水通道蛋白的生理功能 ——水通道基因敲除小鼠表型研究进展

水通道蛋白 (aquaporin, AQP) 是一族细胞膜上选择性高效转运水分子的特异孔道. 自从 Agre 等于 1992 年从红细胞膜发现第一个水通道蛋白 AQP1以来,有关水通道蛋白结构与功能的研究取得了迅速的、系列性的进展 . 已报道的哺乳动物 AQP 家族已有 11 个在蛋白质序列上有同源性成员 (AQP0~AQP10). AQP 在体内各系统组织中广泛表达,除了在与体液分泌和吸收密切相关的多种上皮和内皮细胞高表达外,在一些与体液转运无明显关系的组织细胞如红细胞、白细胞、脂肪细胞和骨骼肌细胞等处也有表达,提示 AQP 可能在多种器官生理和病理中发挥重要作用. 基因打靶技术是研究特定基因在体内生理功能的有力手段. 目前 AQP1、3、4、5 基因敲除和 AQP2 基因点突变的基因敲入小鼠模型 ( 模拟人类常染色体隐性遗传尿崩症 ) 已成功建立并广泛用于表型研究,在 AQP 水通道蛋白生理功能方面获得许多重要进展.     

AQP1 在CD-1 纯系野生型孕鼠胎盘组织的表达

目的:研究水通道蛋白1(Aquaporin 1,AQP1)在小鼠胎盘组织的分布及表达,初步探讨AQP1在羊水循环及母胎液体平衡中的作用.方法:各取四只雌雄成年健康野生型CD1小鼠(wildt ype,AQP1+/+)及AQP1基因敲除小鼠(AQP1-KO,AQP1-/-),将纯合子AQP1基因敲除雌雄小鼠等数量合笼交配,第二日检出阴道拴者记为妊娠第1天(1 gestational day,1GD);野生型小鼠同样合笼记录.分别取两组13GD孕鼠的胎盘组织各一个,应用逆转录-聚合酶链反应(RT-PCR)技术及免疫组织化学技术检测AQP1胎盘组织中的表达,并确定AQP1在小鼠胎盘组织的定位.结果:1.RT-PCR结果表明AQP1在CD-1野生型孕鼠胎盘组织表达,AQP1基因敲除鼠无表达;2.免疫组织化学方法发现AQP1表达于小鼠胎盘血管内皮细胞和滋养细胞,AQP1基因敲除鼠无表达.结论:在mRNA水平和蛋白水平均发现AQP1在CD-1纯系野生型孕鼠胎盘组织的表达,提示AQP1可能在羊水循环及母胎液体平衡中发挥作用.     

水通道蛋白在塔里木兔肺组织中的表达和作用

通过检测塔里木兔Lepus yarcandensis肺脏中水通道蛋白(aquaporin,AQP)1、AQP3及AQP4的表达和分布情况,以探讨水通道蛋白在干旱区动物水液代谢中的作用。采用常规HE染色观察肺组织学结构,采用免疫组织化学检测AQP1、AQP3及AQP4在肺脏中的分布位置及表达。结果表明,AQP1分布在支气管上皮细胞,肺泡间质毛细血管内皮细胞以及肺泡上皮(Ⅰ、Ⅱ型)细胞。AQP3分布在小气管上皮顶质膜和大气道上皮细胞基底膜。AQP4分布在小气管和大气道上皮细胞基底膜。AQP1、AQP3及AQP4在肺中表达的强弱关系为AQP1>AQP4>AQP3。这些结果说明,水通道蛋白在塔里木兔肺泡腔、肺组织间隙及毛细血管腔之间水的转运中很可能起着很重要的作用。同时对吸入空气的湿润和呼出气体中水分的重吸收也具有重要意义。     

