医院从血脂正常的健康人群中按照性别、年龄匹配的原则入选了13例对照。对照组入选标准：年龄18～70岁的健康人，体重指数19-24kg／m^（2）。研究方法1．标本采集：高脂血症患者在治疗前（0周）及治疗结束后（8周），受试者抽血前12小时禁食，采集外周静脉血。用0周和8周采集的静脉血标本用于测定血常规、血生化和血浆蛋白质组学实验。健康人抽血前12小时禁食，采集外周静脉血，血标本处理和实验室检查同高脂血症患者。2．血浆样品分组：从上述人群选取单纯高脂血症患者（不伴有其他心血管危险因素和心血管疾病）（n=19），分组为：匹伐他汀治疗前、治疗后组；洛伐他汀／烟酸治疗前、治疗后组；将高脂血症合并高血压患者治疗前作为一组（n=6）；健康对照组（n=13）。各组间做年龄、性别匹配。3．蛋白质组学实验研究方法：本研究采用三种方法完成蛋白质组学的实验研究：差异凝胶电泳寻找组间有明显差异表达的蛋白质；蛋白印迹和免疫测定方法验证相关蛋白质的差异表达。（1）蛋白质双向电泳及蛋白质鉴定1)将各组中的血浆样品混合：每个样品取17ul；2)去除血浆高丰度蛋白IgG和白蛋白；3)荧光标记：使用DIGE试剂盒（CyDye DIGE Fluor（minimal Dye）Labelling Kit）将每一组的混合血浆样品进行荧光标记；4)双向差异凝胶电泳（DIGE）：按照Ettan DIGE User Manual（AmershamBiosciences）用户手册的方法，使用24cm的预制胶条（Bio-Rad公司）在PROTEAN IEF CELL等电聚焦仪（Bio-Rad公司）上进行一向等电聚焦电泳；使用ETTAN DALT Twelve System电泳仪（Amersham公司）进行二向10％十二烷基硫酸钠-聚丙烯凝胶（10％SDS-PAGE）电泳；5)DIGE凝胶图像分析：使用Typhoon 9410扫描仪在488／520nm，532／580nm，633／670nm波长分别对Cy2，Cy3，Cy5荧光染料标记的凝胶进行扫描。用DeCyder v．5．02软件分析图像和差异点寻找。经内参校正后的蛋白质差异表达倍数（Ratio）≥1．5或≤0．5，且P<0．05者，判定为差异表达的蛋白质。找出两组间有明显差异表达的蛋白质，将差异表达蛋白质在凝胶上做好编号标记；6)胶内酶切和质谱分析：在制备胶上依次切取差异表达蛋白质的胶粒。用胰蛋白酶酶解蛋白质，提取酶解后的肽段，在4800 MALDI TOF／TOF Analyzer （ABI）质谱仪上进行质谱分析。通过GPS检索软件将质谱分析结果在IPI HUMAN 3．23数据库中搜索对应的蛋白质。（2）蛋白印迹杂交：对3个有明显组间差异表达的蛋白质：血浆纤维蛋白原α链、β链和含锌alpha-2糖蛋白进行验证。用Alpha Imager HP分析系统扫描蛋白印迹胶片，统计分析光密度值。（3）血清转铁蛋白和触珠蛋白的测定：用IMMAGE（?）双光路免疫浊度分析仪（Beckman Coulter，Inc．）测定血清的转铁蛋白和触珠蛋白。研究结果：一、服用调脂药前后血脂变化1．基线特征服用匹伐他汀与服用洛伐他汀／烟酸缓释片的高脂血症患者比较，年龄、性别构成、体质指数（BMI）、基线血清TC、TG、LDL-C和HDL-C水平均无统计学差异，见表1。表1高脂血症患者的基线特征（平均值（标准差））注：TC：总胆崮醇，HDL-C：高密度脂蛋白胆固醇；LDL-C：低密度脂蛋白胆固醇：TG：甘油三酯。数据以平均值（标准差）表示。P：服用匹伐他汀与洛伐他汀／烟酸的高脂血症患者比较。2．两种药物调脂疗效匹伐他汀降低TC的幅度大于洛伐他汀／烟酸（2．30±0．64mmol/L vs．1．41±0．92mmol／L；P<0．05）。匹伐他汀降低LDL-C的幅度大于洛伐他汀／烟酸（2．02±0．57mmol／L vs．1．28±0．66mmol／L，P<0．05）。两药降低TG的幅度没有差异（0．59mmol/L vs．0．49mmol／L；P>0．05）。匹伐他汀对血清HDL-C水平没有影响。洛伐他汀／烟酸使HDL-C升高0．16mmol/L（P<0．05）；使apoA=Ⅰ升高0．11mmol／L（P<0．05）；使血清apoB降低0．48mmol／L（P<0．05）；并且使LP（a）降低0．13mmol／L（P<0．05）。二、蛋白质组学研究结果（一）高脂血症患者服用匹伐他汀后表达有改变的血浆蛋白质包括：纤维蛋白原α和β链、触珠蛋白。1．血浆纤维蛋白原α链表达降低（降低74～242％，P<0．05）。2．血浆纤维蛋白原β链前体表达降低（降低134～156％，P<0．05）。3．蛋白印迹杂交验证显示，高脂血症患者服用匹伐他汀后血浆纤维蛋白原α链表达有降低倾向（降低26．4％，P=0．153）；血浆纤维蛋白原β链表达有降低倾向（降低29．7％，P=0．075）。4．血清触珠蛋白水平升高10．1％（P=0．036）。（二）高脂血症患者服用洛伐他汀／烟酸后表达发生改变的血浆蛋白质包括：apoA-Ⅰ前体蛋白、纤维蛋白原α和β链、触珠蛋白和转铁蛋白。1．apoA-Ⅰ前体蛋白的表达升高101％（P=0．025）。2．血浆纤维蛋白原α链表达降低31．6％（P=0．001）。3．血浆纤维蛋白原β链表达降低17．6％（P=0．000）。4．血浆触珠蛋白水平高（升高58～62％，P<0．05）；免疫测定验证表明，高脂血症患者服用洛伐他汀／烟酸后，血清触珠蛋白水平升高9．8％（P=0．042）。5．血浆转铁蛋白升高（升高60％，P=0．033）；免疫测定验证表明，高脂血症患者服用洛伐他汀／烟酸后，血清转铁蛋白水平升高9．0％（P=0．037）。（三）高脂血症患者与健康人比较，表达有差异的血浆蛋白质包括：apoA-Ⅰ前体蛋白、含锌的α_（2）糖蛋白、纤维蛋白原α、β链和触珠蛋白。1．高脂血症患者血浆apoA-Ⅰ前体蛋白的表达比健康人升高67％（P=0．036）。2．高脂血症患者的血浆纤维蛋白原α链表达比健康人升高489％（P=0．002）；血浆纤维蛋白原β链前体表达比健康人升高125～256％（P<0．05）。3．高脂血症患者的血浆含锌的α_（2）糖蛋白（ZAG）的表达比健康人降低（降低58～75％，P<0．05）；蛋白印迹杂交验证显示，高脂血症患者的ZAG表达比健康人降低36．5％（P=0．024）。4．高脂血症患者的血浆触珠蛋白水平比健康人降低59～75％（P<0．05）。5．高脂血症患者与健康人比较，血浆转铁蛋白水平没有差异。研究结论1．本研究提示血浆中的纤维蛋白原、转铁蛋白和触珠蛋白，有可能作为调脂药物（匹伐他汀、洛伐他汀／烟酸）疗效评定的生物标记物。2．从本研究结果推测，不同调脂药的效果评价可能需要不同的生物标记物。3．本研究首次在中国高脂血症患者证实，服用匹伐他汀、洛伐他汀／烟酸能够降低血浆纤维蛋白原水平。4．高脂血症患者服用匹伐他汀和洛伐他汀／烟酸后使血清触珠蛋白水平升高。5．高脂血症患者服用洛伐他汀／烟酸使血清转铁蛋白水平升高。6．高脂血症患者的血浆触珠蛋白和含锌的α_（2）糖蛋白表达水平低于健康人。7．调脂药影响纤维蛋白原、触珠蛋白、转铁蛋白等急性期蛋白的表达，可能是药物降脂外效应的表现。8．应用血浆蛋白质组学的方法，寻找评价药物治疗效应的生物标记物，具有价值。更多还原

