【作者】 秦雪；

【导师】 李瑗； 刘银坤；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2006， 博士

【摘要】 肝细胞性肝癌（hepatocellular carcinoma, HCC）是诊治困难、预后极差的一种恶性肿瘤,全球三大肿瘤死因之一,1990年全世界共有54.1万新发的肝癌病例,50.1万人死于肝癌,其中31.8万（58.5%）新发病例和29.3万（58.8%）肝癌死亡发生在中国。HCC的病因目前认为主要与乙型肝炎病毒（HBV）感染及食品中黄曲霉毒素B₁（AFB₁）污染有关。和其它多数肿瘤一样,HCC的形成是一个多步骤、多阶段、多因素参与的过程,传统的单基因研究模式限制了HCC相关因素的研究发展。近年来迅速发展的蛋白质组学研究,能动态、整体、定量地考察疾病发生发展过程中全部蛋白质种类和数量的变化,对于探讨疾病机理、寻找疾病诊断的特异性标志物和药物治疗的靶标来说,是一种有效的高通量的研究模式,可获得一些传统手段无法得到的新蛋白标志物、新关键分子,极大地丰富了诊断标志物的选择组合及复杂致病机理的阐明。运用该研究策略,建立并优化了适合本实验的双向凝胶电泳（2-DE）技术平台,并利用树鼩可以在实验诱癌过程中经受多次剖腹肝活检手术而长期存活的特点,本研究对HCC形成过程中不同时期肝组织的蛋白质组改变进行动态观察。对AFB₁和AFB₁+人HBV诱发树鼩肝癌发生不同阶段的差异表达蛋白进行筛选,找出在HCC形成过程中一些有潜在作用的差异蛋白质,并对其中的差异蛋白Prx家族在肝癌组织和癌旁组织进行验证,为探讨人HCC发生机理提供重要科学依据。第一部分人工繁育树鼩的生理指标与正常人的对比本部分的研究目的是通过对人工繁育的成年树鼩进行实验室指标的检测,以之与正常人参考值进行比较,分析二者间的关系,探讨树鼩用于研究人疾病动物模型的可行性及优越性。选择人工繁育饲养的成年树鼩22只,同一天内抽取树鼩的抗凝全血和分离血清,随即进行相关实验室指标的检测。结果为,血常规检测,红细胞总数8.19×10¹²/L,高于正常人参考值,白细胞总数2.2×10⁹/L,低于正常人参考值;每份全血标本涂片和染色后镜检观察,树鼩各种血细胞及血小板形态都与正常人相似;白细胞分类计数,树鼩各种细胞百分比与正常人相近;血清标本生化十项指标检测,仅树鼩肌酐一项结果为16.63μmol/L,低于正常人参考值下限44μmol/L,树鼩的血清谷丙转氨酶77.92U/L、谷草转氨酶179.9 U/L分别高于正常人参考值上限40 U/L和45 U/L,其余七项结果符合正常人参考值范围,包括尿素、球蛋白、血清总蛋白、血清白蛋白、血清γ-谷氨酰基转移酶、C-反应蛋白及类风湿因子;检测HBsAg为0.022 ng /ml±0.005,均为阴性。以上各实验室指标检测结果提示,树鼩作为接近人的低等灵长类动物,适合应用于研究人类疾病的动物模型,为进一步利用树鼩模型研究HCC发生机理提供了实验依据。第二部分树鼩肝癌蛋白质组学双向凝胶电泳技术的建立及优化本部分研究的目的是通过建立并优化树鼩肝癌蛋白质组学的双向凝胶电泳技术,提高其分辨率和重复性,并利用该平台进行AFB₁和AFB₁+人HBV诱发树鼩肝癌形成过程中差异表达蛋白质组学的研究。实验以AFB₁和AFB₁+HBV诱发树鼩肝癌形成过程中各时期的肝组织标本,通过对传统的双向凝胶电泳技术的程序及有关环节进行反复改进,尤其对其中的关键因素环节,如样品的处理（单纯的液氮下研磨、液氮下研磨再剧烈振荡）、蛋白定量方法（Lowery法和改良Bradford法）、上样方式（杯上样、泡胀上样）、等电聚焦程序和凝胶染色方法（考马斯亮蓝R-250染色、传统银染、质谱兼容银染）等,进行了一系列的优化。实验结果通过优化获得了较为理想的电泳图谱:图谱蛋白点数明显增多（1228个vs 856个）;图谱分辨率提高,水平条带及纵向拖尾现象明显减少。同时,2-DE的重复性也有大幅度提高:对同组样品在三次不同实验中所得蛋白质斑点数目分别为1241、1192和1216个,IEF方向平均偏差1.73±0.99 mm,SDS-PAGE方向平均偏差1.11±0.75mm。本技术平台的建立为后续工作奠定了基础。第三部分树鼩肝癌发生过程中差异表达蛋白质组学的研究本部分的研究目的是应用2-DE和MALDI-TOF-MS等蛋白质组学技术,比较分析AFB₁与AFB₁+人HBV诱发树鼩肝癌发生过程中肝组织蛋白质表达谱的差异,寻找在树鼩肝癌发生过程中起重要作用的关键蛋白质分子,为探讨人HCC发生机理提供重要科学依据。本研究利用树鼩可以在实验诱癌过程中经受多次剖腹肝活检手术而长期存活的特点,对HCC形成过程中不同时期肝组织、癌前各时期的肝活检组织、肝癌和癌旁组织,以及各同期对照组织蛋白质组改变进行动态观察。由于细胞或组织蛋白质组的复杂性,应用2-DE分离总蛋白时,为保证结果的准确性及精确度,本研究共12个实验组:①AFB₁实验组（A组）,A组45周肝活检组织（A45w）、A组75周肝活检组织（A75w）、A组肝癌组织（ACa）、A组癌旁组织（ACp）;②AFB₁+HBV实验组（C组）,C组45周肝活检组织（C45w）、C组肝癌组织（CCa）、C组癌旁组织（CCp）;③HBV实验组（D组）,D组45周肝活检组织（D45w）、D组105周肝活检组织（D105w）;④正常对照组（E组）,E组45周肝活检组织（E45w）、E组75周肝活检组织（E75w）、E组105周肝活检组织（E105w）,每组6例组织样本的混合总蛋白分别进行3次2-DE以保证试验的重复性。蛋白质均用13cm pH 3-10非线性IPG胶条,采用泡胀上样方式进行等电聚焦电泳,共获得36块2D胶图谱。各组蛋白表达谱中蛋白多集中分布于pI 4-8间的区域,用ImageMaster 2D Platinum 5.0软件将凝胶分类别进行点的匹配分析。每组3块胶,各组平均检测到的蛋白点数如下: A45w组1255±43个,A75w组1197±46个,ACa组1198±50个,ACp组1205±89个;C45w组1233±30个,CCa组1257±35个,CCp组1244±39个;D45w组1100±79个,D105w组1115±61个;E45w组1249±51个,E75w组1179±62个,E105w组1208±36个。筛选出差异2倍及其以上并且在半数胶以上中均存在的蛋白点共206个。从相应的考马斯亮蓝染色的制备胶中挖取差异倍数较大且蛋白斑点较清晰的差异蛋白点160个,进行MALDI-TOF-MS/MS分析。为保证取点的准确性,每个点均从三块胶中挖取后分别进行酶解鉴定,取质谱鉴定结果一致者。