本文選題：脂聯(lián)素 + 脂聯(lián)素受體　； 參考：《第三軍醫(yī)大學》2016年博士論文

【摘要】：研究背景目前研究認為,高血壓的發(fā)病機制涉及多個系統(tǒng)。脂肪組織作為重要的內分泌器官,所分泌的脂肪細胞因子如脂聯(lián)素、瘦素以及生物活性物質如脂肪酸、前列腺素、血管緊張素原等,在心血管疾病的發(fā)生發(fā)展中起到了重要作用。脂肪細胞因子的表達以及功能失調,被認為是導致高血壓發(fā)病的重要原因。在眾多的脂肪細胞因子中,脂聯(lián)素的作用尤其引人注目。脂聯(lián)素與高血壓的關系不僅得到臨床結果的佐證,也得到實驗動物結果的支持。臨床研究發(fā)現(xiàn)血漿脂聯(lián)素水平的改變與高血壓密切相關。脂聯(lián)素敲除小鼠在高鹽誘導下表現(xiàn)為血壓升高,利用腺病毒過表達脂聯(lián)素可以降低肥胖小鼠的血壓。尿鈉重吸收增加,排泄受損是高血壓發(fā)病中的重要部分,但是脂聯(lián)素的腎臟作用、參與高血壓發(fā)生的機制等尚不清楚。脂聯(lián)素受體分為兩個亞型:脂聯(lián)素受體1(Adipo R1)以及脂聯(lián)素受體2(Adipo R2)。Adipo R1和Adipo R2在全身廣泛分布。研究顯示脂聯(lián)素的兩個受體亞型在腎臟均有高表達,并且脂聯(lián)素通過腎臟脂聯(lián)素受體對腎臟的生理和病理調節(jié)起到了重要作用。我們初期研究也發(fā)現(xiàn)脂聯(lián)素受體在RPT細胞中的表達;腎臟灌注脂聯(lián)素發(fā)揮促尿鈉排泄作用,但高血壓狀態(tài)下該作用受損。脂聯(lián)素受體雖不屬于G蛋白偶聯(lián)受體(G protein coupled receptors,GPCRs),但是卻能被G蛋白偶聯(lián)受體激酶(G protein coupled receptors kinases,GRKs)所磷酸化,其受體磷酸化是其功能改變的重要機制之一;既往的研究發(fā)現(xiàn),在眾多的GRK亞型中,GRK4活性增高早于高血壓發(fā)生而出現(xiàn),其在高血壓中的作用受到人們關注。GRK4基因位于人類染色體4q16.3的位置,與原發(fā)性高血壓相關。GRK4變異體出現(xiàn)的機率與血壓增高程度明顯相關。利用GRK4變異體對非洲加納人原發(fā)性高血壓的預測準確率達70%以上,而對日本人原發(fā)性高血壓的預測準確率達94%。我們對多個研究中心的研究結果所做的Meta分析也顯示GRK4變異體與高血壓發(fā)生的關系密切。GRK4變異體三個轉基因小鼠均表現(xiàn)為高血壓,GRK4?142V小鼠為顯性高血壓,A486和R65L小鼠在高鹽負荷的情況下表現(xiàn)為鹽敏感性高血壓。以此我們推測GRK4有可能參與了腎臟脂聯(lián)素受體的磷酸化,從而參與了高血壓的進展。所以,針對性抑制腎臟GRK4的表達可作為增強尿鈉排泄,從而改善高血壓的有效嘗試。綜上所述,我們推測:脂聯(lián)素具有利尿排鈉作用,該作用在高血壓狀態(tài)下喪失;其喪失的原因歸因于脂聯(lián)素受體發(fā)生磷酸化導致受體失活,歸因于腎臟GRK4表達和活性增高;抑制腎臟GRK4具有降血壓作用。研究目的研究脂聯(lián)素對腎臟利尿排鈉的影響及在高血壓發(fā)生發(fā)展中的作用。明確GRK4是否參與調控脂聯(lián)素受體磷酸化從而對血壓進行調節(jié),超聲微泡靶向沉默腎臟GRK4是否對SHR大鼠的血壓和尿鈉產生影響。研究內容和方法1.脂聯(lián)素在腎臟尿鈉排泄中的作用及其機制1.1采用免疫印跡以及免疫熒光染色的方法,觀察脂聯(lián)素2種受體Adipo R1和Adipo R2,在正常血壓大鼠(Wista Kyoto rat,WKY)腎臟及RPT細胞上的表達。1.2利用WKY大鼠,通過單腎灌注不同濃度的脂聯(lián)素,觀察尿流量、尿鈉排泄率、以及血壓的變化以明確脂聯(lián)素是否具有利尿作用。同時采用高溫滅活脂聯(lián)素蛋白活性的方法,以排除蛋白的滲透性利尿作用以及致敏作用。1.3在WKY的RPT細胞上,采用Na+-K+-ATP酶活性檢測方法,觀察脂聯(lián)素作用不同濃度和時間后對Na+-K+-ATP酶活性的影響。1.4實驗首先驗證Adipo R1和Adipo R2的小干擾RNA(si RNA)的干擾效率,然后將Adipo R1和Adipo R2的si RNA轉染至RPT細胞中,觀察在脂聯(lián)素受體受到抑制時,脂聯(lián)素對Na+-K+-ATP酶的作用,以明確何種受體介導脂聯(lián)素的作用。1.5采用Na+-K+-ATP酶活性測定技術,加入磷酸化腺苷酸活化蛋白激酶(Adenosine 5‘-monophosphate-activated protein kinase,AMPK)抑制劑Compound C、一氧化氮合酶(Endothelial nitric oxide synthase,e NOS)抑制劑(NG-nitro-l-arginine methyl ester,L-NAME)初步篩選脂聯(lián)素抑制Na+-K+-ATP酶活性的信號通路。然后,采用免疫印跡法進一步觀察在脂聯(lián)素作用后,磷酸化AMPK及其下游信號分子e NOS的磷酸化表達情況。2.脂聯(lián)素受體表達下調及GRK4調控脂聯(lián)素受體磷酸化導致脂聯(lián)素促尿鈉排泄作用受損2.1采用同WKY大鼠相同濃度梯度的脂聯(lián)素,對SHR進行單腎灌注。觀察尿流量、尿鈉排泄率以及平均動脈壓的改變。2.2采用自發(fā)性高血壓大鼠(Spontaneous hypertensive rat,SHR)的RPT細胞,觀察脂聯(lián)素對Na+-K+-ATP酶的作用。2.3采用實時定量PCR觀察,比較于WKY RPT細胞,脂聯(lián)素兩個受體基因在SHR RPT細胞上的表達情況,同時,免疫印跡方法檢測脂聯(lián)素受體的蛋白表達情況。2.4單腎灌注脂聯(lián)素受體激動劑即小分子化合物Adipo Ron對SHR尿鈉排泄影響,以進一步明確受損的脂聯(lián)素利尿作用來源于脂聯(lián)素受體的改變。2.5根據實驗結果,設計脂聯(lián)素兩個受體的過表達質粒,提取質粒并在SHR的RPT細胞上轉染,觀察脂聯(lián)素受體恢復表達后,脂聯(lián)素對Na+-K+-ATP酶的作用。2.6利用免疫共沉淀的方法,觀察在SHR的RPT細胞上,脂聯(lián)素受體的磷酸化表達情況。