【摘要】：心力衰竭是各種心血管疾病的終末階段,是現(xiàn)代社會主要的致死原因之一。水潴留是心力衰竭發(fā)生發(fā)展的重要病理生理機制,機體內(nèi)多種因素參與了水潴留的形成與發(fā)展,其中腎臟集合管主細胞分布的腎臟水通道蛋白2(Aquaporin2,AQP2)在管腔側(cè)質(zhì)膜分布增加是水潴留形成關鍵性的一環(huán)。AQP2分布于腎臟集合管主細胞管腔側(cè)質(zhì)膜和胞質(zhì)內(nèi)囊泡,其在包括精氨酸加壓素(arginine vasopressin,AVP)、降鈣素(calcitonin)、一氧化氮(NO)等在內(nèi)的多種因素調(diào)節(jié)下而改變其在質(zhì)膜及囊泡內(nèi)的分布比例,被稱為AQP2的短時調(diào)控。Obestatin是近年來發(fā)現(xiàn)的一種由23個氨基酸殘基組成的多肽,GPR39是其可能的受體。以往對其研究主要集中在胃腸道功能以及能量代謝等方面,近幾年來心血管領域也逐漸進入了Obestatin的研究中,目前已發(fā)現(xiàn)其對血壓調(diào)節(jié)、內(nèi)皮損傷以及再灌注損傷等方面具有調(diào)控作用,但對心力衰竭領域仍少有研究。前期研究顯示心衰患者,尤其心腎綜合征患者血漿內(nèi)Obestatin表達升高。另外有研究顯示Obestatin在中樞可抑制口渴,減少飲水進而調(diào)節(jié)機體水平衡,但對于Obestatin在外周對于水平衡的作用尚不明確。結(jié)合GPR39在腎臟的表達以及Obestatin在骨骼肌細胞中自分泌/旁分泌方式的修復作用,我們推測心力衰竭時Obestatin通過內(nèi)分泌及自分泌/旁分泌的方式調(diào)節(jié)AQP2在集合管細胞管腔側(cè)質(zhì)膜的分布比例,進而調(diào)節(jié)腎臟對水的重吸收,參與心衰水潴留的發(fā)生發(fā)展。我們通過構(gòu)建大鼠心力衰竭模型,檢測心衰大鼠循環(huán)血漿及腎臟組織表達Obestatin的變化,在此基礎上,在集合管細胞觀察Obestatin對AQP2的亞細胞分布及蛋白總量及磷酸化水平的影響以明確Obestatin對AQP2的短時調(diào)控作用,并探討其可能的機制。第一部分Obestatin在心梗后心衰大鼠循環(huán)及腎臟的表達目的:研究心力衰竭大鼠循環(huán)及腎臟組織中Obestatin的表達及其可能的生物學意義。方法:采用冠狀動脈結(jié)扎方法制備大鼠心肌梗死后心衰模型,成功構(gòu)建心衰大鼠6只;另5只大鼠只開胸不結(jié)扎為假手術組;5只大鼠作為正常組,分別收集術前及術后8周尿量及飲水量;采用酶聯(lián)免疫吸附法測定大鼠血漿Obestatin濃度,取大鼠腎臟組織行免疫組織化學檢測Obestatin在腎臟的表達并分析累積光密度值比較各組表達差異,同時以Western Blot方法對各組腎臟組織表達的Obestatin進行半定量分析。結(jié)果:心衰組大鼠水潴留量與正常組及假手術組相比明顯增多(22.00±5.44ml vs5.80±2.59ml,P0.01;22.00±5.44ml vs 3.00±4.69ml,P0.01);免疫組化結(jié)果顯示Obestatin在大鼠腎臟腎小管及集合管表達,分析光密度值及Western Blot結(jié)果顯示心衰組大鼠腎臟組織Obestatin表達較正常組及假手術組明顯升高(P0.05)。結(jié)論:Obestatin在心力衰竭大鼠循環(huán)及腎臟表達增高,可能以內(nèi)分泌及自分泌或旁分泌形式參與心衰水潴留的發(fā)生發(fā)展。第二部分Obestatin對腎臟水通道蛋白2的短時調(diào)控作用目的:探究Obestatin對小鼠腎臟內(nèi)髓集合管3上皮細胞(IMCD3)AQP2的短時調(diào)控作用并進一步探索其可能的機制。方法:培養(yǎng)IMCD3,分別給予PBS、不同濃度的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)干預15min、30min、60min,激光共聚焦觀察IMCD3細胞AQP2的分布定位,Western Blot法檢測AQP2及P-AQP2(Ser256)的表達水平,并分析P-AQP2(Ser256)占AQP2的比例。結(jié)果:1、10~(-7)mmol/L Obestatin及10-6mmol/L Obestatin干預15min后激光共聚焦觀察與對照組(PBS)相比均未見AQP2分布改變,作用30min及60min后與對照組相比,可見AQP2在細胞膜分布減少;10~(-7)mmol/L NA-Obestatin作用15min、30min、60min均未見AQP2分布改變;10~(-7)mmol/L d DAVP作用30min后與對照組相比AQP2在細胞膜分布增多,作用15min及60min時未見其分布改變;10~(-7)mmol/L OPC-31260作用15min時與對照組相比AQP2分布未見改變,30min及60min后與對照組相比,可見AQP2在細胞膜分布減少。2、Western Blot檢測蛋白水平可見干預15min后不同濃度Obestatin(10~(-7)mmol/L、10-6mmol/L)組、NA-Obestatin組、d DAVP組及OPC-31260組AQP2表達水平與對照組相比均未見明顯改變;d DAVP組P-AQP2(Ser256)及磷酸化比值較對照組升高(均P0.05),Obestatin、NA-Obestatin及OPC-31260組均未見明顯變化。干預30min后,OPC-31260組AQP2及P-AQP2(Ser256)表達水平與對照組相比明顯降低(均P0.05),磷酸化比值升高(P0.05),余各組AQP2、P-AQP2(Ser256)及磷酸化比值均未見明顯變化。干預60min后,不同濃度Obestatin組、d DAVP組及OPC-31260組AQP2表達水平與對照組相比均明顯下降(均P0.05),NA-Obestatin組未見改變;不同濃度Obestatin組及OPC-31260組P-AQP2(Ser256)表達水平與對照組相比明顯降低(均P0.05),d DAVP組及NA-Obestatin組未見改變未見變化;d DAVP組磷酸化比值與對照組相比明顯升高(P0.05),余各組均未見明顯變化。結(jié)論:Obestatin對IMCD3細胞AQP2具有短時調(diào)控作用,可能通過增加AQP2的內(nèi)吞作用降低其在細胞膜的分布比例。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R541.6

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