鉴定出的差异蛋白点共142个,95种蛋白,包括peroxiredoxin家族（PrxⅠ、PrxⅡ、PrxⅢ、PrxⅣ、PrxⅥ）、细胞色素C氧化酶、谷胱甘肽过氧化物酶Ⅰ、硫氧还蛋白、细胞色素B5、酮己糖激酶、不均一核糖核蛋白、磷酸丙糖异构酶、尼克酰胺N-甲基转移酶、转移抑制基因NM23-H1、翻译控制肿瘤蛋白等。第四部分差异蛋白Prx家族在树鼩及人HCC组织中的表达验证由于第三部分筛选鉴定的结果显示, peroxiredoxin家族（PrxⅠ、PrxⅡ、PrxⅢ、PrxⅣ）在AFB₁诱发树鼩HCC形成过程中,多个成员蛋白均出现明显差异表达:PrxⅠ、PrxⅡ、PrxⅢ在癌组织中表达均较癌前组织明显上调,PrxⅣ则在癌组织中较癌旁组织明显上调,并且由于Prx的生物学功能广泛并与多种肿瘤相关,因此选择该组蛋白作为进一步研究目标。本部分的研究目的是对差异蛋白Prx家族在树鼩HCC发生不同阶段的蛋白水平和mRNA水平的表达差异,以及在人肝癌组织和癌旁组织间的表达差异进行再验证,进一步解析该蛋白的生物学功能,探讨Prx家族蛋白在肝癌发生发展中的作用以及其作用机制。应用Western Blot技术,本部分结果证实PrxⅠ、PrxⅡ、PrxⅢ蛋白表达水平在AFB₁诱发树鼩HCC组织（ACa）明显高于癌前组织（A45w）,略高于正常对照组织（E45w）,PrxⅣ蛋白表达水平亦发生上调,但差异未达2倍。人HCC组织验证结果,PrxⅠ、Ⅱ、Ⅲ、Ⅳ的表达量都略高于癌旁组织,但升高倍数均未达到2倍。运用RT-PCR技术检测,PrxⅠ、Ⅱ、Ⅲ、Ⅳ在树鼩和人的mRNA表达水平升高趋势与蛋白表达水平相同。Prx家族蛋白有望成为一类有应用价值的肝癌标志物蛋白,有可能应用于HCC的诊断、治疗和预后评价。结论1、树鼩作为低等灵长类动物,各项实验室指标检测结果接近正常人,为其用于建立研究人类疾病的动物模型进一步提供了支持依据。2、通过对2-DE相关技术的摸索,建立和优化了适合于本实验的树鼩肝癌蛋白质组学双向凝胶电泳技术平台,为后续研究奠定了基础。3、与对照组各时期的正常肝组织相比,树鼩HCC发生过程中各时期肝组织表现出有一定差异性的蛋白表达谱,表明HCC的发生发展是一个综合的多因素参与的过程。鉴定出的142个,95种差异表达蛋白质,可能参与肝癌的发生发展。4、Prx家族蛋白有望成为一类有应用价值的肝癌标志物蛋白,有可能应用于HCC的诊断、治疗和预后评价。潜在应用价值1、树鼩作为低等灵长类动物,适合应用于研究人类疾病的动物模型。2、建立了树鼩肝癌蛋白质组学双向凝胶电泳技术平台。3、筛检出的差异蛋白有可能成为肝癌发生的诊断标志物及预后判断指标,丰富了肝癌诊疗指标的选择与组合。4、Prx可以作为一种有应用价值的测定肝癌发生发展的标志物。创新点1.建立并优化了树鼩肝癌蛋白质组学双向凝胶电泳技术平台。2.利用树鼩可以在实验诱癌过程中经受多次剖腹肝活检手术而长期存活的特点,对HCC形成过程中不同时期肝组织的蛋白质组改变进行动态分析,筛选出一批与肝癌发生不同阶段相关的差异蛋白。3.分析了与特定病因因素:AFB₁、AFB₁ +人HBV诱发HCC相关的差异表达蛋白。4.分析了肝组织感染HBV不同时期的差异表达蛋白。5.首次证实差异蛋白PrxⅠ、PrxⅡ、PrxⅢ、PrxⅣ在AFB₁诱发的树鼩肝癌发生过程中出现上调。更多还原

【Abstract】 Hepatocellular carcinoma （HCC）, of which the morbidity and mortality are in the forefront among the overall tumors worldwide, is one of the malignancies with difficult in early diagnosis and extremely poor prognosis. Hepatitis B virus （HBV） infection and aflatoxin B₁ （AFB₁） exposure are recognized as the main factors for HCC. The formation of HCC is a multistep, multistage and multifactor process like most other tumors. The conventional strategy of research on single gene has been challenged by the rapidly progress of proteomics, which can be utilized to observe the dynamical changes of nature and quantity of total protein during progression of disease. Proteomics can be used to find new protein markers and key molecules and therefore is an effective and high-throughout method for exploring mechanism of disease and for searching targets of diagnosis or therapy. By optimizing the technical platform of two-dimensional electrophoresis （2-DE）, in the present study we observed dynamically the differentially expressed proteins of liver tissue that were taken at different time points during the hepatocarcinogenesis of tree shrew （Tupaia belangeri chinensis）, a small animal species belonged to low Primates evolutionarily. By taking the advantages that the animal can survive repeated liver biopsies during the experiment and can be infected