人胎肝细胞质膜和人红细胞膜蛋白质组分析

发布时间：2018-05-24 08:48

本文选题：人胎肝 + 细胞质膜　；参考：《中国人民解放军军事医学科学院》2005年博士论文

【摘要】：细胞质膜构成细胞对外界环境的屏障和细胞内外环境交流的界面,镶嵌或连接于其中的蛋白质对维持细胞正常的生理活动具有十分重要的作用,这些蛋白质参与细胞—细胞或细胞—细胞外基质的识别、粘附、连接和通讯,信号的接受和跨膜传导,细胞内外物质的转运等,影响着细胞的生长、发育、分化、病变等。细胞质膜蛋白占到已知药物靶标的70%,因此,细胞质膜已经成为广泛的药物设计的靶标,细胞质膜蛋白质组得到学术界的高度重视。 16-24周孕龄人胎肝处于关键的造血调控和发育分化时期,分析人胎肝细胞质膜蛋白质组有助于对人胎肝的这些生理活动的认识。我们用差速离心和密度梯度离心提取纯化了人胎肝细胞质膜,细胞质膜标志酶—5’单核苷酸酶特异酶活测定显示其富集度达到16倍。用SDS-PAGE-RPLC-ESI-MS/MS和“shotgun”(蛋白三重裂解—SCX—RPLC-ESI—Q-TOF-MS)两条技术路线,我们从人胎肝细胞膜和碱处理细胞膜中鉴定了总共532个非冗余蛋白质,另有82个蛋白group。这是目前已知最大的人类组织细胞质膜表达谱。在人胎肝细胞质膜蛋白质表达谱中,已知明确定位于细胞质膜上的蛋白质126个,占鉴定蛋白总数的24.5%。跨膜蛋白162个,占30.5%,未知蛋白41个。126个细胞质膜蛋白基本覆盖了细胞质膜上所有的功能模块,其中转运体、膜骨架、信号转导、受体、细胞连接、细胞粘附和代谢酶类蛋白所占比例最高,分别为18、15、14、11、10、9和6%。这与人胎肝细胞质膜的结构和生理功能密切相关。另外,在人胎肝细胞质膜上鉴定了一些已报道在细胞质膜上有定位但常见于其它亚细胞结构的蛋白,如细胞质蛋白HSP27和HSP70;内质网蛋白GRP和PDI;线粒体蛋白VDAC-1、HSP60和ATP合成酶;细胞核蛋白Lasp-1,hRNP和组蛋白。这些蛋白质可能参与新的细胞质膜上的生理活动。鉴于在提取人胎肝细胞质膜时不可避免地存在其它内膜系统的污染,对于鉴定的非已知细胞质膜蛋白,我们不能确定是发生真正的定位变化而存在于细胞质膜还是其它亚细胞结构的污染。正常人红细胞只由胞浆和细胞膜构成,因而红细胞膜不会有其它亚细胞结构的污染。所以我们选用正常人红细胞膜作为模式细胞膜进一步对其蛋白质组进行分析。通过与研究人胎肝细胞质膜相同的
[Abstract]:The cytoplasmic membrane forms the barrier of cell to the outside environment and the interface of the communication between the inside and outside of the cell. The protein embedded or connected to it plays a very important role in maintaining the normal physiological activities of the cell. These proteins are involved in the recognition, adhesion, connection and communication of cell-cell or cell-extracellular matrix (ECM), signal reception and transmembrane transduction, and the transport of substances inside and outside the cell, which affect cell growth, development, differentiation, pathological changes and so on. Cytoplasmic membrane proteins account for 70 percent of the known drug targets. Therefore, cytoplasmic membrane has become a widely used target for drug design. Human fetal liver at 16-24 weeks of gestational age is in a critical stage of hematopoietic regulation and differentiation. Analyzing the proteome of plasma membrane of human fetal liver cell is helpful to the understanding of these physiological activities of human fetal liver. The plasma membrane of human fetal hepatocytes was extracted and purified by differential centrifugation and density gradient centrifugation. Using SDS-PAGE-RPLC-ESI-MS/MS and "shotgun" (triple cleavage of protein, SCX-RPLC-ESI-Q-TOF-MS), a total of 532 non-redundant proteins were identified from human fetal liver cell membrane and alkali-treated cell membrane, and 82 protein groups were identified. This is the largest known expression profile of the plasma membrane of human tissue cells. In the plasma membrane protein expression profile of human fetal liver cells, 126 proteins were known to be located on the cytoplasmic membrane, accounting for 24.5% of the total identified proteins. Transmembrane proteins 162 (30.5%), unknown proteins 41. 126 cytoplasmic membrane proteins basically cover all functional modules on the cytoplasmic membrane, including transporters, membrane cytoskeletons, signal transduction, receptors, cell junctions. The proportion of cell adhesion and metabolizing enzyme proteins was the highest, which was 1815, 141110109 and 6g, respectively. This is closely related to the structure and physiological function of the plasma membrane of human fetal hepatocytes. In addition, some proteins, such as cytoplasmic proteins HSP27 and HSP70, endoplasmic reticulum proteins GRP and PDI, mitochondrial proteins VDAC-1HSP60 and ATP synthase, were identified on the plasma membrane of human fetal hepatocytes. Nuclear protein Lasp-1 hRNP and histone. These proteins may be involved in physiological activities on the new cytoplasmic membrane. In view of the inevitable contamination of other intimal systems in the extraction of the plasma membrane of human fetal hepatocytes, the identification of non-known cytoplasmic membrane proteins, We are not sure whether a real localization change occurs in the cytoplasmic membrane or other subcellular structure contamination. Normal human erythrocytes consist only of cytoplasm and cell membrane, so there is no other subcellular structure contamination in erythrocyte membrane. So we selected the normal human erythrocyte membrane as the model cell membrane to further analyze its proteome. The same as the study of the plasma membrane of human fetal hepatocytes
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R341

【引证文献】