【作者】 杨曦；

【导师】 王应雄；

【作者基本信息】 重庆医科大学 ， 遗传学， 2005， 硕士

【摘要】 目的: 胚胎时期的肝脏组织是造血、免疫和肝脏系统干/祖细胞的主要来源,胚胎肝细胞(fetal liver cell,FLC)也被广泛用于多种疾病的临床治疗,但至今对胎肝发育及 FLC 疗法的作用机制认识尚不一致。在胚胎发育过程中,肝脏在形态与生化方面发生了一系列的变化,而这些变化的物质基础是蛋白质。本研究运用蛋白质组相关技术,分析胚胎发育不同阶段肝脏全蛋白质组的变化,有助于从蛋白质分子水平探讨胚胎肝脏发育过程中蛋白质整体变化规律以及胚胎肝脏参与造血、免疫的分子机制,为临床应用 FLC 疗法及先天性肝脏疾病的治疗奠定基础。 方法: 1、建立 NIH 小鼠妊娠动物模型,分离妊娠 12d、13d、14d、16d、18d 小鼠的胚胎肝脏组织,同时分离成鼠(未妊娠的成年母鼠)的肝脏组织,分别于-80℃冻存。 2、用双向凝胶电泳(2-dimensional gel electrophoresis, 2-DE )分别分离 12d、13d、14d、16d、18d 胎鼠及成鼠的肝脏组织蛋白质,考马斯亮蓝染色,PDQuest 软件进行图像分析。 3、对 14 个差异表达蛋白斑点用胰酶进行胶内酶切,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析酶切片断,得到肽质量指纹图(PMF),使用国际互联网上 Matrix Science 公司提供的 PMF鉴定程序(Mascot: Peptide Mass Fingerprint)进行查询,每个查询得到一个分值 (Mowse Score),根据分值计算概率来评价每个检索结果是重庆医科大学硕士研究生学位论文 4否有意义。通常可接受的域值是:一个事件随机发生的概率小于 5%,则该事件为有意义(p<0.05)。结 果:1、2-DE 图谱显示,小鼠肝脏组织蛋白质分子量 (Mw) 主要集中在 14.4~116 kDa 范围内,等电点(pI)主要分布在 pH 4.0~8.0 之间。PDQuest 软件分析图像,检测到 12d、13d、 14d、16d、18d 的胎鼠及成鼠肝脏组织蛋白点分别约为 280 个、466 个、790 个、805 个、850个、925 个。特别对 14d、16d、18d 胎鼠和成鼠图谱进行分析,以 18d的胎肝图谱为参考,14d、16d 胎肝图谱与其匹配率分别为 57%、61%,成鼠肝脏图谱与其匹配率为 69%。2、对 14 个差异候选蛋白点进行 MALDI-TOF-MS 鉴定,经数据库查询,6 个蛋白点获得了有意义的结果(p<0.05),分别为细胞周期依赖激酶调节亚基 1(Cyclin-dependent kinases regulatory subunit 1,CKS-1)、凡科尼蛋白(Fanconi anemia group A protein homolog ,FACAprotein) 、 酪 氨 酸 蛋 白 激 酶 BLK (Tyrosine-protein kinase BLK,p55-BLK)、通用转录因子 3C 多肽 4(General transcription factor 3Cpolypeptide 4, TF3C-delta)、 TBC1 家族因子 13(TBC1 domain familymember 13)、巨噬细胞受体 MARCO (Macrophage receptor MARCO ,MARCO)。其中 CKS-1、FACA protein 在 18d 胎鼠及成鼠肝脏组织中被检测到, TF3C-delt、 p55-BLK 在 14d 胎鼠肝脏组织中有较高表达,TBC1 domain family member 13 在 14d、16d、18d 胎鼠及成鼠肝脏组织中被检测到,Macrophage receptor MARCO 在 14d 胎鼠及成鼠肝脏组织中有较高表达。结 论1、在鼠胚发育中,肝脏经历了一个迅速生长发育的过程,其合成重庆医科大学硕士研究生学位论文 5蛋白的种类和数量都发生了很大的变化,这些变化与胎肝生长发育、结构分化、功能完善密切相关。2、CKS-1属于CKS家族,它能连接细胞周期依赖激酶(CDK)的催化亚基,具有调节功能而无催化功能,是CDK发挥其生物学活性所必需的。CDK 是一类重要的丝氨酸/苏氨酸蛋白激酶,主要生物学作用是启动DNA的复制和诱发细胞的有丝分裂。CKS-1可能通过调节CDK,对胎肝细胞的增殖和生长起着重要的作用。3、凡科尼蛋白(FACA protein)是一种多亚基(包括 PHF9, FANCA,FANCC, FANCE, FANCF 和 FANCG)的 DNA 修复蛋白,主要分布在细胞核,细胞质中也有少量表达,它能进行复制后修复或发挥细胞周期检查点功能,与 DNA 双链的修复和维持正常的染色体稳定性有关。在胚胎肝脏的发育中,该蛋白可能通过对 DNA 进行复制后修复,确保遗传信息的准确传递,从而保证胎肝正常的发生和发育,同时发挥细胞周期检查点功能,促进胎肝细胞迅速的增殖、生长。4、酪氨酸蛋白激酶 BLK 属于酪氨酸蛋白激酶(TPK)家族,SRC亚家族,是包含有 SH2 和 SH3 结构的蛋白,它参与 B 淋巴细胞抗原受体的信号转导。在胚胎肝脏中该蛋白可能通过介导 B 淋巴细胞抗原受体的信号转导,促进胎肝中 B 淋巴细胞迅速的增殖、活化、合成和分泌免疫球蛋白,可能在胎肝免疫功能完善中发挥着重要的作用。5、TF3C-delta是RNA聚合酶Ⅲ转录一些小细胞核和细胞质RNAs所不可缺少,包括 5S RNA、tRNA 以及细胞和病毒起源的腺病毒相关的 RNA(VA),还可与 GTF3C5 一起通过与 BRF1、 POLR3C 和POLR3F 直接作用恢复 TFIIIB 和 RNA 聚合酶的活性。TF3C-delta 对特定底物组蛋白 H3 具有组蛋白乙酰转移酶活性。该蛋白可能通过调节RNA 的转录,促进胎肝细胞核酸和蛋白质的合成,并可能涉及胎肝功更多还原

