【作者】 陈玉琴；

【导师】 俞诗源；

【作者基本信息】 西北师范大学 ， 细胞生物学， 2008， 硕士

【摘要】 目的通过观察9个不同发育时期鸡(Gallus domestiaus)胚胎肾组织的发生过程和5只红腹锦鸡的肾组织,检测表皮生长因子受体(EGFR)、转化生长因子β(TGF-β)、水通道蛋白2(AQP-2)和Bax蛋白在鸡胚肾(小管)发生过程和红腹锦鸡肾组织中的表达,探讨鸟类中肾的发生与退化,后肾发生及分化的规律和特点,同时了解红腹锦鸡肾的结构特征,并研究各种蛋白的生物活性作用及相互之间的调控意义。方法采用组织学方法和电镜技术观察不同发育时期鸡胚胎肾组织的发生过程及红腹锦鸡(Chrysolophus pictus)肾的结构,应用免疫组织化学和体视学半定量检测EGFR、TGF-β(β1、β2、β3)、AQP-2及Bax蛋白在鸡胚肾(小管)发生过程和红腹锦鸡肾中的表达,用IPP专业图像分析软件,对其表达强度进行定量分析。结果(1)鸡胚孵育到第16期在中肾前端附近出现一些中肾小泡。孵育到第18期形成中肾小管。孵育到第26期,中肾小管的盲端内陷,原始的肾小囊和肾血管球形成,中肾小管显著伸长并迂回曲折。孵育到第33~37期体前后部中肾组织均已形成完整的肾单位。第37~46期体前部至后部的中肾组织依次退化。孵育到第26期从泄殖腔附近发出的输尿管芽向生后肾组织侵入生长,生后肾组织产生许多生后肾小泡。第33期出现肾小囊和肾小管,肾小管伸长并发生折叠,出现集合小管、近端小管和远端小管的形态分化。第37~46期肾小体逐渐发育成熟,肾小管继续分化出现细段。(2)第26~46期鸡胚肾的近端小管上皮细胞呈EGFR免疫反应阳性;第26~37期中肾的近端小管上皮细胞和远端小管上皮细胞呈TGF-β免疫反应阳性,第33~40期后肾的近端小管上皮细胞和远端小管上皮细胞呈TGF-β免疫反应阳性,第44~46期近端小管上皮细胞呈TGF-β免疫反应阳性,远端小管上皮细胞呈TGF-β免疫反应阴性;第26~46期鸡胚肾的集合小管上皮细胞呈AQP-2免疫反应阳性;第26~37期中肾近端小管和远端小管上皮细胞呈Bax反应阳性,第33~46期后肾近端小管上皮细胞呈Bax免疫反应阳性。图像分析显示,在鸡胚肾发生过程中EGFR,TGF-β(β1,β2,β3)和Bax蛋白的阳性反应呈现先增强后减弱的变化,AQP-2阳性反应呈持续增强的趋势。(3)红腹锦鸡肾主要由许多肾单位、集合管和少量结缔组织组成。肾单位是肾功能结构的基本单位,它由一个肾小体和一个与其连接的上皮性肾小管构成。血管球由一团迂回盘曲的毛细血管丛构成,结构简单;肾小囊足细胞的突起与毛细血管内皮细胞及基膜紧密相贴,三者构成肾小体的滤过屏障;近端小管由单层立方上皮组成,上皮细胞游离面有许多微绒毛,细胞界限不明显,侧面有许多指状突起彼此交错,质膜内褶较少;远端小管上皮细胞基底面有丰富的质膜内褶;集合管上皮细胞有明细胞和暗细胞两种类型;近端小管上皮细胞呈EGFR、TGF-β免疫反应阳性,集合管上皮细胞呈AQP-2免疫反应阳性。结论(1)鸡的中肾具有排泄功能。鸡后肾的发生与分化存在明显的时间差异。肾单位的分化中,同一胚龄肾组织内可存在不同发育阶段的肾小体,集合小管分化较早,诱导近端小管和远端小管分化,细段分化较迟。(2)中肾肾小管的退化可能与TGF-β和Bax蛋白的大量表达有关。EGFR、TGF-β、AQP-2和Bax蛋白的协同表达调控着后肾肾小管和集合小管的发生及分化成熟。在肾发育过程中集合小管上皮细胞AQP-2表达丰富,AQP-2可能介导胚胎发育过程中水平衡的稳定。EGFR、TGF-β和AQP-2、Bax蛋白在胚胎肾不同发育阶段具有不同的表达规律和特征,发挥着不同的功能,它们对肾单位的构筑、肾小管和集合小管上皮的成熟分化都有重要的调节作用。(3)EGFR、TGF-β、AQP-2可能发挥着不同的功能,对红腹锦鸡肾的肾单位、肾小管和集合管结构的稳定以及肾水的平衡等可能有重要的调节作用。(4)鸡和红腹锦鸡肾结构和功能基本相似。红腹锦鸡肾的近端小管细胞顶部有许多电子密度高的短棒状致密颗粒,底部细胞质被深染,集合管细胞顶部胞质中亦有较多具有分泌功能的囊泡。在鸡肾发育过程中各种蛋白的表达呈现规律性变化,而在红腹锦鸡肾中各种蛋白的表达相对稳定。更多还原