with HBV, the present study aimed at screening differential expressed proteins in various stages of hepatocarcinogenesis induced by AFB₁ or AFB₁+HBV. Subsequently, one group of the screened proteins, peroxiredoxin family （Prxs）, was further confirmed by Western blot and RT-PCR on the tissues samples form tree shrews as well as from HCC patients. The entire study included four parts as following. Part 1. Normal physiological laboratory value of tree shrewTo further confirm the feasibility and superiority of utilizing tree shrew as animal model for studying human disease, this part is to detect the physiological laboratory values of adult tree shrew bred artificially, and to comparer the values with human normal reference.Whole blood sample and separated serum were collected from twenty two adult tree shrews bred artificially in this lab, and then all the laboratory values were determined. Blood routine examination showed that RBC count was 8.19×10¹²/L, which was higher than human normal reference value. Tree shrew WBC count was 2.2×10⁹/L, which was lower than human normal reference. Microscopic examination of whole blood smear from tree shrew showed the morphology of blood cells and platelet were similar to that of human. WBC differential count showed that each cell percentage was near to that of human. Assay of ten biochemical values of serum samples showed that seven values including urea, globulin, serum total protein, serum albumin, serumγ-GT, C-reactive protein and rheumatoid factor accorded with the range of human reference. While serum creatinine of tree shrew as 16.63μmol/L was lower than 44μmol/L of human reference, serums GPT as 77.92 U/L and GOT as 179.9 U/L were higher than the upper limit of human reference of 40 U/L and 45 U/L respectively. The result of HBsAg examination showed all of the animals were negative, with the value of 0.022 ng/ml±0.005.Analysis of these laboratory values mentioned above suggests that tree shrew, as a low grade primate related closely to humankind, is suitable as animal model for studying human disease. Part 2. Establish and optimize 2-DE technique for proteomic study on tree shrew liver samplesFor achieving a high quality on resolution and a steady result, effort was made to establish and optimize the 2-DE technique that should be adaptive for proteomic analysis on tree shrew liver samples. The conventional protocol of 2-DE was modified, especially on some critical steps such as sample processing （grinding and homogenating）, quantitative methods of protein （Lowery method and modified Bradford method）, loading modes （cup-loading and swelling-loading）, IEF procedure, gel stain methods （Coomassie brilliant blue R-250 stain, traditional silver stain, MS compatible silver stain）, and etc.The protein spots acquired