V2R和AQPs在双峰驼肾脏的表达研究

发布时间：2018-05-25 01:11

本文选题：双峰驼(Camelus + bactrianus)　；参考：《兰州大学》2016年博士论文

【摘要】：【目的】双峰驼(Camelus bactrianus)对干旱和半干旱地区极端恶劣生境耐受性极强,是能够较好地适应极度缺水环境而生存、繁衍和生产的典型哺乳动物,这与其特殊的水代谢能力密切相关。哺乳动物抗利尿激素V2型受体(V2 vasopressin receptor,V2R)通过介导分布于肾小管各段的水通道蛋白(Aquaporins,AQPs)来调控肾小球滤液中水的重吸收和尿液的浓缩能力,从而维持机体的水分和电解质平衡。双峰驼对干旱缺水环境的适应机制是人们普遍关注的问题,本文试图通过对双峰驼肾脏V2R、AQP2、AQP3和AQP4的表达进行研究,结合其肾脏结构指数、超微结构特征以及生物信息学特点,并与生活在低海拔黄牛(Bos taurus)对照,系统探究双峰驼对缺水环境适应的结构和分子基础及其相互之间调控的分子机制。【方法】1.组织学方法:固定充分的双峰驼和黄牛肾脏置于通风橱中,整体观察并解剖其结构特征,记录并拍照。测定肾脏结构指数,包括髓质厚度的百分数(Percentage of medulla thickness,PMT)、髓质面积的百分数(Percentage of medulla area,PMA)、髓质相对厚度(Relative medulla thickness,RMT)、髓质相对面积(Relative medulla area,RMA)、乳头厚度的百分数(Percentage of papilla thickness,PPT)和乳头面积的百分数(Percentage of papilla area,PPA);组织学切片和HE染色,扫描和透射电镜观察。2.石蜡切片免疫组织化学反应:选择双峰驼和黄牛不同切片的相同部位,在不同视野下进行数码显微拍照。应用Image-Pro Plus6.0软件对所拍得的照片进行免疫阳性反应产物(AQP2、AQP3、AQP4、V2R和AVP)光密度值(Integrated optical density,IOD)测定。3.QRT-PCR反应:应用Oligo 6.0软件分别设计定量引物(AQP2、AQP3、AQP4和V2R)和β-actin内参引物,以反转录产物cDNA为模板进行PCR反应。4.生物信息学方法:运用生物信息学部分软件分别对V2R和AQP2进行氨基酸序列比对、三级结构预测、疏水/亲水性、跨膜结构及潜在磷酸化位点进行比较分析。【结果】1.双峰驼和黄牛肾脏髓质与皮质的平均厚度比例分别约为4/1和1.5/1。髓质部可进一步分为染色较深的外髓和较浅的内髓,双峰驼内髓厚度是其外髓的2倍,但黄牛内外髓质厚度基本相当。双峰驼肾脏六项结构指数(pmt、pma、rma、ppa、rmt和ppt)都高于低海拔黄牛,且存在极显著差异(p0.001),尤其是rma和ppa尤甚。扫描电镜下,发现黄牛皮质浅-中层肾小体数目(9.0±1.58)高于双峰驼(4.6±1.52),且差异极显著(p0.01)。与黄牛相比,双峰驼皮质和外髓质肾小管和集合管上皮细胞中线粒体体积较小,但数量居多。然而,在内髓质,双峰驼和黄牛集合管上皮细胞中线粒体体积基本一致,但前者数量较后者为多。2.与黄牛相比,双峰驼外髓集合管(outermedullarycollectingduct,omcd)和内髓集合管(innermedullarycollectingduc,imcd)上皮细胞顶质膜及其胞内囊泡aqp2显示更强的免疫阳性产物,且各阳性细胞之间iod值的差异均极显著(p0.01)。双峰驼皮质集合管(corticalcollectingduct,ccd)、omcd和imcd基底膜aqp3的表达量均高于黄牛,且omcd和imcd差异极显著(p0.01)。双峰驼肾脏ccd、omcd和imcd上皮细胞基底膜aqp4的iod值均低于黄牛,且ccd和imcd差异显著(p0.01)。此外,v2r在双峰驼外髓(outermedulla,om)和内髓(innermedulla,im)的表达量均高于黄牛,且差异极显著(p0.01);相反,v2r在双峰驼皮质(cortex,ct)的iod值低于黄牛,且差异极显著(p0.01)。双峰驼和黄牛avp在视上核(supraopticnucleus,son)大细胞神经元的表达量基本是一致的;而在室旁核(paraventricularnucleus,pvn),双峰驼avp的表达量明显高于黄牛。3.实时定量rt-pcr检测结果发现,双峰驼和黄牛肾脏aqp2和v2rmrna的表达趋势在ct-om-im基本是一致的,但双峰驼在im转录水平明显高于黄牛(p0.001)。aqp3mrna的转录水平在双峰驼肾脏各个部位均极显著高于黄牛(p0.001)。然而,aqp4mrna的转录水平在双峰驼肾脏各个部位均极显著低于黄牛(p0.001)。4.双峰驼和黄牛v2r序列同源性较高,且在核心区高度保守;v2r磷酸化位点分析发现,双峰驼和黄牛分别有17和12个潜在的磷酸化位点,且差异性主要体现在色氨酸(ser)残基上。此外,aqp2磷酸化位点在双峰驼和黄牛中也存在差异,分别有10和8个色氨酸(ser)或苏氨酸(thr)磷酸化位点。【结论】1.双峰驼肾脏形态学上的特殊性(即宽髓质/长髓袢)和肾小管上皮细胞中较多数目的线粒体可能是其形成高渗透压梯度的结构基础。2.双峰驼长期对干旱环境的适应导致其具有基础水平较高的V2R,后者介导产生一系列的信号级联,最终导致肾脏皮质、外髓质和内髓质集合管上皮细胞AQP2和AQP3的mRNA和蛋白质水平出现上调,从而为加强其对原尿中水分的重吸收以最有效的方式保留机体水分提供了分子基础。
[Abstract]:[Objective] Camelus bactrianus (Bactrian Bactrian camel) is highly tolerant to extreme habitats in arid and semi-arid areas. It is a typical mammal that can survive, reproduce and produce well in the extreme water shortage environment. This is closely related to its special water metabolism ability. The mammalian animal antidiuretic hormone V2 receptor (V2 vasopressin receptor) V2R) can regulate the reabsorption of water and the concentration of urine in the glomerular filtrate by mediating the water channel protein (Aquaporins, AQPs) distributed in the various segments of the renal tubules, thus maintaining the balance of water and electrolyte in the body. The adaptation mechanism of Bactrian camels to the drought and water shortage environment is a common concern. This article attempts to pass the Bactrian camel kidney. Study on the expression of dirty V2R, AQP2, AQP3 and AQP4, combined with its renal