AQP1在CD-1纯系野生型孕鼠胎盘组织的表达

目的：研究水通道蛋白1（Aquaporin 1,AQP1）在小鼠胎盘组织的分布及表达,初步探讨AQP1在羊水循环及母胎液体平衡中的作用。方法：各取四只雌雄成年健康野生型CD1小鼠（wild type,AQP1＋/＋）及AQP1基因敲除小鼠（AQP1-KO,AQP1-/）-,将纯合子AQP1基因敲除雌雄小鼠等数量合笼交配,第二日检出阴道栓者记为妊娠第1天（1 gestational day,1GD）;野生型小鼠同样合笼记录。分别取两组13GD孕鼠的胎盘组织各一个,应用逆转录-聚合酶链反应（RT-PCR）技术及免疫组织化学技术检测AQP1胎盘组织中的表达,并确定AQP1在小鼠胎盘组织的定位。结果：1.RT-PCR结果表明AQP1在CD-1野生型孕鼠胎盘组织表达,AQP1基因敲除鼠无表达;2.免疫组织化学方法发现AQP1表达于小鼠胎盘血管内皮细胞和滋养细胞,AQP1基因敲除鼠无表达。结论：在mRNA水平和蛋白水平均发现AQP1在CD-1纯系野生型孕鼠胎盘组织的表达,提示AQP1可能在羊水循环及母胎液体平衡中发挥作用。     

斜纹夜蛾水通道蛋白1（AQP1）基因的克隆、分子特性和表达分析

水通道蛋白(aquaporin, AQP)是生物体中一种重要的跨膜通道蛋白, 它通过一些中性小分子化合物的运输, 从而参与了昆虫对食物中水分再吸收、抗寒和抗干燥等重要生理机制。为研究斜纹夜蛾中水通道蛋白的基因特征和时空表达特征, 本研究利用同源克隆和RACE技术获得了斜纹夜蛾水通道蛋白1（AQP1）基因的两个转录异构体, 并将其分别命名为SL-AQP1A （GenBank登录号： KC999953）和SL-AQP1B （GenBank登录号： KC999954）,其中SL-AQP1B比SL-AQP1A在推导的编码区5′端连续性缺失81个碱基, 而其他序列完全一致; 同源分析显示推导的SL-AQP1与家蚕为代表的水通道蛋白1具有较高的同源性。拓扑学和三级结构模拟显示其有经典的6个全跨膜结构域、2个半跨膜结构域、2个保守的NPA (asparagine-proline-alanine, 天冬氨酸-脯氨酸-丙氨酸)结构基序及选择性水孔构件ar/R （aromatic arginine, 芳香族精氨酸）。qRT-PCR结果显示, SL-AQP1整体时空表达差异性十分明显, 并且SL-AQP1B的表达量显著高于SL-AQP1A; SL-AQP1在卵期和预蛹期有较高的表达量, 在中肠、马氏管、血淋巴、消化腺中有相对较高的表达量, 暗示其在斜纹夜蛾中存在重要的渗透压调节作用。本文结果为进一步研究水通道蛋白在斜纹夜蛾中的作用提供了一定的分子基础。     

白术配伍人参前后挥发油调控慢性萎缩性胃炎大鼠AQP 3、4表达的比较研究

为探究参术药对配伍治疗慢性萎缩性胃炎的增效机制,本文就白术配伍人参前后挥发油成分物质基础变化及对慢性萎缩性胃炎大鼠胃组织水通道蛋白3(AQP 3)、水通道蛋白4(AQP 4)的影响进行了比较研究。采用水蒸气蒸馏法提取人参白术配伍前后挥发油,以GC-MS法表征挥发油化学特征,并随机将大鼠分成空白组、模型组、白术挥发油组及配伍挥发油组,使用MNNG建立慢性萎缩性胃炎模型,给药后观察各组胃黏膜组织显微、超微结构变化以及AQP 3、AQP 4的表达。结果显示,白术配伍人参后挥发油的提取量升高,经GC-MS测定,配伍后苍术酮相对含量降低,新增少量人参挥发油成分。显微及超微观察显示,模型组大鼠胃组织黏膜折叠皱起,黏膜层和固有层腺体萎缩严重,胃小凹形态改变,上皮细胞破损,炎症细胞大面积浸润,且病理评分明显高于正常组(P<0.05);各药物组与模型组相比,均缓解或改善上述病理结果,且配伍挥发油组表现更优。免疫组化结果显示,模型组大鼠胃黏膜AQP 3、AQP 4表达明显低于空白组(P<0.05);相较模型组,各药物组对胃黏膜AQP 3、AQP 4蛋白的表达有增加趋势或明显增加,且配伍挥发油组...     

