1.鼻咽癌细胞系HNE1细胞膜蛋白质组分析和膜蛋白数据库的构建 2.肝脏星状细胞（HSC）细胞膜蛋白质组分析
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摘要
生物膜是细胞结构的重要基础，大部分细胞器都是以生物膜为基础构建的，它是细胞内外，以及各亚细胞器间物质与信息交换场所，也是细胞内外因子识别的靶标所在场所。膜蛋白质组是在特定的时空里，细胞中所有膜蛋白的总和。膜蛋白质组的研究具有很重要的意义，因为它不仅有助于对细胞信号传导，细胞与细胞间的相互作用，离子以及溶液的传递的研究，同时也为疫苗研究，病理研究提供了新的方法与手段。
鼻咽癌(Nasopharyngeal carcinoma,NPC)是高发于我国南方的一种恶性肿瘤，其发病率和死亡率均居世界首位。本文以鼻咽癌细胞系HNE1为材料，以贴壁的方式对其进行培养，满瓶后将细胞收集以低渗破膜与蔗糖密度梯度离心相结合的方法分离纯化得到质膜，并以扫描电镜对其进行了分析，证实了分离的质膜材料的可靠性。然后以裂解液抽提出质膜的全蛋白，用固相pH梯度双向聚丙烯酰胺凝胶电泳对膜蛋白进行分离，并以银染显色，得到了清晰的HNE1膜蛋白图谱。经Bio-Rad公司的PDQUEST软件进行图象分析，检测到600个左右的蛋白质点。将胶上的蛋白质点切下经脱色，原位还原和烷基化处理后，以胰蛋白酶进行酶解，再用MALDI-TOF MS和ESI-Q-TOF分析，得到了质量较好的肽质谱指纹图(PMF)和MS／MS数据，然后将其在MS-Fit中的genepeptide数据库和MASCOT的Swissprot中进行搜索从而对蛋白质点进行鉴定。我们对其膜蛋白双向电泳图谱的部分蛋白质点进行了鉴定。其中有196个蛋白点得到了归属，这些蛋白质中大部分为质膜蛋白质(92个)如Na~+-K~+-ATP酶，热休克蛋白27，整联蛋白β-8亚基，G蛋白偶联受体，丝氨酸-苏氨酸特异性蛋白磷酸酶，酪氨酸蛋白激酶受体，INT-1蛋白和载脂蛋白受体2前体等重要的膜上标志性蛋白。其中的囊纤维化跨膜传导调节物，属于整合膜蛋白，与膜上的氯离子的转运密切相关；而酪氨酸蛋白激酶受体，则是一种重要的Ⅰ类膜蛋白，与细胞和细胞之间的识别有着密切的联系。
多年来，人们进行了大量的实验及临床研究，以探讨肝纤维化
一一一一一一一一一一一一一一一二竺鲤乳一一一-___
的可逆性。西方医学界公认肝纤维化是可逆的，而肝硬化是不可逆
的。以分子生物学手段研究发现，中药复方861对血小板生长因子
(PDGF)诱导的HSC增殖有很强的抑制作用，因此，本文以肝星状
细胞为材料进行细胞膜蛋白质组学分析，以探求复方861的抗纤维
化作用的机理。当肝星状细胞贴壁培养丰度达到70%左右时更换培养
基，向其中加入lmg/ml的复方861合剂作用48小时，分为给药组
和对照组分别培养(作用剂量和作用时间都是经过了不同大小和长
短的比较之后优化选择的)。满瓶后将细胞收集以低渗破膜的方法分
离纯化得到质膜，并以扫描电镜对其进行了分析，从电镜照片上可
见到经低渗后，细胞膜破裂，细胞内容物流出而细胞膜有的完全破
碎，有的形成血影状的空泡，从而证实了材料的可靠性。然后以裂
解液抽提出质膜蛋白，用固相pH梯度聚丙烯酞胺凝胶电泳对膜蛋白
进行分离，获得了分辨率和重复性俱佳的双向凝胶电泳图谱。运用
Bi。一Rad公司的PDQUEST软件进行图象分析，检测到600个左右的蛋
白质点。并对给药组和对照组进行了差异表达分析，找到了一些具
有表达量差异的蛋白质点。选取全部的有无差异点和部分上调和下
调的胶上的蛋白质点切下，经脱色，原位还原和烷基化处理后，以
胰蛋白酶进行酶解，再用基质辅助激光解吸电离飞行时间质谱
(MALD工一TOF MS)得到肤质谱指纹图，然后将PMF数据在MS一Fit中
的genepeptide数据库中进行搜索从而对蛋白质点进行鉴定。初步
鉴定出胶原质IV的Q亚基，低密度脂蛋白，Ly49c等一些与肝纤
维化密切相关的蛋白质，其中胶原质IV的Q亚基属于H类膜蛋
白，其功能与肝纤维化相关而且在给药组明显下调。这些蛋白质表
达量的变化可能有助于我们对复方861合剂的抗肝纤维化的重要作
用机理的了解。
The plasma membrane proteins, as the "doorbells" and "doorways" of the cell, play crucial roles to the cell function. The proteomic definition of plasma membrane proteins is an important initial step in searching for novel tumor marker proteins expressed during the different stages of cancer progression.