2.7脂聯(lián)素受體激動劑Adipo Ron灌注GRK4?142V轉基因小鼠(相比較于GRK4?WT小鼠,該小鼠的GRK4活性增強),觀察脂聯(lián)素受體參與的利尿作用是否受損。2.8篩選有效的GRK4 si RNA。觀察在SHR RPT細胞中,干擾GRK4表達后,脂聯(lián)素受體磷酸化程度。3.GRK4超聲微泡靶向治療改善SHR脂聯(lián)素受體磷酸化并調節(jié)尿鈉排泄與血壓3.1利用5-羧基熒光素(5-Carboxyfluorescein,FAM)標記的超聲微泡對GRK4si RNA進行包裹。3.2將超聲微泡注射到SHR,在腎臟進行超聲微泡靶向破壞技術(Ultrasound-targeted microbubble destruction,UTMD)釋放出GRK4 si RNA。20天后,觀察SHR血壓以及24小時(hour,h)尿量、尿鈉排泄率,以及腎功能指標和腎纖維化指標。3.3取UTMD傳輸GRK4 si RNA至SHR大鼠腎臟,采用免疫共沉淀方法,觀察脂聯(lián)素受體磷酸化情況。研究結果1.脂聯(lián)素受體Adipo R1以及Adipo R2在腎臟近曲小管均有表達,特別是腎臟皮質,在RPT細胞表達豐富。Adipo R1的蛋白條帶為43Kd,Adipo R2的蛋白條帶為44Kd。2.脂聯(lián)素對WKY大鼠具有促尿量和尿鈉排泄的作用,該作用呈現(xiàn)濃度依賴性3.脂聯(lián)素對WKY RPT細胞上的Na+-K+-ATP酶活性產生抑制作用,該作用呈現(xiàn)濃度和依賴性。4.為研究脂聯(lián)素作用的特異性,采用si RNA在WKY RPT細胞中敲低Adipo R1和Adipo R2,結果發(fā)現(xiàn)脂聯(lián)素對Na+-K+-ATP酶活性的抑制作用消失。5.Na+-K+-ATP酶活性檢測發(fā)現(xiàn),加入AMPK阻斷劑和e NOS阻斷劑后,脂聯(lián)素對Na+-K+-ATP酶活性的抑制作用消失。同時,脂聯(lián)素增加了磷酸化AMPK以及e NOS的表達,同時采用AMPK阻斷劑后,脂聯(lián)素對e NOS磷酸化增強的作用消失,說明脂聯(lián)素通過AMPK-e NOS信號途徑對WKY RPT細胞上的Na+-K+-ATP酶活性產生抑制作用。6.脂聯(lián)素的利尿作用在SHR中受損。脂聯(lián)素對Na+-K+-ATP酶的抑制作用在SHR的RPT細胞中同樣受損。脂聯(lián)素的作用得到了脂聯(lián)素受體激動劑Adipo Ron的佐證:即:Adipo Ron具有利尿作用,該作用在SHR喪失。7.SHR腎臟脂聯(lián)素受體表達量低于對照WKY大鼠,但其受體的表達量改變難以解釋其功能喪失,因為過表達Adipo R1和Adipo R2后,其利尿作用仍不能恢復,說明還有其它因素參與。由于脂聯(lián)素受體的磷酸化影響受體功能,我們的研究發(fā)現(xiàn)SHR腎臟脂聯(lián)素受體磷酸化水平升高,提示高磷酸化水平可能是脂聯(lián)素受體功能下降的原因。8.為驗證GRK4是否參與調控脂聯(lián)素受體磷酸化過程,我們采用GRK4?142V轉基因小鼠灌注脂聯(lián)素受體激動劑,發(fā)現(xiàn)脂聯(lián)素受體參與調節(jié)的利尿作用消失。在SHR的RPT細胞中采用GRK4 si RNA干擾后,脂聯(lián)素受體Adipo R1和Adipo R2的磷酸化程度減弱。這說明GRK4參與調控了脂聯(lián)素受體的磷酸化,而脂聯(lián)素促尿鈉排泄作用的受損和脂聯(lián)素受體在SHR中發(fā)生磷酸化相關。9.采用微泡包裹GRK4 si RNA,在SHR的腎臟進行靶向定位超聲破碎微泡,釋放出的GRK4 si RNA對GRK4產生抑制作用,腎臟脂聯(lián)素受體的磷酸化水平減弱,SHR大鼠尿鈉排泄增加,血壓下降。同時,SHR腎功能未受到影響,腎纖維化得到改善。結論脂聯(lián)素具有促腎臟尿鈉排泄作用,在高血壓狀態(tài)下,這一功能受損;檢測發(fā)現(xiàn)脂聯(lián)素受體表達下調,但過表達受體后并不能完全恢復脂聯(lián)素功能。受體的磷酸化可能在其中發(fā)揮重要的作用;GRK4活性增高是脂聯(lián)素受體高磷酸化的原因;UTMD傳輸GRK4 si RNA有效抑制腎臟GRK4表達,在SHR可發(fā)揮降低血壓、利尿排鈉作用。
[Abstract]:Research background current research suggests that the pathogenesis of hypertension involves multiple systems. Adipose tissue is an important endocrine organ, and the secreted adipocytokines such as adiponectin, leptin and bioactive substances such as fatty acids, prostaglandins, angiotensinogen and so on, play an important role in the development of cardiovascular diseases. The expression and dysfunction of adipocytokines are considered to be an important cause of hypertension. In many adipocytokines, the role of adiponectin is especially attractive. The relationship between adiponectin and hypertension is not only supported by clinical results, but also supported by experimental animal results. Plasma adiponectin is found in clinical research. Level changes are closely related to hypertension. Adiponectin knockout mice are elevated in high salt induced blood pressure. Using