牦牛AQP4基因CDS序列的克隆及其生物信息学特征分析

为了研究高原动物对青藏高原高寒、低氧等极端生境的适应机理,进一步探讨高原动物对高原反应——高原脑水肿抗性的分子机理,运用基因克隆与生物信息学相关技术和方法,对牦牛脑AQP4(水通道蛋白4,AQP4)基因CDS全长序列进行克隆、基因序列比对及其生物信息学特征分析。结果表明,牦牛AQP4的CDS含有一个966 bp的开放阅读框,编码322个氨基酸;牦牛AQP4基因编码蛋白分子量34.69 k D,理论等电点(p I)7.59,其编码蛋白含有6次跨膜结构,属于疏水性蛋白;二级结构主要由α-螺旋、延伸及无规则卷曲构成;AQP4基因编码产物氨基酸同源性及系统进化分析发现,牦牛AQP4基因编码氨基酸序列与黄牛、绵羊等物种间同源性较高,系统进化情况与其亲缘关系远近一致。     

HgCl2胁迫对小麦幼苗水分利用效率和叶绿素含量的影响

水通道蛋白(aquaporin,AQP)是植物体内水分吸收和运输的通道.以小麦品种石4185的幼苗为材料,在正常供水供养条件下,采用不同浓度的AQP抑制剂HgCl2对其根部进行胁迫处理,研究植株水分利用效率等性状的变化,以明确水通道蛋白与水分利用效率(WUE)的关系.结果表明:(1)小麦水通道蛋白在受到HgCl2抑制后,其幼苗的单株生物量和单株WUE(生物学产量干重/耗水量)显著低于对照处理,并且随着HgCl2浓度的增加呈逐渐下降趋势.(2)处理后植株24 h5、d和14 d的单株耗水量均显著低于对照组.(3)HgCl2胁迫处理后5 min内小麦叶片叶绿素含量持续下降,处理24 h的含量均远低于初始值,并且随胁迫时间延长处理叶片逐渐变黄.研究发现,HgCl2在抑制小麦幼苗水通道蛋白的同时,也对其光合作用相关酶和蛋白质体在短时间内产生一定影响,从而阻碍幼苗正常生长.     

水通道蛋白AQP1,3,4,5在双峰驼肺中的表达

目的研究水通道蛋白AQPs在双峰驼肺中的表达情况,探讨双峰驼适应极干旱荒漠环境的呼吸生理机制。方法运用常规形态学统计方法和石蜡切片HE染色法对双峰驼肺组织形态结构进行统计与分析,免疫组化方法对双峰驼肺中AQPs的表达进行定位分析。结果双峰驼气管长且弯曲,肺较致密且含水量较黄牛高。免疫组化检测显示,在双峰驼肺中有AQP1、AQP3、AQP4和AQP5四种AQPs表达。其中,AQP1主要表达于肺毛细血管网、淋巴管以及气管上皮细胞顶膜面;AQP3主要表达于气管上皮基底细胞质膜上;AQP4主要分布于整个气管上皮杯状细胞基底侧细胞膜和肺泡Ⅱ型上皮细胞;AQP5表达于气管粘膜下腺腺体细胞管腔面和肺泡Ⅰ型上皮细胞膜上。结论呼吸道和肺组织形态学特征表明双峰驼对干旱沙漠环境具有很好的适应性,AQPs在双峰驼肺中的强烈表达,与气道润化、气道水平衡、气道表面液体层、肺内液体转运和肺内水平衡等生理过程有关,为其适应极干旱荒漠环境提供了分子生物学依据。     

人胸膜间皮细胞水通道蛋白1～10 mRNA的表达

体外培养人胸膜间皮细胞(HPMC),检测人胸膜间皮细胞水通道蛋白1～10mRNA的表达,探讨其在胸腔内液体平衡中的意义.从胸腔积液中分离人胸膜间皮细胞,进行培养,用形态学和免疫组化染色进行细胞鉴定.用RT-PCR检测水通道蛋白1～10(AQP1～10)mRNA在人胸膜间皮细胞上的表达.成功建立人胸膜间皮细胞体外培养模型,鉴定证实为间皮细胞,人胸膜间皮细胞上AQP1～10mRNA均有表达,AQP1、AQP9、AQP10表达丰富.人胸膜间皮细胞存在AQP1～10mRNA的表达,结合已知水通道蛋白的功能,证实人胸膜间皮细胞参于胸腔内液体转运.     

