【作者】 李永明；

【导师】 麻彤辉；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2006， 博士

【摘要】 水通道蛋白(aquaporin,AQP)是一类存在于细胞膜上的水通透性蛋白,在哺乳动物各种组织细胞中广泛分布,自从九十年代红细胞膜上的水通道蛋白(AQP1)被发现以来,目前在哺乳类已发现至少有13个成员(AQP0~12),大量研究表明,AQP在多种器官的生理和病理中发挥重要作用。近期研究发现的水通道蛋白在细胞迁移和血管新生中的重要作用,为细胞迁移的分子机制增加了新的内容,也为水通道基因功能研究开启了一个新的领域。肿瘤细胞的迁移是肿瘤浸润和转移的关键步骤。目前已知水通道蛋白在多种类型的肿瘤细胞中表达,水通道蛋白在肿瘤细胞迁移中的作用目前国内外均未见报道,我们推测水通道蛋白可能在肿瘤细胞迁移活动中发挥重要作用。本研究发现水通道蛋白介导的细胞膜高水通透性促进肿瘤细胞迁移。对高转移性SMMC-7221人肝癌细胞系进行系统的RT-PCR、免疫荧光和免疫印迹分析,发现水通道蛋白AQP1表达。进一步分析发现SMMC-7221细胞系含细胞膜具有高水通透性(SMMC-7221hPf)和低水通透性(SMMC-7221lPf)的两个亚群,分别与细胞膜AQP1表达量相一致。SMMC-7221hPf细胞的穿孔迁移速率和平面迁移率均显著高于SMMC-7221lPf,而细胞的贴附和增殖能力没有显著差别。AQP1腺病毒感染SMMC-7221lPf细胞提高了细胞膜AQP1表达、细胞膜水通透性和细胞迁移速率。上述结果显示,水通道蛋白AQP1介导的细胞膜高水通透性促进SMMC-7221人肝癌细胞细胞的迁移并且可能参与肿瘤的浸润和转移过程。为了进一步研究水通道蛋白在细胞迁移活动中的作用,我们利用基因打靶技术制作了对氯离子高度敏感的EYFP-V163S绿色荧光蛋白转基因小鼠。通过对EYFP-V163S转基因小鼠各组织器官体内原位灌注并监测其荧光的动态变化,我们可以直接测定各个组织细胞膜的水通透性;同时我们可以体外培养表达EYFP-V163S的各组织细胞,研究水通道蛋白在这些不同类型细胞迁移活动中的表现特征;另外,我们还可以在小鼠活体动物模型中研究水通道蛋白在白细胞参与抗肿瘤、抗感染过程中的作用及水通道蛋白在胚胎发育、组织损伤修复等诸多与细胞迁移相关的重要生理病理活动中的作用。更多还原

【Abstract】 Aquaporins (AQPs) are membrane water channels that play pivotal roles in physiological and pathophysiological processes in diverse mammalian organs. Thirteen mammalian AQP have been molecularly identified since AQP1 was first cloned from the membrane of erythrocyte in 1990. Recent studies uncovered an important role of aquaporins in cell migration, and migration-associated cell function such as angiogenesis, wound healing, and neutrophil motility, provided a new molecular mechanism of cell migration and opend a new field of aquaporin gene function .Migration of tumor cells is a crucial step in tumor invasion and metastasis. Previous studies demonstrated the expression of AQPs in many types of tumor cells. The role of aquaporins in tumor cell migration has not been reported. We hypothesize that aquaporins also play a critical role in tumor cell migration. Here we provide evidence that aquaporin expression is involved in tumor cell migration. RT-PCR, Immunofluorescence and Western blot analysis demonstrated AQP1 protein expression on the plasma membrane of SMMC-7221 human hepatoma cells. SMMC-7221 cell clones with high (SMMC-7221hPf) and low (SMMC-7221lPf) water permeability were identified by functional assays with corresponding high and low AQP1 expression. Cell migration rate was remarkably higher in SMMC-7221hPf cells as compared to SMMC-7221lPf cells, assessed by Boyden chamber and wound healing assays, whereas cell growth and adhesion were not different. Adenovirus-mediated AQP1 expression in SMMC-7221lPf cells increased their water permeability and migration rate. These results provide the first evidence that aquaporin-mediated membrane water permeability enhances tumor cell migration and may be associated with tumor invasion and metastasis.To further investigate the functions of aquaporin water channels in cell migration, we established the transgenic mouse model expressing the fluorescent protein EYFP-V163S sensitive to Cl-. The establishment of EYFP-V163S transgenic mouse model will bring it into realization that measure directly water and Cl- channel function in different tissues by perfusing in vivo tissues with halides and detecting dynamically the change of fluorescent. At the same time, cells from tissues of EYFP-V163S transgenic mouse could be cultured as cell models for studing the roles of aquaporin water channels in cell migration.更多还原