Nasopharyngeal carcinoma (NPC) is a commonly occurring tumor in southern China and south east Asia, occurring as one of the top ten cancers in frequency. This project was initiated with the purpose of separating and identifying the membrane protein of a human nasopharyngeal carcinoma cell line. We purified the crude plasma membrane and validated by electron microscopy observation. After two dimensional gel electrophoresis separation, silver staining, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and ESI-Q-TOF analyses, tryptic peptide masses were searched for matches in the SWISS-PROT, NCBI and Mascot databases. Over 196 spots were identified using this approach (including 92 known membrane proteins). These proteins include those involved in regulation of gene expression, affected the signal pathway, altered the cell metabolism, inhibit or accelerate cell proliferation, induce the cell apoptosis and intervened the tumor invasion. This study of the NPC proteome, c
oupled with similar proteome analyses of the whole cell, the normal nasopharyngeal tissues, and other nasopharyngeal carcinoma cell lines, defines the proteome of the NPC plasma membrane, which are valuable resources in studies on the differential protein expressions of human nasopharyngeal carcinoma.
Herbal compound 861(Cpd861) is an effective compound in therapy against liver fibrosis and early hepatocirrhosis. To elucidate the antifibrotic mechanism of herbal compound 861, we employed proteomics techniques to study only the membrane proteins change in cultured rat hepatic stellate cells treated with Cpd861. Twenty five proteins that
change in cultured rat hepatic stellate cells treated with Cpd861. Twenty five proteins that were up- and down-regulated were identified unambiguously, and the functional implications of 18 of these proteins are discussed in some detail.
鼻咽癌(Nasopharyngeal carcinoma,NPC)是高发于我国南方的一种恶性肿瘤，其发病率和死亡率均居世界首位。本文以鼻咽癌细胞系HNE1为材料，以贴壁的方式对其进行培养，满瓶后将细胞收集以低渗破膜与蔗糖密度梯度离心相结合的方法分离纯化得到质膜，并以扫描电镜对其进行了分析，证实了分离的质膜材料的可靠性。然后以裂解液抽提出质膜的全蛋白，用固相pH梯度双向聚丙烯酰胺凝胶电泳对膜蛋白进行分离，并以银染显色，得到了清晰的HNE1膜蛋白图谱。经Bio-Rad公司的PDQUEST软件进行图象分析，检测到600个左右的蛋白质点。将胶上的蛋白质点切下经脱色，原位还原和烷基化处理后，以胰蛋白酶进行酶解，再用MALDI-TOF MS和ESI-Q-TOF分析，得到了质量较好的肽质谱指纹图(PMF)和MS／MS数据，然后将其在MS-Fit中的genepeptide数据库和MASCOT的Swissprot中进行搜索从而对蛋白质点进行鉴定。我们对其膜蛋白双向电泳图谱的部分蛋白质点进行了鉴定。其中有196个蛋白点得到了归属，这些蛋白质中大部分为质膜蛋白质(92个)如Na~+-K~+-ATP酶，热休克蛋白27，整联蛋白β-8亚基，G蛋白偶联受体，丝氨酸-苏氨酸特异性蛋白磷酸酶，酪氨酸蛋白激酶受体，INT-1蛋白和载脂蛋白受体2前体等重要的膜上标志性蛋白。其中的囊纤维化跨膜传导调节物，属于整合膜蛋白，与膜上的氯离子的转运密切相关；而酪氨酸蛋白激酶受体，则是一种重要的Ⅰ类膜蛋白，与细胞和细胞之间的识别有着密切的联系。
多年来，人们进行了大量的实验及临床研究，以探讨肝纤维化
一一一一一一一一一一一一一一一二竺鲤乳一一一-___
的可逆性。西方医学界公认肝纤维化是可逆的，而肝硬化是不可逆
的。以分子生物学手段研究发现，中药复方861对血小板生长因子
(PDGF)诱导的HSC增殖有很强的抑制作用，因此，本文以肝星状
细胞为材料进行细胞膜蛋白质组学分析，以探求复方861的抗纤维
化作用的机理。当肝星状细胞贴壁培养丰度达到70%左右时更换培养
基，向其中加入lmg/ml的复方861合剂作用48小时，分为给药组
