Obestatin对心力衰竭大鼠肾脏水通道蛋白2短时调控作用的研究

发布时间：2019-05-17 23:53

【摘要】：心力衰竭是各种心血管疾病的终末阶段,是现代社会主要的致死原因之一。水潴留是心力衰竭发生发展的重要病理生理机制,机体内多种因素参与了水潴留的形成与发展,其中肾脏集合管主细胞分布的肾脏水通道蛋白2(Aquaporin2,AQP2)在管腔侧质膜分布增加是水潴留形成关键性的一环。AQP2分布于肾脏集合管主细胞管腔侧质膜和胞质内囊泡,其在包括精氨酸加压素(arginine vasopressin,AVP)、降钙素(calcitonin)、一氧化氮(NO)等在内的多种因素调节下而改变其在质膜及囊泡内的分布比例,被称为AQP2的短时调控。Obestatin是近年来发现的一种由23个氨基酸残基组成的多肽,GPR39是其可能的受体。以往对其研究主要集中在胃肠道功能以及能量代谢等方面,近几年来心血管领域也逐渐进入了Obestatin的研究中,目前已发现其对血压调节、内皮损伤以及再灌注损伤等方面具有调控作用,但对心力衰竭领域仍少有研究。前期研究显示心衰患者,尤其心肾综合征患者血浆内Obestatin表达升高。另外有研究显示Obestatin在中枢可抑制口渴,减少饮水进而调节机体水平衡,但对于Obestatin在外周对于水平衡的作用尚不明确。结合GPR39在肾脏的表达以及Obestatin在骨骼肌细胞中自分泌/旁分泌方式的修复作用,我们推测心力衰竭时Obestatin通过内分泌及自分泌/旁分泌的方式调节AQP2在集合管细胞管腔侧质膜的分布比例,进而调节肾脏对水的重吸收,参与心衰水潴留的发生发展。我们通过构建大鼠心力衰竭模型,检测心衰大鼠循环血浆及肾脏组织表达Obestatin的变化,在此基础上,在集合管细胞观察Obestatin对AQP2的亚细胞分布及蛋白总量及磷酸化水平的影响以明确Obestatin对AQP2的短时调控作用,并探讨其可能的机制。第一部分Obestatin在心梗后心衰大鼠循环及肾脏的表达目的:研究心力衰竭大鼠循环及肾脏组织中Obestatin的表达及其可能的生物学意义。方法:采用冠状动脉结扎方法制备大鼠心肌梗死后心衰模型,成功构建心衰大鼠6只;另5只大鼠只开胸不结扎为假手术组;5只大鼠作为正常组,分别收集术前及术后8周尿量及饮水量;采用酶联免疫吸附法测定大鼠血浆Obestatin浓度,取大鼠肾脏组织行免疫组织化学检测Obestatin在肾脏的表达并分析累积光密度值比较各组表达差异,同时以Western Blot方法对各组肾脏组织表达的Obestatin进行半定量分析。结果:心衰组大鼠水潴留量与正常组及假手术组相比明显增多(22.00±5.44ml vs5.80±2.59ml,P0.01;22.00±5.44ml vs 3.00±4.69ml,P0.01);免疫组化结果显示Obestatin在大鼠肾脏肾小管及集合管表达,分析光密度值及Western Blot结果显示心衰组大鼠肾脏组织Obestatin表达较正常组及假手术组明显升高(P0.05)。结论:Obestatin在心力衰竭大鼠循环及肾脏表达增高,可能以内分泌及自分泌或旁分泌形式参与心衰水潴留的发生发展。第二部分Obestatin对肾脏水通道蛋白2的短时调控作用目的:探究Obestatin对小鼠肾脏内髓集合管3上皮细胞(IMCD3)AQP2的短时调控作用并进一步探索其可能的机制。方法:培养IMCD3,分别给予PBS、不同浓度的Obestatin(10~(-7)mmol/L、10-6mmol/L)、NA-Obestatin(10~(-7)mmol/L)、d DAVP(10~(-7)mmol/L)及OPC-31260(10~(-7)mmol/L)干预15min、30min、60min,激光共聚焦观察IMCD3细胞AQP2的分布定位,Western Blot法检测AQP2及P-AQP2(Ser256)的表达水平,并分析P-AQP2(Ser256)占AQP2的比例。结果:1、10~(-7)mmol/L Obestatin及10-6mmol/L Obestatin干预15min后激光共聚焦观察与对照组(PBS)相比均未见AQP2分布改变,作用30min及60min后与对照组相比,可见AQP2在细胞膜分布减少;10~(-7)mmol/L NA-Obestatin作用15min、30min、60min均未见AQP2分布改变;10~(-7)mmol/L d DAVP作用30min后与对照组相比AQP2在细胞膜分布增多,作用15min及60min时未见其分布改变;10~(-7)mmol/L OPC-31260作用15min时与对照组相比AQP2分布未见改变,30min及60min后与对照组相比,可见AQP2在细胞膜分布减少。2、Western Blot检测蛋白水平可见干预15min后不同浓度Obestatin(10~(-7)mmol/L、10-6mmol/L)组、NA-Obestatin组、d DAVP组及OPC-31260组AQP2表达水平与对照组相比均未见明显改变;d DAVP组P-AQP2(Ser256)及磷酸化比值较对照组升高(均P0.05),Obestatin、NA-Obestatin及OPC-31260组均未见明显变化。干预30min后,OPC-31260组AQP2及P-AQP2(Ser256)表达水平与对照组相比明显降低(均P0.05),磷酸化比值升高(P0.05),余各组AQP2、P-AQP2(Ser256)及磷酸化比值均未见明显变化。干预60min后,不同浓度Obestatin组、d DAVP组及OPC-31260组AQP2表达水平与对照组相比均明显下降(均P0.05),NA-Obestatin组未见改变;不同浓度Obestatin组及OPC-31260组P-AQP2(Ser256)表达水平与对照组相比明显降低(均P0.05),d DAVP组及NA-Obestatin组未见改变未见变化;d DAVP组磷酸化比值与对照组相比明显升高(P0.05),余各组均未见明显变化。结论:Obestatin对IMCD3细胞AQP2具有短时调控作用,可能通过增加AQP2的内吞作用降低其在细胞膜的分布比例。
[Abstract]:Heart failure is the terminal stage of various cardiovascular diseases, and is one of the main causes of death in modern society. Water retention is an important pathophysiological mechanism of the development of heart failure, and many factors in the body are involved in the formation and development of water retention. The distribution of the renal aquaporin 2 (Aquaporin2, AQP2) in the main cell of the renal collection tube is a key part of the water retention formation. AQP2 is distributed in the main cell tube cavity side plasma membrane and the