structural index, ultrastructural features and bioinformatics characteristics, and compared with living at low altitude yellow cattle (Bos taurus), systematically explored the structure and molecular basis of the adaptation of the Bactrian camel to the water shortage environment and the molecular mechanism of its regulation between each other. [Methods] 1. histology Methods: a fully fixed Bactrian camel and a yellow cow kidney were placed in a ventilator, and the structural features were observed and recorded and recorded and photographed. The index of renal structure, including the percentage of Percentage of medulla thickness, PMT, the percentage of medulla area (Percentage of medulla area, PMA), and the relative thickness of medulla (Relative MED) Ulla thickness, RMT), the relative area of the medulla (Relative medulla area, RMA), the percentage of the thickness of the nipple (Percentage of papilla thickness, PPT) and the percentage of the area of the nipple; tissue section and staining, scanning and transmission electron microscope observation of the immunohistochemical reaction of the paraffin section: select the Bactrian camel and the immunochemical reaction of the paraffin section. The same parts of different sections of yellow cattle were photographed under different field of vision. Image-Pro Plus6.0 software was used to measure the photo density value (Integrated optical density, IOD) of the immunoreactive products (AQP2, AQP3, AQP4, V2R and AVP) to determine the.3.QRT-PCR reaction: the quantitative primers were designed with 6 software. 2, AQP3, AQP4 and V2R) and beta -actin primers, using the reverse transcription product cDNA as the template for PCR reaction.4. bioinformatics: using the bioinformatics software, the amino acid sequence alignment, the three grade structure prediction, the hydrophobicity / hydrophile, the transmembrane structure and the potential phosphorylation sites were compared. [results] 1. The average thickness ratio of medulla and cortex in the kidney of the Bactrian camel and the Yellow Cattle kidney can be further divided into 4/1 and 1.5/1. medullary parts to be further divided into the deep outer pulp and the shallow intramedullary medulla, the inner pulp thickness of the Bactrian camel is 2 times that of the outer pulp, but the inner and outer medulla thickness of the yellow cattle is basically equal. The six structural indexes of the kidney of the Bactrian camel (PMT, PMA, RMA, PPA, RMT and PPT) are all high In low altitude cattle, there were very significant differences (p0.001), especially RMA and PPA. Under scanning electron microscope, the number of renal corpuscles in the superficial middle layer of the Yellow Cattle cortex (9 + 1.58) was higher than that of the Bactrian camel (4.6 + 1.52), and the difference was very significant (P0.01). Compared with the yellow cattle, the mitochondria of the cortex of the Bactrian camel and the outer medulla tubule and the collecting tube epithelial cells were more than the yellow cattle. However, in the inner medulla, the volume of mitochondria in the epithelial cells of the collection tube of the Bactrian camel and the yellow cattle is basically the same, but the number of the former is more.2. than that of the yellow cattle, and the epithelia of the epithelial cells of the outermedullarycollectingduct, omcd and innermedullarycollectingduc (IMCD) epithelial cells of the Bactrian camel and the inner medulla collection tube (innermedullarycollectingduc, IMCD) and the apical membrane of the epithelial cells of the Bactrian camel and the inner medulla of the inner medulla (IMCD) and the epithelia of the epithelia of the camel The intracellular vesicle AQP2 showed a stronger immunoreactive product, and the difference of IOD values between the positive cells was very significant (P0.01). The expression of AQP3 in the cortex collection tube (corticalcollectingduct, CCD), omcd and IMCD in the basal membrane of the Bactrian camel was higher than that of the yellow cattle, and the difference between omcd and IMCD was very significant (P0.01). The IOD value of membrane AQP4 is lower than that of yellow cattle, and the difference between CCD and IMCD is significant (P0.01). In addition, the expression of V2R in the outer pulp (outermedulla, OM) and the inner pulp (innermedulla, IM) of the Bactrian camel is higher than that of the Yellow Cattle, and the difference is very significant (P0.01). On the contrary, the V2R in the cortex of the Bactrian camel is lower than the yellow cattle, and the difference is very significant. Bactrian camel and cattle are very significant. In the paraventricular nucleus (supraopticnucleus, son), the expression of large cell neurons is basically the same; while in the paraventricular nucleus (paraventricularnucleus, PVN), the expression of AVP in Bactrian camel is significantly higher than that of.3. real-time quantitative RT-PCR detection results. The expression trend of AQP2 and v2rmrna in the kidney of Bactrian camel and yellow cow kidney is basically identical in ct-om-im, but in ct-om-im. The transcriptional level of the peak camel at IM was significantly higher than that of the yellow cattle (p0.001).Aqp3mrna in all parts of the kidney of the Bactrian camel (p0.001). However, the transcriptional level of AQP4mRNA was significantly lower in each part of the kidney of the Bactrian camel than in the yellow cattle (p0.001).4. Bactrian camel and the Yellow Cattle V2R sequence, and was highly conserved at the core area. V2R phosphorylation site analysis showed that there were 17 and 12 potential phosphorylation sites in Bactrian camels and yellow cattle, and the difference was mainly on tryptophan (SER) residues. In addition, AQP2 phosphorylation sites were also different in Bactrian camels and Huang Niuzhong, 10 and 8 tryptophan (SER) or threonine (THR) phosphorylation sites respectively. [Conclusion] 1. Shuangfeng The morphological particularity of the kidney of the camel (namely the broad medullary / long pulp loop) and the renal tubular epithelial cells may be the structural basis for the formation of the high osmotic pressure gradient. The long-term adaptation of the.2. Bactrian camel to the drought environment leads to its high basic level of V2R, which mediates a series of signal cascades that eventually lead to the kidney. The mRNA and protein levels of AQP2 and AQP3 in the epithelial cells of the medulla and medulla of the medulla and medulla are up-regulated, thus providing a molecular basis for strengthening the reabsorption of water in the primary urine to preserve the body's moisture in the most effective way.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S824

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