Distribution of AQP2 and AQP3 water channels in human tissue microarrays

SummaryThe objective of this investigation was to use semi-quantitative immunohistochemistry to determine the distribution and expression levels of AQP2 and AQP3 proteins in normal human Tissue MicroArrays. Expression of the vasopressin regulated AQP2 was observed in a limited number of tissues. AQP2 was prominent in the apical and subapical plasma membranes of cortical and medullary renal collecting ducts. Surprisingly, weak AQP2 immunoreactivity was also noted in pancreatic islets, fallopian tubes and peripheral nerves. AQP2 was also localized to selected parts of the central nervous system (ependymal cell layer, subcortical white matter, hippocampus, spinal cord) and selected cells in the gastrointestinal system (antral and oxyntic gastric mucosa, small intestine and colon). These findings corroborate the restricted tissue distribution of AQP2. AQP3 was strongly expressed in many of the human tissues examined particularly in basolateral membranes of the distal nephron (medullary collecting ducts), distal colon, upper airway epithelia, transitional epithelium of the urinary bladder, tracheal, bronchial and nasopharyngeal epithelium, stratified squamous epithelial cells of the esophagus, and anus. AQP3 was moderately expressed in basolateral membranes of prostatic tubuloalveolar epithelium, pancreatic ducts, uterine endometrium, choroid plexus, articular chondrocytes, subchondral osteoblasts and synovium. Low AQP3 levels were also detected in skeletal muscle, cardiac muscle, gastric pits, seminiferous tubules, lymphoid vessels, salivary and endocrine glands, amniotic membranes, placenta and ovary. The abundance of basolateral AQP3 in epithelial tissues and its expression in many non-epithelial cells suggests that this aquaglyceroporin is a major participant in barrier hydration and water and osmolyte homeostasis in the human body.http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html, NCBI AceView, July 2003     

版纳微型猪近交系AQP3克隆、序列分析及组织表达

目的 克隆版纳微型猪近交系aquaporin 3(AQP3)基因,并利用生物信息学方法分析其序列特征,研究其在猪各组织中的表达情况.方法 从版纳微型猪近交系脾脏中提取总RNA,利用RT-PCR方法扩增猪AQP3编码区序列,将纯化的片段与pMD18-T载体连接,转化宿主菌DH 5α,筛选阳性克隆进行测序.并采用半定量RT...     

Seasonal effect in expression of AQP1, AQP3 and AQP5 in skin of Murrah buffaloes

Aquaporins are transmembrane protein channels which are known to help the passage of water and solutes across the cell membranes. AQP1, AQP3 and AQP5 are isoforms of aquaporin known to aid in transepithelial water movement. AQP3 is also known to aid in glycerol transport. The present study was conducted to investigate the role of AQP1, AQP3 and AQP5 in thermoregulation of buffaloes by probing the expression of the genes in skin of buffaloes during different season viz. winter, spring and summer. The skin tissue samples were collected from the neck region of Murrah buffaloes (n = 12) and analyzed for gene expression by RT-PCR and immunolocalization. The physiological responses including respiration rate, rectal temperature and neck skin temperature observed during summer were significantly higher than winter and spring seasons. The study revealed the expression of AQP1, AQP3 and AQP5 genes in skin samples. The relative mRNA expressions of AQP1, AQP3 and AQP5 in skin relative to spring season were 1.41 ± 0.47, 1.95 ± 0.22 and 6.77 ± 1.02 folds during summer which were significantly higher than other seasons. The up-regulation of the expression of the studied AQPs were concomitant with the increase in physiological responses including skin temperature and sweating rate during summer. During summer season, AQP1 were mostly immunolocalized in the walls of skin blood capillaries, while AQP3 were observed mostly in the epidermal layer of the skin. The immunolocalization of AQP5 were mostly observed in the secretory glands of skin. The up-regulation of AQP1, AQP3 and AQP5 in skin during summer season indicates their role in thermoregulation of buffaloes.     

MiR-874 promotes intestinal barrier dysfunction through targeting AQP3 following intestinal ischemic injury

Intestinal ischemic injury is a significant clinical problem arising from diseases or as a complication of abdominal surgery. Our previous study showed aquaporin 3 is involved in intestinal barrier impairment. Here, we revealed that intestinal ischemia induced a time-dependent increase of miR-874 expression and a time-dependent decrease of AQP3 expression, and the level of miR-874 expression was inversely related to AQP3 protein expression. In addition, miR-874 promoted the paracellular permeability in vitro through targeting 3′UTR of AQP3. Two of the tight junction proteins, Occludin and Claudin-1, were found to be involved in miR-874-induced intestinal barrier dysfunction.     

