模擬失重條件對大鼠腎小管水通道蛋白2表達影響的研究
發(fā)布時間：2018-10-18 10:29
【摘要】：水通道蛋白(Aquaporins，AQPs)是水分子跨越細胞的快速轉(zhuǎn)運通道，廣泛分布于機體組織細胞，主要介導細胞內(nèi)外的水分子運輸，對維持細胞內(nèi)水平衡具有重要作用。AQPs的發(fā)現(xiàn)不僅從分子水平揭示水跨膜轉(zhuǎn)運調(diào)節(jié)的機制，而且也揭示水平衡在遺傳性及獲得性疾病時的病理生理機制。它在全身各個器官組織中都有表達，以腎臟分布種類最多[1]，研究表明至少有AQPl、2、3、4、6、7、8、11這8種水通道蛋白在腎臟表達，這些選擇性的表達在腎單位細胞質(zhì)膜和腎小血管的水通道蛋白在尿濃縮機制中起著至關重要的作用[2]。現(xiàn)已明確AQP1-4是腎臟重吸收水、濃縮尿液、維持機體水平衡的主要分子基礎。其中AQP2是腎臟集合管柱狀上皮細胞內(nèi)最重要的水通道蛋白[3]，也是迄今為止發(fā)現(xiàn)的唯一在細胞內(nèi)分布受抗利尿激素（ADH）調(diào)控的水通道蛋白。目前已證實AQP2在腎小管濃縮、重吸收功能中起著關鍵的作用[4]。本課題就模擬失重條件下大鼠腎小管水通道蛋白2的表達變化展開相關研究。 目的：利用尾吊模型，模擬失重條件，探討在模擬失重條件下大鼠腎小管水通道蛋白2表達的變化，并結(jié)合腎臟的電鏡超微結(jié)構(gòu)，抗利尿激素的表達以及腎功能的改變，研究失重條件下水通道蛋白的表達變化與腎小管功能損傷的關系。方法：采用尾部懸吊法建立失重模型。48只健康雄性wistar大鼠分為對照組及尾吊3天組、尾吊5天組、尾吊7天組、尾吊2周組、尾吊4周組。適應性飼養(yǎng)1周后開始尾吊。分別于觀察終點處死各組大鼠，處死前留取尿標本，檢測尿滲透壓。斷頭取血，離心后取血漿檢測腎功能及血清抗利尿激素。剝離腎臟組織，分離腎皮質(zhì)和髓質(zhì)。電鏡觀察腎小管超微結(jié)構(gòu)。免疫熒光法及western bolt方法檢測AQP2的表達。酶聯(lián)免疫吸附法（ELISA）檢測血清中抗利尿激素的水平。結(jié)果：1.電鏡下尾吊組大鼠腎臟主要表現(xiàn)為腎小管不同程度受損，其中以尾吊5天組大鼠腎小管損傷最嚴重。2.免疫熒光結(jié)果顯示尾吊組大鼠腎臟AQP2表達的熒光較對照組均升高，亮度增強，其中以尾吊5天組大鼠腎臟AQP2表達條帶最亮。3.western bolt結(jié)果顯示實驗組AQP2的表達較對照組均升高，差異有統(tǒng)計學意義（P＜0.05），其中尾吊5天組大鼠腎臟AQP2表達最明顯。4.尾吊5天組尿滲透壓顯著升高，與其他各組比較差異有統(tǒng)計學意義（P＜0.01）。各組間ADH及腎功能指標差異無統(tǒng)計學意義（P＞0.05）。5.AQP2蛋白表達量與尿滲透壓呈正相關（r=0.468，P=0.003）。結(jié)論：模擬失重條件能引起大鼠腎小管損傷，腎小管AQP2表達上升，尿滲透壓改變，各種改變均以尾吊5天組最明顯。而尾吊對腎功能、ADH的影響并沒有統(tǒng)計學差異。
[Abstract]:Aquaporin (Aquaporins,AQPs) is a rapid transport channel of water molecules across cells. It is widely distributed in tissues and cells, and mainly mediates the transport of water molecules inside and outside cells. The discovery of AQPs not only reveals the mechanism of water transmembrane transport regulation at molecular level, but also reveals the pathophysiological mechanism of water balance in hereditary and acquired diseases. It is expressed in all organs and tissues of the whole body, most of which are found in kidneys. Studies have shown that at least eight aquaporins, AQPl,2,3,4,6,7,8,11, are expressed in the kidneys. These selective expressions play an important role in the mechanism of urinary concentration in renal unit cytoplasmic membrane and renal microvascular aquaporins [2]. AQP1-4 has been identified as the main molecular basis for reabsorbing water, concentrating urine and maintaining water balance. Among them, AQP2 is the most important aquaporin in the columnar epithelial cells of renal collecting tube, and it is the only aquaporin which is regulated by the antidiuretic hormone (ADH). It has been confirmed that AQP2 plays a key role in renal tubule concentration and reabsorption [4]. In this study, we studied the changes of renal tubular aquaporin 2 expression in rats under simulated weightlessness. Objective: to investigate the changes of renal tubular aquaporin-2 expression in rats under simulated weightlessness by tail suspension model and the ultrastructure of kidney, the expression of anti-diuretic hormone and the changes of renal function. To study the relationship between the expression of aquaporin in weightlessness and renal tubular dysfunction. Methods: the weightlessness model was established by tail suspension. 48 healthy male wistar rats were divided into three groups: control group, tail suspension group for 3 days, tail suspension group for 5 days, tail suspension group for 7 days, tail suspension group for 2 weeks, and tail suspension group for 4 weeks. Adaptive feeding for 1 week after the start of tail suspension. The rats were killed at the end of observation. Urine samples were collected before death and urine osmotic pressure was measured. Blood was taken off the head and plasma was collected after centrifugation to detect renal function and serum antidiuretic hormone. The renal cortex and medulla were separated. The ultrastructure of renal tubules was observed by electron microscope. The expression of AQP2 was detected by immunofluorescence and western bolt. The level of antidiuretic hormone in serum was detected by enzyme linked immunosorbent assay (ELISA). The result is 1: 1. The renal tubules were damaged in different degree in the tail suspension group under electron microscope, and the renal tubule injury was the most serious in the tail suspension group for 5 days. 2. The results of immunofluorescence showed that the fluorescence of AQP2 expression in the kidney of the tail suspension group was higher than that of the control group, and the brightness was enhanced. The expression of AQP2 in the kidney of the tail suspension group was the brightest, and the 3.western bolt result showed that the expression of AQP2 in the experimental group was higher than that in the control group. The difference was statistically significant (P < 0. 05), and the expression of AQP2 was the most obvious in the tail suspension group for 5 days (P < 0. 05). The urine osmotic pressure of the tail suspension group was significantly higher than that of the other groups (P < 0.01). There was no significant difference in ADH and renal function among different groups (P > 0. 05). The expression of 5.AQP2 protein was positively correlated with urinary osmotic pressure (r = 0. 468, P < 0. 003). Conclusion: simulated weightlessness can induce tubular injury, increase the expression of renal tubule AQP2 and change the urine osmotic pressure. But the effect of tail suspension on renal function, ADH has no statistical difference.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R692.6