from 2-DE gel by the modified method increased greatly （1228 vs 856）, along with the enhanced spot-resolution, as well as with the decreased horizontal band and vertical tail. The protein spots from same sample that run repeatedly three times of 2-DE were 1241, 1192 and 1216 respectively, with the mean shiftings as 2.0±0.56mm at IEF direction and 2.3±0.61mm at SDS-PAGE direction. Establishment of this technical platform provided the foundation for following research.Part 3. Proteomic study on differential expression during tree shrew’s hepatocarcinogenesisTo search the critical protein molecules responsible for tree shrew hepatocarcinogenesis, and to explore the mechanism of HCC. This part compared and analyzed protein profiles of tree shrew HCC induced by AFB₁ and AFB₁+HBV, by 2-DE and MALD1-TDF MS proteomic techniques.Twelve categories of liver samples were collected from differently treated animals at different time points during the experiment. Samples from the animals in group A that were treated with AFB₁ and developed HCC at late stage of the experiment, named A45w, A75w, ACa and ACp respectively, referring the samples were biopsies at 45th week （A45w） or 75th week （A75w） of the experiment before HCC appeared, and the HCC samples （ACa） or HCC-surrounding samples （ACp） were from the identical animals. Similarly, the samples from the animals in group C that were not only treated with AFB₁ but also infected with HBV and developed HCC at late stage of the experiment were named C45w, CCa and CCp respectively. The samples from the animals in group D that were only infected with HBV, had no HCC developed until the experiment ended at 165th week, were named D45w and D105w respectively. The samples from the control animals in group E that treated nothing else but only breed normally were named E45w, E75w and E105w respectively. The mixed total protein from samples in each category was assayed repeatedly with 2-DE method. Thirteen centimeters non-linear IPG gel strip with pH 3-10 was adopted. The IEF electrophoresis was carried out by applying the swelling sample, and pictures from 36 blocks of 2-DE gels were acquired.Profile of proteins was similar among the different categories of sample as most protein spots distributed inside the field of pI 4-8. ImageMaster 2-DE Platimum 5.0 software was utilized to carry out analysis for each well-matched gel dot between the comparable categories. The mean detected numbers of protein spot in each categories were as follows: 1255±43 in A45w, 1197±46 in A75w, 1198±50 in ACa, 1205±89 in ACp; 1233±30 in C45w, 1257±35 in CCa, 1244±39 in CCp; 1100±79 in D45w, 1115±61 in D105w; 1249±51 in E45w, 1179±62 in E75w, 1208±36 in E105w. Two hundred and six protein points which had double or more density and appeared in