發(fā)布時間：2018-05-25 01:11

  本文選題：雙峰駝(Camelus + bactrianus)　； 參考：《蘭州大學》2016年博士論文

【摘要】：【目的】雙峰駝(Camelus bactrianus)對干旱和半干旱地區(qū)極端惡劣生境耐受性極強,是能夠較好地適應極度缺水環(huán)境而生存、繁衍和生產(chǎn)的典型哺乳動物,這與其特殊的水代謝能力密切相關。哺乳動物抗利尿激素V2型受體(V2 vasopressin receptor,V2R)通過介導分布于腎小管各段的水通道蛋白(Aquaporins,AQPs)來調控腎小球濾液中水的重吸收和尿液的濃縮能力,從而維持機體的水分和電解質平衡。雙峰駝對干旱缺水環(huán)境的適應機制是人們普遍關注的問題,本文試圖通過對雙峰駝腎臟V2R、AQP2、AQP3和AQP4的表達進行研究,結合其腎臟結構指數(shù)、超微結構特征以及生物信息學特點,并與生活在低海拔黃牛(Bos taurus)對照,系統(tǒng)探究雙峰駝對缺水環(huán)境適應的結構和分子基礎及其相互之間調控的分子機制。【方法】1.組織學方法:固定充分的雙峰駝和黃牛腎臟置于通風櫥中,整體觀察并解剖其結構特征,記錄并拍照。測定腎臟結構指數(shù),包括髓質厚度的百分數(shù)(Percentage of medulla thickness,PMT)、髓質面積的百分數(shù)(Percentage of medulla area,PMA)、髓質相對厚度(Relative medulla thickness,RMT)、髓質相對面積(Relative medulla area,RMA)、乳頭厚度的百分數(shù)(Percentage of papilla thickness,PPT)和乳頭面積的百分數(shù)(Percentage of papilla area,PPA);組織學切片和HE染色,掃描和透射電鏡觀察。2.石蠟切片免疫組織化學反應:選擇雙峰駝和黃牛不同切片的相同部位,在不同視野下進行數(shù)碼顯微拍照。應用Image-Pro Plus6.0軟件對所拍得的照片進行免疫陽性反應產(chǎn)物(AQP2、AQP3、AQP4、V2R和AVP)光密度值(Integrated optical density,IOD)測定。3.QRT-PCR反應:應用Oligo 6.0軟件分別設計定量引物(AQP2、AQP3、AQP4和V2R)和β-actin內參引物,以反轉錄產(chǎn)物cDNA為模板進行PCR反應。4.生物信息學方法:運用生物信息學部分軟件分別對V2R和AQP2進行氨基酸序列比對、三級結構預測、疏水/親水性、跨膜結構及潛在磷酸化位點進行比較分析�！窘Y果】1.雙峰駝和黃牛腎臟髓質與皮質的平均厚度比例分別約為4/1和1.5/1。髓質部可進一步分為染色較深的外髓和較淺的內髓,雙峰駝內髓厚度是其外髓的2倍,但黃牛內外髓質厚度基本相當。雙峰駝腎臟六項結構指數(shù)(pmt、pma、rma、ppa、rmt和ppt)都高于低海拔黃牛,且存在極顯著差異(p0.001),尤其是rma和ppa尤甚。掃描電鏡下,發(fā)現(xiàn)黃牛皮質淺-中層腎小體數(shù)目(9.0±1.58)高于雙峰駝(4.6±1.52),且差異極顯著(p0.01)。與黃牛相比,雙峰駝皮質和外髓質腎小管和集合管上皮細胞中線粒體體積較小,但數(shù)量居多。然而,在內髓質,雙峰駝和黃牛集合管上皮細胞中線粒體體積基本一致,但前者數(shù)量較后者為多。2.與黃牛相比,雙峰駝外髓集合管(outermedullarycollectingduct,omcd)和內髓集合管(innermedullarycollectingduc,imcd)上皮細胞頂質膜及其胞內囊泡aqp2顯示更強的免疫陽性產(chǎn)物,且各陽性細胞之間iod值的差異均極顯著(p0.01)。雙峰駝皮質集合管(corticalcollectingduct,ccd)、omcd和imcd基底膜aqp3的表達量均高于黃牛,且omcd和imcd差異極顯著(p0.01)。雙峰駝腎臟ccd、omcd和imcd上皮細胞基底膜aqp4的iod值均低于黃牛,且ccd和imcd差異顯著(p0.01)。此外,v2r在雙峰駝外髓(outermedulla,om)和內髓(innermedulla,im)的表達量均高于黃牛,且差異極顯著(p0.01);相反,v2r在雙峰駝皮質(cortex,ct)的iod值低于黃牛,且差異極顯著(p0.01)。雙峰駝和黃牛avp在視上核(supraopticnucleus,son)大細胞神經(jīng)元的表達量基本是一致的;而在室旁核(paraventricularnucleus,pvn),雙峰駝avp的表達量明顯高于黃牛。3.實時定量rt-pcr檢測結果發(fā)現(xiàn),雙峰駝和黃牛腎臟aqp2和v2rmrna的表達趨勢在ct-om-im基本是一致的,但雙峰駝在im轉錄水平明顯高于黃牛(p0.001)。aqp3mrna的轉錄水平在雙峰駝腎臟各個部位均極顯著高于黃牛(p0.001)。然而,aqp4mrna的轉錄水平在雙峰駝腎臟各個部位均極顯著低于黃牛(p0.001)。4.雙峰駝和黃牛v2r序列同源性較高,且在核心區(qū)高度保守;v2r磷酸化位點分析發(fā)現(xiàn),雙峰駝和黃牛分別有17和12個潛在的磷酸化位點,且差異性主要體現(xiàn)在色氨酸(ser)殘基上。此外,aqp2磷酸化位點在雙峰駝和黃牛中也存在差異,分別有10和8個色氨酸(ser)或蘇氨酸(thr)磷酸化位點�！窘Y論】1.雙峰駝腎臟形態(tài)學上的特殊性(即寬髓質/長髓袢)和腎小管上皮細胞中較多數(shù)目的線粒體可能是其形成高滲透壓梯度的結構基礎。2.雙峰駝長期對干旱環(huán)境的適應導致其具有基礎水平較高的V2R,后者介導產(chǎn)生一系列的信號級聯(lián),最終導致腎臟皮質、外髓質和內髓質集合管上皮細胞AQP2和AQP3的mRNA和蛋白質水平出現(xiàn)上調,從而為加強其對原尿中水分的重吸收以最有效的方式保留機體水分提供了分子基礎。
[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.
【學位授予單位】：蘭州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S824

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