intracytoplasmic vesicle of the kidney collection tube, and the AQP2 changes the distribution proportion of the plasma membrane and the vesicle under the regulation of various factors including arginine vasopressin (AVP), calcitonin (calcitonin), nitric oxide (NO), and the like, Is known as the short-time regulation of AQP2. Obestatin is a polypeptide that has been found in recent years, consisting of 23 amino acid residues, and the GPR39 is its possible receptor. In the past few years, the research on the functions of the gastrointestinal tract and energy metabolism has also gradually entered the study of Obestatin in recent years, and has been found to have a regulatory role in the aspects of blood pressure regulation, endothelial injury, and reperfusion injury. But there is still a rare study in the field of heart failure. In the earlier study, the expression of Obestatin in the plasma of patients with heart failure, especially in the heart and kidney syndrome, was increased. In addition, it is shown that Obestatin can inhibit the thirst in the central nervous system, reduce the drinking water and adjust the water balance of the body, but it is not clear to the effect of the Obestatin on the water balance at the periphery. In combination with the expression of GPR39 in the kidney and the repair of Obestatin in the autocrine/ paracrine mode of skeletal muscle cells, we hypothesized that in the case of heart failure, Obestatin regulates the distribution of AQP2 in the plasma membrane of the tube cavity of the collection tube by the way of endocrinology and autocrine/ paracrine secretion. So as to regulate the reabsorption of the water and participate in the development of the water retention of the heart failure. We established a model of rat heart failure to detect the changes of the expression of Obestatin in the circulating plasma and the kidney of the rats with heart failure. On the basis of this, the effect of Obestatin on the subcellular distribution of AQP2 and the total protein and the level of phosphorylation of AQP2 was observed in the collection tube, and the short-time control effect of Obestatin on AQP2 was determined. And to explore its possible mechanism. Objective: To study the expression and possible biological significance of the first part of Obestatin in the circulation of heart failure rats and the expression of the kidney. Methods: The model of heart failure after myocardial infarction was prepared by the method of coronary artery ligation.6 rats with heart failure were successfully constructed. An enzyme-linked immunosorbent assay was used to determine the concentration of Obestatin in the rat, and the expression of Obestatin in the kidney was detected by immunohistochemistry in the kidney of the rat. The expression of Obestatin in each group was compared by Western Blot method. Results: Compared with the normal group and the sham operation group, the amount of water retention of the rats in the heart failure group increased significantly (22.00 to 5.44 ml vs5.80, 2.59 ml, P 0.01, 22.00, 5.44 ml vs 3.00% 4.69 ml, P 0.01), and the results of the immunohistochemistry showed that the Obestatin was expressed in the renal tubular