adenovirus to express adiponectin can reduce blood pressure in obese mice. The increase in urine sodium reabsorption and impaired excretion is an important part of hypertension, but the renal function of lipoprotein is involved in the pathogenesis of hypertension. The adiponectin receptor is divided into two subtypes: the adiponectin receptor 1 (Adipo R1) and the adiponectin receptor 2 (Adipo R2).Adipo R1 and Adipo R2 are widely distributed throughout the body. The study shows that the two receptor subtypes of adiponectin are highly expressed in the kidney, and the adiponectin is regulated by the renal adiponectin receptor for the kidney's physiology and pathology. Our initial study also found the expression of adiponectin receptors in RPT cells; renal perfusion adiponectin played a role in promoting urinary sodium excretion, but the effect was impaired in hypertension. Although the adiponectin receptor was not a G protein coupled receptor (G protein coupled receptors, GPCRs), it could be found to be G protein coupled receptor kinase (G PR). The phosphorylation of otein coupled receptors kinases, GRKs) is one of the important mechanisms of its receptor phosphorylation. Previous studies have found that in many GRK subtypes, the increase of GRK4 activity is earlier than the occurrence of hypertension, and its role in hypertension is concerned with the location of the.GRK4 gene located in the human chromosome 4q16.3. The incidence of primary hypertension related.GRK4 variants was significantly associated with increased blood pressure. The predictive accuracy of GRK4 variants for essential hypertension in African Garner people was more than 70%, while the accuracy of the Japanese primary hypertension was up to 94%. and the Meta analysis of the results of multiple research centers was also shown by 94%.. The relationship of GRK4 variant with hypertension is closely related to the occurrence of hypertension in three transgenic mice with.GRK4 variant. GRK4? 142V mice are dominant hypertension, and A486 and R65L mice are salt sensitive hypertension under high salt load conditions. We speculate that GRK4 may be involved in the phosphorylation of renal adiponectin receptors. It is associated with the progress of hypertension. Therefore, the targeted inhibition of the expression of GRK4 in the kidney can be used as an effective attempt to enhance urinary sodium excretion and improve hypertension. In summary, we speculate that adiponectin has diuretic excretion of sodium, which is lost in high blood pressure, and its loss of origin is attributed to the phosphorylation of adiponectin receptors to receptors. Inactivation is attributed to the increase of GRK4 expression and activity in the kidney, and the inhibition of renal GRK4 has the effect of lowering blood pressure. The