【作者】 李丽华；

【导师】 麻彤辉；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2009， 博士

【摘要】 水通道蛋白(Aquaporin,AQP)是广泛存在于细胞膜上转运水的特异性孔道。大量研究表明,水通道蛋白在许多体液转运的生理及病理过程包括尿浓缩、外分泌腺功能、眼球房水代谢、脑脊液分泌和吸收以及脑水肿形成中发挥重要作用。近期的一些研究显示水通道蛋白还参与细胞的一些重要生命活动包括细胞增殖、迁移和凋亡等。肝胆系统的主要生理功能不仅是生成胆汁,而且还可以修饰和浓缩胆汁。正常情况下胆汁中含有98%以上的水。如何保证高效的水的转运,对于维持肝胆系统的正常生理功能是十分重要的。目前已知在肝胆系统中有7种水通道表达:AQP0,AQP1,AQP4,AQP5,AQP8,AQP9,AQP11,其分布遍及肝细胞、胆管上皮细胞、胆囊上皮细胞和血管内皮细胞。AQP0主要表达于肝细胞中央静脉周围区域;AQP1广泛存在肝脏血管内皮细胞、胆管上皮细胞和胆囊上皮细胞;AQP8分别在肝细胞毛细胆管膜和胆囊上皮细胞表达;AQP9主要集中在肝细胞的窦基底膜。肝胆系统中广泛分布着水通道蛋白AQPs,但到目前为止,究竟诸多AQPs在胆汁形成、修饰及浓缩过程中起到什么样的作用?一些研究仍然处在间接的、推测水平。本课题利用AQP8和AQP1基因敲除小鼠模型,系统地研究了水通道蛋白AQP8和AQP1在小鼠“原始”胆汁的形成和胆囊内胆汁浓缩过程中的作用。我们首先引取了小鼠的生理水平下的胆汁。与野生型小鼠相比,AQP8基因敲除小鼠的胆汁分泌量明显减少。为了避免胆管对胆汁形成修饰作用,我们使用胆盐刺激小鼠,加快胆汁分泌的速度,结果仍显示,AQP8基因敲除小鼠的胆汁分泌量明显少于野生型小鼠。胆汁成分分析,基础状态下的两组小鼠的胆汁无明显差异,但是胆盐刺激后,AQP8基因敲除小鼠胆汁内的总胆汁酸与谷胱甘肽的含量明显高于野生型小鼠的,表明水通道蛋白AQP8参与了胆汁的形成过程。为了更进一步确定AQP8在“原始”胆汁形成中的作用,本课题组进行了细胞水平的实验研究,分离了肝细胞对(Couplets),发现两组小鼠的肝细胞数量及形态无差异,以及肝细胞对在肝细胞中的比例也无不同。利用荧光染料可被肝细胞分泌进入毛细胆管的原理,分析了肝细胞的胆汁分泌情况,荧光结果显示,与野生型小鼠的肝细胞对比较,在AQP8基因敲除小鼠的肝细胞对中,有很少的胆汁被分泌进入毛细胆管。另外,胆囊可以重吸收肝胆汁中的大量水分,在食物消化时,胆囊收缩,将浓缩的胆汁排入十二指肠以助于食物的消化和吸收,提示胆囊上存在着高效转运水的水通道蛋白AQPs。从形态学上观察,两组小鼠的胆囊结构无明显差异。但通过RT-PCR、Real-time PCR和免疫荧光分析发现水通道蛋白AQP1在野生型小鼠的胆囊上皮细胞上高表达,本研究首次利用calcein荧光技术测量小鼠胆囊的跨上皮水转运,结果发现,AQP1基因敲除小鼠的胆囊跨上皮水转运速度明显慢于野生型小鼠,提示AQP1是胆囊水转运的主要途径。而且野生型小鼠胆囊的跨上皮水转运速度是目前已知哺乳动物中跨上皮水转运速度最快的。此外,利用本实验室可以做显微手术的优势,将小鼠的胆囊里层外翻,结合荧光染料calcein-AM,测量胆囊上皮细胞顶质膜的水通透性。分析结果显示,与野生型小鼠相比,AQP1基因敲除小鼠的胆囊上皮细胞顶质膜的水通透性显著降低。根据结果我们估计,胆囊上皮细胞膜上存在约4000 /um2个AQP1,占20%质膜蛋白。由此可见,胆囊上皮的水转运是跨细胞转运,而不是细胞旁转运。但胆囊胆汁成分分析表示,两组小鼠的胆汁成分含量基本相似。表明在胆囊胆汁浓缩过程中还存在其他的机制,需进一步研究。综上所述,水通道蛋白AQP8表达定位于肝脏并在肝胆汁形成过程中发挥了重要的作用,水通道蛋白AQP1介导了胆囊上皮细胞膜的高水通透性,但在胆囊胆汁浓缩中并无重要作用。更多还原

