【作者】 林源吉；

【导师】 张铭；

【作者基本信息】 浙江大学 ， 细胞生物学， 2010， 博士

【摘要】 细胞膜(Cell membrane)是细胞内外环境交流的界面,也是细胞与周围环境以及细胞与细胞之间进行物质交换和信息传递的重要通道。细胞膜表面蛋白是细胞膜的重要组成部分,它们参与了细胞的识别和免疫应答、信号传导和调控、细胞内物质及能量的传递等重要功能,对细胞的生存、生长、分裂和分化都至关重要。细胞膜蛋白质组学是研究细胞膜蛋白质组成成分、表达水平以及修饰情况,进而了解细胞信号传导、细胞与细胞间的相互作用、离子及溶液传递规律等,同时也能为疫苗、病理研究等临床应用领域提供新的理论基础。细胞膜蛋白质在临床药物研究和开发中占整个已知药物靶标的70%,而细胞膜的功能基本上都是由占细胞总蛋白质20%-25%的细胞膜蛋白质来执行的。膜蛋白具有疏水性强、相对丰度较低等特点,同时多数的膜蛋白存在着各种各样的翻译后修饰,如糖基化、磷酸化、甲基化等,造成其具有不均一性。因此,分离和鉴定膜蛋白一直是膜蛋白质组学研究的难点。本研究利用生物素标记的方法,通过使用一种新型的纳米乳胶颗粒材料Latex作为亲和素的载体,在活细胞状态下,进行标记、分离和提纯。纳米级颗粒的使用,在相同总体积的情况下,较通常使用的微米级或是毫米级颗粒大幅提高了载体的表面积,增加了亲和素的结合量,同时摒弃了长时间超速离心法的费时费力,提高了分离纯化效率。我们运用该材料并进行适当优化后应用于肿瘤细胞、人和小鼠胚胎干细胞以及人类精子的细胞膜蛋白质组学研究上,都充分证明其高效性。以肿瘤细胞K562及其耐药株K562／A02细胞为例,我们运用该方法共检测到1294和1415种蛋白,其中膜及相关蛋白分别为645和726种,是目前已有报道中一次性检测到该细胞膜蛋白最多的方法。与此同时,我们还首次在K562中发现了8种肿瘤标志蛋白以及大量的低丰度、未知的和功能不明确的蛋白,体现了该方法的高灵敏度,且亲水、疏水蛋白均可的广泛适用性,为今后对细胞膜表面蛋白的深入研究提供了良好的技术平台和理论基础。慢性髓细胞样白血病(chronic myeloid leukemia, CML),是一种起源于骨髓多能造血干细胞的恶性增殖性疾病,年发病率维持在每10万人中有1-2例。K562细胞株是人类建立的第一株髓系白血病细胞株,是目前公认的最经典的白血病研究模型之一,而K562／A02细胞株是用阿霉素(adriamycin, ADM)逐步筛选K562细胞所建立的一株具有典型多药耐药特征(multidrug resistance, MDR)细胞株。本文通过蛋白质组学相关技术,对K562及其多药耐药株K562／A02细胞质膜进行蛋白质组学层面的分离、鉴定及比较。仅K562细胞的膜蛋白中,我们就发现114个与信号转导有关,119个与代谢相关,95个与通道及运输有关,51个与囊泡运输有关,23个为粘附和连接蛋白,17个是氧化还原酶类,26个为伴侣分子,19个是与细胞骨架相关,17个与修饰作用有关,27个和蛋白降解有关,以及74个未知功能的膜相关蛋白,这是目前已知的对该细胞膜蛋白最完整、最全面的-次分类和总结。我们又经过比较蛋白质组学的研究,发现并验证了K562／A02特异性的跨膜蛋白201种,也是已有报道中一次性发现K562／A02特异性跨膜蛋白最多的。这些研究结果都为寻找新的白血病耐药相关蛋白提供了全新的研究方向。在对K562及其耐药株K562／A02细胞膜进行蛋白质组学比较研究的基础上我们就K562／A02特异性的膜表面分子——尼古丁型乙酰胆碱受体alpha3 (nAChRα3)与该细胞耐药性之间的关系进行了深入的研究。我们发现,加入尼古丁后含有alpha3受体的A02细胞耐药性比未加的对照组提高了约1.1倍,两者差异极显著(P<0.01),而不含该受体的K562细胞基本没有变化。进一步研究发现,尼古丁通过alpha3受体后,提高细胞内ERK的表达,即通过ERK途径,提高了细胞的存活能力。这是首次在K562／A02细胞上发现alpha3受体,也是第一次将该受体与细胞的耐药性相联系,并初步探明了其作用途径,这一研究结果为白血病多药耐药发生机制提供了一条新的研究线索。更多还原