purpose of this study is to investigate the effect of adiponectin on renal diuretic sodium and its role in the development of hypertension. Whether GRK4 participates in the regulation of the phosphorylation of adiponectin receptors and regulates blood pressure, and the ultrasound microbubble target is silent on the kidney GRK4 Influence of blood pressure and urine sodium in SHR rats. Research contents and methods 1. the role of adiponectin in renal urinary sodium excretion and its mechanism 1.1 use immunoblotting and immunofluorescence staining to observe the expression of 2 kinds of adiponectin receptor Adipo R1 and Adipo R2 in normal blood pressure rats (Wista Kyoto rat, WKY) and RPT cells. 1.2 WKY rats were used to infuse different concentrations of adiponectin through single kidney to observe the urine flow, urine sodium excretion rate, and blood pressure changes to determine whether adiponectin has diuretic effect. At the same time, the method of high temperature inactivation of adiponectin protein activity was used to remove the osmotic urination effect and the sensitization of.1.3 on the WKY RPT cells. The effect of Na+-K+-ATP enzyme activity detection method was used to observe the effect of adiponectin on the activity of Na+-K+-ATP enzyme after different concentration and time..1.4 experiment first verified the interference efficiency of small interfering RNA (Si RNA) of Adipo R1 and Adipo R2, and then transfected Adipo R1 and Adipo into the cells, and observed the adiponectin receptor when the adiponectin receptor was inhibited. The role of Na+-K+-ATP enzyme to identify the receptor mediates the action of adiponectin.1.5 using Na+-K+-ATP enzyme activity determination technique, adding phosphorylated adenylate activated protein kinase (Adenosine 5 '-monophosphate-activated protein kinase, AMPK) inhibitor Compound C, one oxygenated nitrogenase (Endothelial nitric) inhibition NG-nitro-l-arginine methyl ester (L-NAME) preliminarily screened the signal pathway of adiponectin inhibiting the activity of Na+-K+-ATP enzyme. Then, the phosphorylation of phosphorylated AMPK and its downstream signal molecule e NOS after the action of adiponectin, the expression of.2. adiponectin receptor down regulation and GRK4 regulation of the adiponectin receptor after the action of adiponectin were further observed. Phosphorylation was damaged by adiponectin induced urinary sodium excretion. 