more than fifty percent gels were screened out.One hundred sixty protein spots with different expression were dug out from the corresponding prepared Coomassie brilliant blue stain gels and one hundred forty-two spots were identified by MALDI-TOF MS. To assure the accuracy of blots sampling, each spot dug out from three blocks of gel was identified and only identical data in MS was selected. The identified differential proteins included the members of Peroxiredoxin family （PrxⅠ, PrxⅡ, PrxⅢ, and PrxⅣ）, cytochrome C oxidase, glutathione peroxidaseⅠ, thioredoxin, cytochrome B5, ketohexose kinase, heterogeneous ribose nucleoprotein, phosphotriose isomerase, nicotinamide N-methyl transferase, metastasis suppressed gene NM23-H1, translation controlled tumor protein, and others.Part 4. Verify tests on differential expression of peroxiredoxins among liver samples from tree shrews and from HCC patientsThe members in peroxiredoxin family （PrxⅠ, PrxⅡ, PrxⅢand PrxⅣ）, which were among the differentially expressed protein screened from Part 3 and were known had widespread biological functions concerning many other tumors, were studied further to confirm the 2-DE results that showed the expressions of PrxⅠ, PrxⅡ, and PrxⅢin HCC tissue were up-regulated greatly compared to precancerous tissue, and the expression of PrxⅣwas up-regulated prominently in HCC tissue compared to HCC-surrounding liver tissue. By Western blot and RT-PCR assays, the expression levels of PrxⅠ, PrxⅡ, PrxⅢand PrxⅣwere examined on the samples from tree shrew as well as from HCC patients.Results confirmed that the expressions of PrxⅠ, PrxⅡ, PrxⅢ, and PrxⅣwere not only up-regulated in tree shrew HCC but also in human HCC tissue in both protein level and mRNA level, which indicated that the proteins in Prx family might play important role in hepatocarcinogenesis and could be hopefully applied as a biomarker for HCC diagnosis and/or treatment. ConclusionTree shrew, a lower grade primate related closely with humankind evolutionarily, is proved suitable as animal model to research human disease because most of its laboratory values were found similar to that of human beings.The modified 2-DE technical platform for proteomic analysis on liver tissues of tree shrews provides the foundation for research. Profiles of differentially expressed protein screened from the tissues taken at different time points during tree shrew hepatocarcinogenesis induced by AFB₁ or AFB₁+HBV suggests the progression of HCC is concerned with many factors. At least some of the proteins among the 142 identified ones might play important roles in the HCC initiation, development and/or progression. Particularly, proteins in Prx family might be applied as a biomarker for HCC diagnosis, treatment and/or prognosis-evaluation.更多还原