和对照组分别培养(作用剂量和作用时间都是经过了不同大小和长
短的比较之后优化选择的)。满瓶后将细胞收集以低渗破膜的方法分
离纯化得到质膜，并以扫描电镜对其进行了分析，从电镜照片上可
见到经低渗后，细胞膜破裂，细胞内容物流出而细胞膜有的完全破
碎，有的形成血影状的空泡，从而证实了材料的可靠性。然后以裂
解液抽提出质膜蛋白，用固相pH梯度聚丙烯酞胺凝胶电泳对膜蛋白
进行分离，获得了分辨率和重复性俱佳的双向凝胶电泳图谱。运用
Bi。一Rad公司的PDQUEST软件进行图象分析，检测到600个左右的蛋
白质点。并对给药组和对照组进行了差异表达分析，找到了一些具
有表达量差异的蛋白质点。选取全部的有无差异点和部分上调和下
调的胶上的蛋白质点切下，经脱色，原位还原和烷基化处理后，以
胰蛋白酶进行酶解，再用基质辅助激光解吸电离飞行时间质谱
(MALD工一TOF MS)得到肤质谱指纹图，然后将PMF数据在MS一Fit中
的genepeptide数据库中进行搜索从而对蛋白质点进行鉴定。初步
鉴定出胶原质IV的Q亚基，低密度脂蛋白，Ly49c等一些与肝纤
维化密切相关的蛋白质，其中胶原质IV的Q亚基属于H类膜蛋
白，其功能与肝纤维化相关而且在给药组明显下调。这些蛋白质表
达量的变化可能有助于我们对复方861合剂的抗肝纤维化的重要作
用机理的了解。
The plasma membrane proteins, as the "doorbells" and "doorways" of the cell, play crucial roles to the cell function. The proteomic definition of plasma membrane proteins is an important initial step in searching for novel tumor marker proteins expressed during the different stages of cancer progression.
Nasopharyngeal carcinoma (NPC) is a commonly occurring tumor in southern China and south east Asia, occurring as one of the top ten cancers in frequency. This project was initiated with the purpose of separating and identifying the membrane protein of a human nasopharyngeal carcinoma cell line. We purified the crude plasma membrane and validated by electron microscopy observation. After two dimensional gel electrophoresis separation, silver staining, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and ESI-Q-TOF analyses, tryptic peptide masses were searched for matches in the SWISS-PROT, NCBI and Mascot databases. Over 196 spots were identified using this approach (including 92 known membrane proteins). These proteins include those involved in regulation of gene expression, affected the signal pathway, altered the cell metabolism, inhibit or accelerate cell proliferation, induce the cell apoptosis and intervened the tumor invasion. This study of the NPC proteome, c
oupled with similar proteome analyses of the whole cell, the normal nasopharyngeal tissues, and other nasopharyngeal carcinoma cell lines, defines the proteome of the NPC plasma membrane, which are valuable resources in studies on the differential protein expressions of human nasopharyngeal carcinoma.
Herbal compound 861(Cpd861) is an effective compound in therapy against liver fibrosis and early hepatocirrhosis. To elucidate the antifibrotic mechanism of herbal compound 861, we employed proteomics techniques to study only the membrane proteins change in cultured rat hepatic stellate cells treated with Cpd861. Twenty five proteins that
change in cultured rat hepatic stellate cells treated with Cpd861. Twenty five proteins that were up- and down-regulated were identified unambiguously, and the functional implications of 18 of these proteins are discussed in some detail.
引文
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