2.1 the same concentration gradient of adiponectin was used in the same WKY rats. Single kidney perfusion was performed on SHR. The urine flow, urinary sodium excretion rate and the change of mean arterial pressure were observed in.2.2 of spontaneously hypertensive rats (Spontaneous hypertensive rat, SHR), and the adiponectin to Na+-K+-ATP was observed. The effect of enzyme.2.3 was observed by real-time quantitative PCR, compared with WKY RPT cells and the expression of adiponectin two receptor genes on SHR RPT cells, and the immunoblotting method was used to detect the protein expression of adiponectin receptor,.2.4 single kidney perfusion adiponectin receptor agonist, that is, the effect of Adipo Ron on SHR urine sodium excretion. One step is to identify the impaired adiponectin diuretic effect from the changes in the adiponectin receptor.2.5. According to the experimental results, the overexpressed plasmids of the two adiponectin receptors were designed, the plasmids were extracted and transfected on the SHR RPT cells. The effect of adiponectin on the Na+-K+-ATP enzyme was observed and the effect of adiponectin on the Na+-K+-ATP enzyme was observed by the method of immunoprecipitation. On the RPT cells of SHR, the phosphorylation of adiponectin receptor,.2.7 adiponectin receptor agonist Adipo Ron perfusion GRK4? 142V transgenic mice (compared with GRK4? WT mice, the GRK4 activity of this mouse is enhanced). After disturbing the expression of GRK4, the phosphorylation of adiponectin receptor.3.GRK4 ultrasound microbubble targeting therapy improves the phosphorylation of SHR adiponectin receptors and regulates the urinary sodium excretion and blood pressure 3.1 using 5- carboxyl fluorescein (5-Carboxyfluorescein, FAM) labeled ultrasound microbubbles for GRK4si RNA to be encapsulated.3.2 into SHR and ultrasound microbubbles in the kidney Ultrasound-targeted microbubble destruction (UTMD) released GRK4 Si RNA.20 after GRK4 Si RNA.20 days, and observed the blood pressure of SHR and the urine volume of 24 hours (hour, H), urine sodium excretion rate, renal function index and renal fibrosis index.3.3 UTMD transfer to kidney, using immunoprecipitation method to observe the adiponectin receptor phosphorus. Results 1. the expression of adiponectin receptor Adipo R1 and Adipo R2 was expressed in the renal proximal tubule, especially in the renal cortex. The protein strip of.Adipo R1 expressed in RPT cells was 43Kd, and the protein strip of Adipo R2 was the effect of 44Kd.2. adiponectin on urinary excretion and urine volume of WKY rats. This effect showed concentration dependence. Sex 3. adiponectin inhibits the activity of Na+-K+-ATP enzyme on WKY RPT cells, which presents a concentration and dependent.4. as a specificity of the study of the action of adiponectin. Si RNA knocks low Adipo R1 and Adipo R2 in WKY RPT cells. The inhibitory effect of adiponectin on the activity of the enzyme is found to be disappearing. After adding AMPK blockers and E NOS blockers, the inhibitory effect of adiponectin on the activity of Na+-K+-ATP enzyme disappeared. At the same time, adiponectin increased the expression of phosphorylated AMPK and E NOS, and the effect of adiponectin