【Abstract】 BackgroundStatins are most effective lipid-modulating drugs in the world. Japanese studieshave proved that pitavastatin has great efficacy in reducing serum cholesterol andlow-density lipoprotein cholesterol. Lovastatin/niacin is a broad-spectrum agent,which can improve the lipids profile as a whole. However, there is no data of usingthese drugs in Chinese population up to now.Since plasma protein biomarkers may be used as sensors of drug therapy, wehypothesize that lipid-modulating drugs may influence the plasma proteomicexpression. Therefore, the plasma protein biomarkers may be helpful to assess theeffects of the lipid-modulating drugs. Proteomics technique has been a prospectingtechnology in screening biomarkers. However,the proteomic studies inhyperlipidemia and its drug-therapy are very limited.Objectives:1. In order to explore the hyperlipidemia-related differentially-expressed proteins in the plasma by comparing the proteomics profile of hyperlipidemia patients and healthy volunteers.2. To explore the differentially-expressed proteins in the plasma related to lipid-modulating drug therapy by comparing the proteomics profile of hyperlipidemia patients before and after lipid-modulating therapy.3. To provide clues and evidence for biomarkers research in lipid modulating therapy. Study populationsThe hyperlipidemia populations were obtained from two clinical trials. The firstwas pitavastatin phaselI clinical trial and the second was lovastatin/niacin phaselIclinical trial. A total of 15 and 36 hyperlipidemia patients were randomizedly enrolledin the pitavastatin trial and lovastatin/niacin trial respectively during August 2005 toDecember 2006. Inclusion criteria of the studies: 1. Men or women aged at 18～70years old who had primary hyperlipidemia. 2. Patient with serum LDL-C(?)140～160mg/dl(3.6～4.13mmol/dL) and TG(?)400mg/dl(4.52mmol/dL). The studies wereimproved by the Independent ethics committee of Fuwai Hospital. Written informedconsent was obtained from each participant before the study.Lipid-modulating therapy: The patients of pitavastatin trial received 2mgpitavastin and the patients of lovastatin/niacin trial received 10mg/500mg every nightfor 8 weeks.Healthy control population was obtained from Fuwai Hospital. 13 healthyvolunteers were included by matching the sex, age.Methods of proteomic study:Blood samplingHyperlipidemia patients: Peripheral venous blood samples for proteomic studywere collected at before (0 week) and after lipid-modulating therapy (eighth week) inthe morning after an overnight fast. Routine blood biochemistry was measured bothbefore and after the therapy. Serum concentrations of total cholesterol (TC),high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol(LDL-C) and triglycerides (TG) were measured using a Beckman Synchrom CX5(Fullerton, California,USA).Healthy control: Peripheral venous blood samples for proteomic study werecollected in the morning after an overnight fast. Routine blood biochemistry wasmeasured in the healthy population. Sampling divisionThe blood samples were divided into 6 groups: groups before and afterpitavastin treatment, groups before and after lovastatin/niacin treatment, patients withboth hyperlipidemia and hypertension, healthy control group.Procedure of proteomic studyEach EDTA-anticoagulated blood sample(4ml) was centrifuged at 4℃(3000rpmfor 10 minutes) and the supernatant was collected. The plasma samples of each groupwere pooled and then the albumin and IgG were removed using the ProteoExtractAlbumin/IgG Removal Kit (CALBIOCHEM). Prior to 2-DE, samples were labeledwith fluorescent cyanine dyes for 2-D DIGE, Cy2, Cy3, and Cy5(CyDye DIGE FluorLabeling