【Abstract】 The cell membrane, also called the plasma membrane or plasmalemma, is one biological membrane separating the interior of a cell from the outside environment. The cell membrane surrounds all cells and it is selectively-permeable, controlling the movement of substances in and out of cells. The cell membrane is also the channel of materials exchange and signal transduction between cell to cell or cell to the outside environment. The cell membrane proteins are one of important parts for cell membrane, they participate in cell recognition and immune response, signal transduction, the inner materials and energy transmission. It is important for cell’s existence, proliferation, separation and differentiation.The proteome is the entire complement of proteins, and proteornics is the large-scale study of proteins, particularly their structures and functions. Plasma membrane proteomics is one part of the proteomics. The researches of plasma membrane proteomics focus on not only the membrane protein composition, expression, modification and enriching the knowledge of the cell signal transduction, the interactions of cell to cell, and materials transmission, but also providing the theoretical basis for clinical application on vaccine and pathology. The plasma membrane protein researches are particularly important in drug discovery. Currently, they account for nearly 70% of all known pharmaceutical drug targets are membrane proteins. And the functions of membrane are controlled by the membrane proteins which account for 20-25% of total cellular proteins.Although the pure membrane proteins are the basic of membrane protein research, they have been underrepresented in proteomics studies, due to their low abundance and poor solubility of many lipid proteins. The post-translational modification also makes the membrane proteins heterogeneous, like glycosylation, phosphorylation, methylation and so on. All of them make it difficult to purify the membrane proteins. To circumvent these problems, in this study, we employed the biotin-avidin system, using the nano-particles as the carrier, and substantially increased the surface area of the carrier and the binding capacity of streptavidin compared with other general beads or particles. This original technology abandons long time ultracentrifugation and improves the efficiency of separation and purification of membrane proteins. We applied this method on tumor cells, mouse embryonic stem cells, human embryonic stem cells and human sperms cell membrane proteome studies. As an example of suspension tumor cells, we found 1294 and 1415 proteins in K562 and K562/A02 cells including 645 and 726 cell membrane or membrane relative proteins. It is one of the most efficient methods in the recent membrane protein researches. Meanwhile, we also found 8 cancer markers in K562 cells and a series of low abundance, function unknown or lipid proteins at the first time. That reflects the high sensitivity and broad applicability of the method for the future study of cell membrane proteins and also provides a reliable technology platform and theoretical basis.Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by the malignant expansion of bone marrow stem cell with an annual incidence of 1 to 2 cases per 100 000 people. K562 cells were the first human immortalized myelogenous leukemia line to be established. K562/A02 is a multi-drug resistant (MDR) cell line which builted up from K562 cells induced by adriamycin (ADM). This MDR cell line can resistant vincristine, homoharringtonine (HHT) and the other antineoplastic drugs which have the anthracene nucleus. The mechanism of MDR includes metabolism, signal transduction, RNA modification, cell proliferation and viability. In this study, we used the proteomics techniques, comparative analysis of the plasma membrane between K562 and K562/A02. Only in K562 cell’s membrane proteins, we discovered 114 signaling transmission,119 metabolism,95 channel or transporter,51 vesicle transport,23 adhesion or junction,17 oxidoreductase,26 chaperone,19 cytoskeleton associated,17 modification,27 protein degradation,62 other membrane relative proteins, and 74 uncharacterized proteins. Further more, we categorized and gathered statistics of all the K562/A02 special membrane protein data, and confirmed them. It gave a new direction to find new drug resistance associated proteins.On the base of comparative analysis of membrane protein between K562 and K562/A02, in this study, we used RNAi technique, and found out the relationship between K562/A02 special protein neuronal acetylcholine receptor alpha 3 (nAChRa3) and multi-drug resistance. Utilizing nicotine to stimulate K562/A02, will enhance 1.1 times of the resistant capacity to ADM, compared with the control group(P＜0.01). Further studies revealed the nAChRa3 can promote the expression of ERK, and then increase the cell viability. This is the first time to discover the nAChRa3 in K562/A02 cell line and also the first time to relate this receptor to drug resistance. This consequence gave a new clue to clarify the mechanisms of multi-drug resistance in CML.更多还原