on the enhancement of E NOS phosphorylation was disappearing after the use of AMPK blockers. The diuretic effect of the inhibitory effect of a+-K+-ATP enzyme activity on.6. adiponectin is damaged in SHR. The inhibition of adiponectin to Na+-K+-ATP enzyme is also damaged in SHR RPT cells. The role of adiponectin is supported by the adiponectin receptor agonist Adipo Ron: Adipo Ron has diuretic use, and this effect is in SHR.7.SHR kidney adiponectin. The receptor expression was lower than that of the control WKY rats, but the change in the receptor expression was difficult to explain the loss of function, because the diuretic effect of Adipo R1 and Adipo R2 was still not restored, indicating that there were other factors involved. Our study found that SHR renal adiponectin receptor phosphoric acid phosphate receptor phosphorylation was affected by the phosphorylation of adiponectin receptors. The high phosphorylation level suggests that the level of high phosphorylation may be the cause of the decline in the function of adiponectin receptor.8. to verify whether GRK4 participates in the process of regulating the phosphorylation of adiponectin receptors. We use GRK4? 142V transgenic mice to perfusion the adiponectin receptor agonists and find that the adiponectin receptor participates in the diuretic action of the joint, and is used in the RPT cells of SHR. After GRK4 Si RNA interference, the phosphorylation of adiponectin receptor Adipo R1 and Adipo R2 weakens. This indicates that GRK4 participates in the regulation of phosphorylation of adiponectin receptors, impaired adiponectin excretion and phosphorylation associated with adiponectin receptors in SHR,.9. using microbubbles encapsulated GRK4 Si, and targeted targeting ultrasound in the kidneys. The GRK4 Si RNA released by the broken microbubbles inhibited GRK4, the phosphorylation level of the renal adiponectin receptor weakened, the urine sodium excretion and blood pressure decreased in SHR rats. Meanwhile, the renal function of SHR was not affected, and the renal fibrosis was improved. Conclusion adiponectin has the effect of promoting renal sodium excretion, and the function is impaired in hypertension. It was found that the expression of adiponectin receptor was downregulated, but the receptor did not fully recover the function of adiponectin after the over expression receptor. The phosphorylation of the receptor may play an important role. The increase of GRK4 activity is the cause of the high phosphorylation of adiponectin receptor; UTMD transmission GRK4 Si RNA effectively inhibits the expression of GRK4 in the kidney, and reduces the blood pressure and diuretic sodium in SHR in SHR. Effect.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R544.1

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