【Abstract】 Objective Haematogenesis, immune and stem /progenitor cells of the liversystem are mainly from the liver tissue of the embryo. Fetal liver cells werecommonly used in the clinic treatment of various diseases. Up to now, it isnot clear about the molecular mechanism of the liver embryogeny and theFLC therapeutics. Liver experiences changes in morphology andbiochemistry depend on protein expression during these stages. To discussthe whole rule of change about protein in the process of development onthe level of protein molecule and to find out the molecule base of fetal liveron haematogenesis and immune, We analyzed the proteome from the liverin the different developmental stages of embryo by comparative proteomics.Our aim is to established the base of action in the clinic application of FLCand treatment of congenital disease of liver.Methods1. Found the NIH mouse pregnant animal model, livers were separated from embryo mouse E12d, E13d, E14d, E16d, E18d and adult mouse, then immediately stored in -80℃.2. The livers were separated in the first dimension by isoelectric focusing in linear pH 3~10 immobilized pH gradient (IPG). The separation in the second dimension was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie Blue Staining of two-dimensional gel electrophoresis gels wereanalysised by PDQuest Software.3. Target protein spots were excised from the gel and the digested with trypsin. The MALDI-TOF mass spectrometer was operated in the reflector mode. A mass list of peptides was obtained for the target protein digest. Then peptide mass fingerprint was submitted to an appropriate software (Mascot: Peptide Mass Fingerprint) for identify the protein (http://www.matrixscience.com/cgi/search_form.).Results1. 2-D PAGE profile show that these proteins in the liver were located mostly in the range of mass 14.4~75.4 kDa and isoelectric points (pI) 4~8. 280,466,790,805,850 and 925 protein spots with a molecular mass were catalogued from Coomassie Brilliant Blue-stained gels of the E12d,E13d,E14d,E16d,E18d fetal liver and adult liver respectively. When E18d gel acted as master,The number of matched protein spots E14d,E16d and adult gel was about 57%,61% ,69% respectively.2. Fourteen differentially expressed protein spots were analysed by MALDI -TOF-MS. Six protein spots of them were identified clearly by sequence database searching. They are Cyclin-dependent kinases regulatory subunit 1 (CKS-1),Fanconi anemia group A protein homolog (FACA protein),Tyrosine-protein kinase BLK (p55-BLK),General transcription factor 3C polypeptide 4 (TF3C-delta), TBC1 domain family member 13 , Macrophage receptor MARCO (Macrophage receptor with collagenous structure). CKS-1 and FACA protein were detected in gels of E18 fetal liver and adult mouse liver, p55-BLK,TF3C-delta were significantly higher expressed on gel of E14d fetal liver, TBC1 domain family member 13 was detected in gels of E14d, 16d, E18d fetal liver and adult mouse liver, Macrophage receptor MARCO was significantlyhigher expressed in gels of E14d fetal liver and adult mouse liver.Conclusion1. The liver go through an evident change in the expression of proteins during the embryo development,the patterns and amount of proteins are increasing. These changes are relevant with the development, structure differentiation and function perfection of fetal liver.2. CKS-1 belongs to the CKS family. It can bind to the catalytic subunit of the cyclin dependent kinases(CDK) and is essential for their biological function. CDK is an important serine/threonine protein kinase. The main function of CDK is to start up the DNA copy and place a premium on cell mitosis. CKS-1 may play a key role in proliferation, growth of the fetal liver cell by regulation CDK.3. FACA protein belongs to a multisubunit complex and mainly expressed in karyon, and at lower levels in cytoplasm. It is the DNA repairing protein that may operate in a post-replication repairing or a cell cycle checkpoint function. It may be related with DNA repair and the mainten更多还原