【Abstract】 Members of the AQP family are distributed in many cell types in different organ systems where they play important roles in various physiological functions and pathological conditions including urinary concentrating function, exocrine glandular fluid secretion, brain edema formation, regulation of intracranial and intraocular pressure, skin hydration, fat metabolism, tumor angiogenesis,cell migration and apoptosis.The major function of hepatobiliary system includes bile formation and modification by secretory and absorptive processes in the epithelial cells of intrahepatic bile ducts and gallbladder. Aquaporin (AQP)-mediated transepithelial water transport mechanism may play an important role in bile formation. To date, 13 AQPs have been found in mammals, with many numbers (AQP0, AQP1, AQP4, AQP5, AQP8, AQP9, AQP11) being localized to the hepatobiliary system in the plasma membranes of hepatocytes, the epithelial cell of bile ducts , the epithelial cells of gallbladder and intrahepatic vascular endothelial cells. In the present study, we focus on the expression and potential physiological roles of AQP1 and AQP8 in bile secretion and concentration.We collected bile from wild type and AQP8 null mice. Compare with wild type mice, bile flow was significantly decreased in AQP8 null mice under both basal and bile salt-stimulated conditions. Bile composition analysis showed no difference in the total bile acid and glutathione concemtration under physiological condition between wild type and AQP8 null mice. But after bile salt stimulation, total bile acid and glutathione concentration in bile were significantly increased in AQP8 null mice compared with wild type mice. Those data indicated that AQP8-facilitated canalicular membrane water transport is involved in hepatic bile secretion.To further confirm the reduced hepatic bile secretion in AQP8-/- liver, we isolated hepatocyte couplets from wild type and AQP8 null mice. Although the couplets number and morphology are similar in both genotypes, fluorescence assay showed significantly decreased bile secretion into canalicular space of hepatocyte couplets in AQP8 null mice compare with wild type mice. These results provide direct evidence that AQP8-mediated water transport playa an important role in hepatic bile secretion.Water transport across gallbladder epithelium is driven by osmotic gradients generated from active salt absorption and secretion. Aquaporin (AQP) water channels have been proposed to facilitate transepithelial water transport in gallbladder and to modulate bile composition. We found strong AQP1 immunofluorescence at the apical membrane of mouse gallbladder epithelium.Transepithelial osmotic water permeability (Pf) was measured in freshly isolated gallbladder sacs from the kinetics of luminal calcein self-quenching in response to an osmotic gradient. Pf was very high (0.12 cm/s) in gallbladders from wildtype mice, cAMP-independent, and independent of osmotic gradient size and direction. Although gallbladders from AQP1 knockout mice had similar size and morphology to those from wildtype mice, their Pf was reduced by ~10-fold. Apical plasma membrane water permeability was greatly reduced in AQP1-deficient gallbladders, as measured by cytoplasmic calcein quenching in perfluorocarbon-filled, inverted gallbladder sacs. However, neither bile osmolality nor bile salt concentration differed in gallbladders from wildtype vs. AQP1 knockout mice. Our data indicate constitutively high water permeability in mouse gallbladder epithelium involving transcellular water transport through AQP1. The similar bile salt concentration in gallbladders from AQP1 knockout mice argues against a physiologically important role for AQP1 in mouse gallbladder.In conclusion, our results provide evidence for AQP8-mediated water transport across hepatocyte canalicular membrane plays an important role in hepatic bile secretion. AQP1 creates high osmotic water permeability in mouse gallbladder epithelium, but does not affect bile concentration.更多还原