Expression and nephron segment-specific distribution of major renal aquaporins (AQP1-4) in Equus caballus, the domestic horse

Aquaporins (AQPs) play fundamental roles in water and osmolyte homeostasis by facilitating water and small solute movement across plasma membranes of epithelial, endothelial, and other tissues. AQP proteins are abundantly expressed in the mammalian kidney, where they have been shown to play essential roles in fluid balance and urine concentration. Thus far, the majority of studies on renal AQPs have been carried out in laboratory rodents and sheep; no data have been published on the expression of AQPs in kidneys of equines or other large mammals. The aim of this comparative study was to determine the expression and nephron segment localization of AQP1-4 in Equus caballus by immunoblotting and immunohistochemistry with custom-designed rabbit polyclonal antisera. AQP1 was found in apical and basolateral membranes of the proximal convoluted tubules and thin descending limbs of the loop of Henle. AQP2 expression was specifically detected in apical membranes of cortical, medullary, and papillary collecting ducts. AQP3 was expressed in basolateral membranes of cortical, medullary, and papillary collecting ducts. Immunohistochemistry also confirmed AQP4 expression in basolateral membranes of cells lining the distal convoluted and connecting tubules. Western blots revealed high expression of AQP1-4 in the equine kidney. These observations confirm that AQPs are expressed in the equine kidney and are found in similar nephron locations to mouse, rat, and human kidney. Equine renal AQP proteins are likely to be involved in acute and chronic regulation of body fluid composition and may be implicated in water balance disorders brought about by colic and endotoxemia.     

大耳猬血液、尿无机离子等指标测定及肾脏水通道蛋白1、2的表达

测定了大耳猬血清及尿中多种无机离子和尿素氮等指标,并应用免疫组织化学方法观察了AQP1、AQP2在肾脏的表达.大耳猬血清钠、氯含量较高;而尿液中以钾、钠、氯及尿素氮含量较高.尿液中主要离子浓度高于血清,较为浓缩,尿素氮、钾排泄能力较强.AQP1免疫反应阳性表达于近曲小管上皮和髓袢细段,AQP2主要表达于集合管上皮细胞.因此,AQP1、AQP2可能在大耳猬肾脏水重吸收及尿液浓缩过程中具有重要作用.     

Membrane trafficking of AQP5 and cAMP dependent phosphorylation in bronchial epithelium

Phosphorylation pathway has been identified as an important step in membrane trafficking for AQP5. We generated stably transfected BEAS-2B human bronchial epithelial cells with various over-expression constructs on permeable support. In stable cells with wild-type AQP5 and S156A (AQP5 mutant targeting PKA consensus sequence), AQP5 expression was predominantly polarized to the apical membrane, whereas stable cells with N185D (AQP5 mutant targeting second NPA motif), mainly localized to the cytoplasm. Treatment with H89 and/or chlorophenylthio-cAMP (cpt-cAMP) did not affect membrane expression of AQP5 in any of three stable cells. In cells with wild-type AQP5 and N185D, AQP5s were phosphorylated by PKA, while phosphorylation of AQP5 was not detected in cells with S156A. These results indicate that, in AQP5, serine156 may be phosphorylated by PKA, but membrane expression of AQP5 may not be regulated by PKA phosphorylation. We conclude that AQP5 membrane targeting can include more than one mechanism besides cAMP dependent phosphorylation.     

Localization of AQP5/AQP8 chimeras in MDCK-II cells: exchange of the N- and C-termini

AQP5 and AQP8 possess targeting/retention motifs which mediate their localization to the apical and basolateral membranes, respectively, of polarized MDCK-II cells. As targeting/retention motifs have been localized to the N- or C-termini of other AQPs, we sought the location of such motifs in AQPs 5 and 8 by exchanging their corresponding N- or C-termini and examining the expression, localization, and function of the resultant chimeras. We did not detect the expression of constructs in which the C-terminus of AQP5 was replaced by the C-terminus of AQP8. Substitution of the N-terminus of AQP8 for the N-terminus of AQP5 generated a construct which was trapped intracellularly and did not significantly facilitate transepithelial fluid movement. In contrast, modifications of the N- and C-termini of AQP8 were better tolerated. Substitution of either AQP8 terminus by the corresponding AQP5 terminus generated constructs which localized to basolateral membranes and facilitated transepithelial fluid movement. Our results suggest that, unlike the other AQP targeting/retention signals reported thus far, an AQP8 basolateral targeting/retention motif might reside between the two cytosolic termini.