Kit, Minimal Dye) following the manufacturer’s protocols. The internalstandard was prepared by combining equal quantities of each of all the tested samples.Six DIGE gels were performed to analyze hypercholesterolemia samples (before andafter treatment) or control samples. The first-dimension isoelectric focusing wasperformed using an immobilized drystrips (24 cm, pH 3-10 NL, Bio-Rad) andPROTEAN IEF CELL (Bio-Rad).The Cy2, Cy3, and Cy5 components of each gelwere individually imaged with a Typhoon 9410 scanner (Amersham Biosciences).DeCyder v. 5.02 (GE Healthcare) was used for a differential gel analysis. A totalof 950～1050 protein spots were analyzed. Protein relative abundance ratios largerthan +1.5 or smaller than—1.5 were set as a threshold indicating significantchanges. The Student’s t-test was used to calculate significant differences in therelative abundance of individual protein spot between different groups.A preparative gels was run for MS identification. The proteins of interest, asdefined by the 2-D DIGE/DeCyder analysis, were excised from the CBB-stained gelfor an in-gel tryptic digestion. Each gel-piece was incubated with the digestion andextraction solutions and then evaporated to dryness for MS analysis. Peptides fromeach gel were mixed with MALDI matrix and its MALDI PMF was analyzed using a4800 MALDI-TOF/TOF Analyzer (Applied Biosystems). Protein identification byPMF was performed using the MASCOT search engine (version 1.9, Matrix Science).The IPI human database v3.23 (http://www.ebi.ac. uk/IP/IPIhelp.html) was used for the protein searching.Results1. There was not dignificant difference of baseline levels of plasma TC、TG、LDL-C and HDL-C between the two groups of hyperlipidemia patients.2. Compared to heslthy population, the plasma fibrinogen was raised expression in hyperlipidemia patients. Both pitavastatin and lovastatin/niacin therapy decreased the plasma fibrinogen level.3. The expression of plasma alpha-2-glycoprotein, zinc, a fat mobilization factor, was lower in hyperlipidemia patients than that in healthy population.4. The expression of plasma haptoglobin was lower in hyperlipidemia patients than that in healthy population. However, both pitavastatin and lovastatin/niacin therapy increased the plasma haptoglobin level.5. Lovastatin/niacin therapy increased the plasma transferrin level of hyperlipidemia patients.Conclusion1. In Chinese population, both pitavastatin and lovastatin/niacin therapy can decrease the plasma fibrinogen level in hyperlipidemia patients, which indicated that the drugs had anticoagulation effect.2. The expression of plasma haptoglobin was lower in hyperlipidemia patients than that in healthy population. It indicated that plasma anti-oxidative ability was depressed in hyperlipidemia patients. However, both pitavastatin and lovastatin/niacin therapy can increase the plasma haptoglobin level, indicating that the drugs had anti-oxidative effectrs.3. Lovastatin/niacin therapy can increase the plasma transferrin level of hyperlipidemia patients.4. Therefore, plasma fibrinogen, haptoglobin and transferrin may be therapy biomarkers of these lipid-modulating drugs. These proteins are all acute-phase proteins, indicating that the effects on acute-phase proteins may be the pleitropic effects of the drugs.5. Proteomic technology is helpful for exploring the biomarkers of drug-therapy.更多还原