【作者】 刘学庆；

【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: 心脏发育是一复杂的过程,涉及许多蛋白质的参与。本研究运用蛋白质组相关研究技术,分析妊娠 14d、16d 和 18d 胎鼠心脏蛋白质组,重点研究 14d、16d、18d 胎心蛋白质差异表达,从而发现不同发育时期蛋白的动态变化,探讨心脏发育的分子机制及先天性心脏病的发病机制。 方法: 1、建立 NIH 小鼠妊娠动物模型,分离妊娠 14d、16d 和 18d 胎鼠心脏,-80℃冰箱保存。 2 、 运 用 二 维 聚 丙 烯 酰 胺 凝 胶 电 泳 (two-dimensional polyacrylamide gel electrophoresis,2-DE)技术,分离妊娠 14d, 16d和 18d 不同时期的胎鼠心脏蛋白质组,考马斯亮蓝染色,PDQuest 软件进行图像分析。 3、对 14 个妊娠 14d、16d、18d 胎心差异表达候选蛋白斑点进行胶内酶切,基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser disorption/ionization-time of flight-mass spectrometry, MALDI-TOF-MS)分析酶切片段,得到肽指纹图(peptide massea fingerprint, PMF),利用 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%重庆医科大学硕士研究生学位论文 4(p<0.05),则该事件为有意义。结果:1、2-DE 图谱显示,胎鼠心脏蛋白质分子量(Mw)主要集中在 20至 116 之间,等电点(pI)主要分布在 pH4.0-8.5 之间。PDQuest 软件分析图像,结果显示妊娠 14d 胎鼠心脏大约有 565 个蛋白点、16d 胎鼠心脏大约有 711 个蛋白点、18d 大约有 773 个蛋白质点。其中妊娠 16d胎鼠心脏蛋白点与 14d 相比,约有 22 个蛋白点有 2 倍以上的上调,8个蛋白点下调。妊娠 18d 胎心与 14d 相比约有 39 个蛋白点有 2 倍以上的上调,11 个点有超过 2 倍以上的下调。2、选择 14 个差异表达候选蛋白质点的 PMF 在国际互联网上查询,有四个蛋白质点获得有意义的结果(p <0.05),分别为热休克蛋白 60(Heat shock protein 60,Hsp60)、细胞角蛋白 G6(cytoskeletal 6G )和 两 个 蛋 白 点 均 鉴 定 为 丝 分 裂 原 活 化 蛋 白 激 酶 激 酶 激 酶 4(Mitogen-activated protein kinase kinase kinase4,MAPKKK4 或MEKK4)。结论:1、在小鼠胚胎的发育中,胚胎心脏的分化和功能的完善需要大量的蛋白质参与。2、Hsp60 是一个分子量为 60kDa 的蛋白质,普遍存在于原核细胞和真核细胞中,主要存在于细胞质和线粒体基质中,其一是起分子伴侣(moleculer chaperonin )的作用,即对新生蛋白质进行折叠、转运和定位。当机体受到协迫时,热休克蛋白大量合成,以恢复变性蛋白或凝集蛋白的天然构像;其二调控细胞分裂和细胞凋亡。在心脏发育不同时期的表达,对心脏细胞生长和发育以及功能的完善和实现具有重要作用。3 、 MAPKKK4 是 参 与 构 成 细 胞 主 要 信 号 传 递 系 统 MAPK更多还原

【Abstract】 Objective The development of heart involve intricacy process. A number of proteins were involved in the process. In the present study, we analyed the proteome of fetal heart tissues from Day 14d, 16d and 18d in NIH mice. Specially, we studied differential protrome of fetal heart of mice on 14d, 16d and 18d of pregnancy by using comparative proteomics. The aim of this work is to acquire knowledge about the mechanism in the development of heart. Methods 1. Found the NIH mouse pregnant animal model, the fetal heart tissue of 14d, 16d and 18d were separated from embryo of NIH mice, then immediately stored in -80℃ untill processed. 2. The proteins of pregnant 14d, 16d and 18d were separated by two-dimensional gel electrophoresis. The two-dimensional gels were stained by Coomassie Blue and subjeted to analyse by PDQuest software. 3. Differential protein spots were excised from the gels and digested with trypsin. The peptides were analysed by MALDI-TOF-MS, the peptides mass fingerprint(PMF) was obtained. PMF were analysed by Moscot software. Results 1. 2-D PAGE profile showed that the proteins in the fetal heart were located mostly in the range of mass 20-116 kda and isoelectric points(pI) at 4.0-8.5. Analysing from the profile by PDQuest software, there are 565 protein spots in pregnant 14d, 711 protein spots in pregnant 16d, 773 protein spots in pregnant 18d. Comparing between pregnant 14d and 16d, at least 22 protein spots were up-regulated by over two fold and 8 protein spots were down-regulated. Comparing between pregnant 14d and 18d, at least 39 protein spots were up-regulated and 11 protein spots were down-regulated by over two fold respectively. 2. PMF of 14 differential protein spots were searched by database. 4 protein spots of them were identified unambiguously. They are Heat shock protein 60 (Hsp60), cytoskeletal 6G, mitogen-activated protein kinase kinase kinase4(MAPKKK4 or MEKK4). Among them , another 2 protein spots were identified as MAPKKK, but they were not isoform. Conclusions 1. during the development of fetal heart of mice, a lot of proteins were participated in this process. 2. Molecular weight of Hsps 60KDa. It exist mainly in cytoplasm and mitochondria and play a role of molecular chaperonin, viz. protein folding, transportation orientation. When the body is stressed, the synthesis of Hsp60 can be markedly induced to stabilize and refold proteins. Hsp60 is benefited for the function of heart as it is expressed in the different time of embryo. 3. MAPKKK4 or MEKK4 is one of the members of the major systems participating in the cellular signal transduction of MAPK. It can activate signal pathway of JNK and regulate consequently cell division and apoptosis. By a series of signal transduction, it play an important role for cell growth, differentiation, development and death. 4. Cytoskeleted 6G participate in the forming of hair. It is possibly pollution of protein.更多还原