本文編號：2278854
[Abstract]:Aquaporin (Aquaporins,AQPs) is a rapid transport channel of water molecules across cells. It is widely distributed in tissues and cells, and mainly mediates the transport of water molecules inside and outside cells. The discovery of AQPs not only reveals the mechanism of water transmembrane transport regulation at molecular level, but also reveals the pathophysiological mechanism of water balance in hereditary and acquired diseases. It is expressed in all organs and tissues of the whole body, most of which are found in kidneys. Studies have shown that at least eight aquaporins, AQPl,2,3,4,6,7,8,11, are expressed in the kidneys. These selective expressions play an important role in the mechanism of urinary concentration in renal unit cytoplasmic membrane and renal microvascular aquaporins [2]. AQP1-4 has been identified as the main molecular basis for reabsorbing water, concentrating urine and maintaining water balance. Among them, AQP2 is the most important aquaporin in the columnar epithelial cells of renal collecting tube, and it is the only aquaporin which is regulated by the antidiuretic hormone (ADH). It has been confirmed that AQP2 plays a key role in renal tubule concentration and reabsorption [4]. In this study, we studied the changes of renal tubular aquaporin 2 expression in rats under simulated weightlessness. Objective: to investigate the changes of renal tubular aquaporin-2 expression in rats under simulated weightlessness by tail suspension model and the ultrastructure of kidney, the expression of anti-diuretic hormone and the changes of renal function. To study the relationship between the expression of aquaporin in weightlessness and renal tubular dysfunction. Methods: the weightlessness model was established by tail suspension. 48 healthy male wistar rats were divided into three groups: control group, tail suspension group for 3 days, tail suspension group for 5 days, tail suspension group for 7 days, tail suspension group for 2 weeks, and tail suspension group for 4 weeks. Adaptive feeding for 1 week after the start of tail suspension. The rats were killed at the end of observation. Urine samples were collected before death and urine osmotic pressure was measured. Blood was taken off the head and plasma was collected after centrifugation to detect renal function and serum antidiuretic hormone. The renal cortex and medulla were separated. The ultrastructure of renal tubules was observed by electron microscope. The expression of AQP2 was detected by immunofluorescence and western bolt. The level of antidiuretic hormone in serum was detected by enzyme linked immunosorbent assay (ELISA). The result is 1: 1. The renal tubules were damaged in different degree in the tail suspension group under electron microscope, and the renal tubule injury was the most serious in the tail suspension group for 5 days. 2. The results of immunofluorescence showed that the fluorescence of AQP2 expression in the kidney of the tail suspension group was higher than that of the control group, and the brightness was enhanced. The expression of AQP2 in the kidney of the tail suspension group was the brightest, and the 3.western bolt result showed that the expression of AQP2 in the experimental group was higher than that in the control group. The difference was statistically significant (P < 0. 05), and the expression of AQP2 was the most obvious in the tail suspension group for 5 days (P < 0. 05). The urine osmotic pressure of the tail suspension group was significantly higher than that of the other groups (P < 0.01). There was no significant difference in ADH and renal function among different groups (P > 0. 05). The expression of 5.AQP2 protein was positively correlated with urinary osmotic pressure (r = 0. 468, P < 0. 003). Conclusion: simulated weightlessness can induce tubular injury, increase the expression of renal tubule AQP2 and change the urine osmotic pressure. But the effect of tail suspension on renal function, ADH has no statistical difference.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R692.6
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