【Abstract】 Objective To explore the development and degeneration of mesonephros,the patternsand characteristics about metanephric development and differentiation of chicks andstructural characteristics in Chrysolophus pictus kidney,mesonephros and metanephrosin chicks at 9 different incubation stages and 5 Chrysolophus pictus kidney wereobserved. Through measuring the expression of EGFR, TGF-β, AQP-2, Bax duringdevelopment of renal tubules in chick embryo and in Chrysolophus pictus kidneyexplore the role and regulating significance of them.Methods Electromicroscopic and histological methods were applied.The expressionof EGFR,TGF-β,AQP-2,Bax were measured by immunohistochemistry combined withstereological methods in chicks kidney at different incubation stages and inChrysolophus pictus kidney.The expression intensity of them was analyzed by IPP.Results (1)Some mesonephric vesicles appeared in the vicinity of the anterior of themesonephric ducts at the 16th stage.At the 18th stage mesonephric tubules tookshape.At the 26th stage mesonephric tubules’blind ends invaginated,resulting inprimitive renal sacs and glomerulus,mesonephric tubules elongated distinctly andcurved back and forth.All mesonephric tissue had produced integrated nephric unitsduring the 33rd-37th stages.During the 37th-46th stages mesonephric tissue fromanterior to posterior of the embryos degenerated in turn.At the 26th stage ofincubation,ureteric bud from cloaca grew into metanephogenic tissue which producedmany metanephrogenic vesicles. At the 33rd stage renal tubules and renal sacsappeared,next renal tubules lengthened apparently and folded regularly,which presentedmorpholotical differentiation of collecting convoluted tubules,proximal convolutedtubules and distal convoluted tubules.During the 37th-46th stages renal corpuscledeveloped and matured,renal tubules produced functional differentiationgradually,presented morpholotical differentiation the neck part of tubules. (2)Theepithelial cells of proximal tubules in chick embryo appeared EGFR immunoreaction atthe 26th-46th stages.At the 26th-37th stages the epithelial cells of proximal tubules and distal tubules of mesonephros appeared TGF-βimmunoreaction,the epithelial cells ofproximal tubules and distal tubules of metanephros appeared TGF-βimmunoreaction atthe 33rd-40th stages,at the 40th-46th stages the epithelial cells of proximal tubules ofmetanephros appeared TGF-βimmunoreaction but the epithelial cells of distal tubulesdid not appeare TGF-βimmunoreaction.The epithelial cells of collecting tubules inchick embryo appeared AQP-2 immunoreaction at the 26th-46th stages.The epithelialcells of proximal tubules and distal tubules of mesonephros appeared Baximmunoreaction at the 26th-37th stages,the epithelial cells of proximal tubules ofmetanephros appeared Bax immunoreaction at the 33rd-46th stages.Images showed theexpression of EGFR, TGF-β(β1,β2,β3) and Bax first displayed up then down,butexpression of AQP-2 always displayed up during development of chick embryo.(3)The kidney of Chrysolophus pictus was made from many nephrons,collecting ductsand a little connective tissue.The nephron was the structural and functional unit ofkidney,made from a renal corpuscle and a epithelial renal tubule.The renal glomeruluswas made from the convoluted capillary and its structure was simple.The podocytes ofthe renal capsule combined with the endothelium cell and basal membrance,whichtogether formed the filtration barrier of the glomerulus.The proximal tubule was linedby a simple cuboidal epithelium with a well-developed brush border (microvilli).Thelateral borders of these cells had many lateral extensions. The complex interdigitationsof the lateral extensions of neighbouring cells interlocked them with one another.Theplasma membrane infoldings were not abundant in the epithelial cells of proximaltubule,but the plasma membrane infoldings were abundant in the epithelial cells ofdistal tubule.The epithelial cells of collecting duct had light cells and dark cells.EGFRand TGF-βappeared to be expressed in the epithelial cells of proximal tubules. AQP-2was present in the collecting ducts.Conculsion (1)Mesonephros of chick embryo has excretory function. Developmentand differentiation evidently vary in time at different sections of mesonephros.Duringdevelopment and differentiation of nephric units renal corpuscles of differentdeveloping sections appeare at the same stage.The differentiation of collectingconvoluted tubules is earlier,which induces the differentiation of proximal and distal convoluted tubules.The differentiation of neck part of tubules is later.(2)Degeneration of renal tubules of mesonephros might be related with the excessiveexpression of TGF-βand Bax. Development,differentiation and maturation of renaltubules and collecting tubules of metanephros might be due to the corporate expressionof EGFR, TGF-β, AQP-2 and Bax.Expression of AQP-2 is abundant in the epithelialcells of collecting tubules,which related with the balance of water during developmentand maturation of renal tubules in chick embryo.In different stages the expression ofEGFR,TGF-β,AQP-2,Bax are different,which hints they have different function.Theyare very important for forming nephron,maturation and differentiation of renal tubulesand collecting tubules. (3)EGFR,TGF-β,AQP-2 have different function.They are veryimportant for structural stable of nephron,renal tubules and collecting ducts and waterbalance in Chrysolophus pictus kidney. (4)The structure and function of chick andChrysolophus pictus’s kidney are similar.There is dense electron cosh shape grains atthe top cells of proximal tubules，but cytoplasm is deeply stained at the bottom ofproximal tubules in Chrysolophus pictus kidney,there is vesicles with excreting at thetop cells of collecting ducts.During development of chick kidney interrelatedpolypeptides expression present rule changes,but interrelated polypeptides expressionpresent comparatively stabilization in Chrysolophus pictus kidney.更多还原