and collection tubes of the rat kidney. The results of optical density and Western Blot show that the expression of Obestatin in the kidney of the heart failure group is higher than that in the normal group and the sham operation group (P0.05). Conclusion: The expression of Obestatin in heart failure rats is increased, and it is possible to take part in the development of water retention of heart failure in the form of endocrine and autocrine or paracrine. Objective: To investigate the short-term regulatory effect of the second part of Obestatin on the renal aquaporin-2, and to investigate the short-term regulatory effect of Obestatin on the AQP2 in the rat kidney, and to further explore its possible mechanism. Methods: The expression levels of AQP2, AQP2 and P-AQP2 (Ser256) were measured by Western Blot method, and the expression level of AQP2 and P-AQP2 (Ser256) was detected by Western Blot method, and the ratio of P-AQP2 (Ser256) to AQP2 was analyzed. Results:1,10 ~ (-7) mmol/ L Obestatin and 10-6mmol/ L Obestatin had no change in the distribution of AQP2 compared with the control group (PBS), and the distribution of AQP2 in the cell membrane was reduced after 30 min and 60 min, and the distribution of AQP2 was not found in 10-(-7) mmol/ L NA-Obestatin for 15 min,30 min and 60 min. Compared with the control group, AQP2 had no change in the distribution of the cell membrane, and the distribution of AQP2 was not changed at the time of 15 min and 60 min after the effect of 10 ~ (-7) mmol/ L d DAVP and the distribution of AQP2 was not changed at 15 min and 60 min. Compared with the control group, the distribution of AQP2 was less than that in the control group, and the distribution of AQP2 in the cell membrane was reduced after 30 min and 60 min. The expression levels of AQP2 in different concentrations of Obestatin (10 ~ (-7) mmol/ L,10-6mmol/ L), NA-Obestatin group, d-DAVP group and OPC-31260 group were not significantly changed after 15 min, and the level of P-AQP2 (Ser256) and P-AQP2 (Ser256) and the phosphorylation ratio of DDAVP group were higher than that of the control group (all P0.05), and no significant changes were observed in the group of Obestatin, NA-Obestatin and OPC-31260. After 30 min of intervention, the expression level of AQP2 and P-AQP2 (Ser256) in the OPC-31260 group was significantly lower than that in the control group (P0.05). After 60 min of intervention, the expression level of AQP2 in different concentrations of Obstatin group, d-DAVP group and OPC-31260 group was significantly lower than that in the control group (all P0.05). The expression level of P-AQP2 (Ser256) in the different concentration of Obestatin group and the OPC-31260 group was significantly lower than that of the control group (all P0.05), and no change was found in the d-DAVP group and the NA-Obststatin group. The phosphorylation ratio of d-DAVP group was significantly higher than that of the control group (P0.05). Conclusion: It is possible to decrease the distribution of AQP2 in the cell membrane by increasing the endocytosis of AQP2.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.6

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