模拟失重条件对大鼠肾小管水通道蛋白2表达影响的研究

发布时间：2018-10-18 10:29

【摘要】：水通道蛋白(Aquaporins，AQPs)是水分子跨越细胞的快速转运通道，广泛分布于机体组织细胞，主要介导细胞内外的水分子运输，对维持细胞内水平衡具有重要作用。AQPs的发现不仅从分子水平揭示水跨膜转运调节的机制，而且也揭示水平衡在遗传性及获得性疾病时的病理生理机制。它在全身各个器官组织中都有表达，以肾脏分布种类最多[1]，研究表明至少有AQPl、2、3、4、6、7、8、11这8种水通道蛋白在肾脏表达，这些选择性的表达在肾单位细胞质膜和肾小血管的水通道蛋白在尿浓缩机制中起着至关重要的作用[2]。现已明确AQP1-4是肾脏重吸收水、浓缩尿液、维持机体水平衡的主要分子基础。其中AQP2是肾脏集合管柱状上皮细胞内最重要的水通道蛋白[3]，也是迄今为止发现的唯一在细胞内分布受抗利尿激素（ADH）调控的水通道蛋白。目前已证实AQP2在肾小管浓缩、重吸收功能中起着关键的作用[4]。本课题就模拟失重条件下大鼠肾小管水通道蛋白2的表达变化展开相关研究。目的：利用尾吊模型，模拟失重条件，探讨在模拟失重条件下大鼠肾小管水通道蛋白2表达的变化，并结合肾脏的电镜超微结构，抗利尿激素的表达以及肾功能的改变，研究失重条件下水通道蛋白的表达变化与肾小管功能损伤的关系。方法：采用尾部悬吊法建立失重模型。48只健康雄性wistar大鼠分为对照组及尾吊3天组、尾吊5天组、尾吊7天组、尾吊2周组、尾吊4周组。适应性饲养1周后开始尾吊。分别于观察终点处死各组大鼠，处死前留取尿标本，检测尿渗透压。断头取血，离心后取血浆检测肾功能及血清抗利尿激素。剥离肾脏组织，分离肾皮质和髓质。电镜观察肾小管超微结构。免疫荧光法及western bolt方法检测AQP2的表达。酶联免疫吸附法（ELISA）检测血清中抗利尿激素的水平。结果：1.电镜下尾吊组大鼠肾脏主要表现为肾小管不同程度受损，其中以尾吊5天组大鼠肾小管损伤最严重。2.免疫荧光结果显示尾吊组大鼠肾脏AQP2表达的荧光较对照组均升高，亮度增强，其中以尾吊5天组大鼠肾脏AQP2表达条带最亮。3.western bolt结果显示实验组AQP2的表达较对照组均升高，差异有统计学意义（P＜0.05），其中尾吊5天组大鼠肾脏AQP2表达最明显。4.尾吊5天组尿渗透压显著升高，与其他各组比较差异有统计学意义（P＜0.01）。各组间ADH及肾功能指标差异无统计学意义（P＞0.05）。5.AQP2蛋白表达量与尿渗透压呈正相关（r=0.468，P=0.003）。结论：模拟失重条件能引起大鼠肾小管损伤，肾小管AQP2表达上升，尿渗透压改变，各种改变均以尾吊5天组最明显。而尾吊对肾功能、ADH的影响并没有统计学差异。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.6

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