本文選題：心腎綜合征 + DOCA-Salt小鼠　； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：一、研究背景：心腎綜合征(Cardiorenal Syndrome, CRS)是同時(shí)累及心臟和腎臟的一大組疾病的統(tǒng)稱,包括心臟(或腎臟)的急性或慢性功能不全導(dǎo)致腎臟(或心臟)的急性或慢性功能不全,或者共同因素同時(shí)導(dǎo)致心臟和腎臟的結(jié)構(gòu)和功能損害。心功能和腎功能之間有著緊密聯(lián)系,心腎同時(shí)受累,大大增加了疾病的診治難度和死亡率1,2。根據(jù)2010年ADKI (Acute Dialysis Quality Initiative)共識(shí),心腎綜合征共分為5型3。CRS的病理生理機(jī)制尚未完全闡明,各型CRS在病生理機(jī)制上存在共同特征,可分為血流動(dòng)力學(xué)機(jī)制與非血流動(dòng)力學(xué)機(jī)制4。其中血流動(dòng)力學(xué)機(jī)制的核心是水鈉潴留和容量負(fù)荷增加,而非血流動(dòng)力學(xué)機(jī)制包括腎素-血管緊張素-醛固酮系統(tǒng)(Renin-Angiotensin-Aldosterone System, RAAS)和交感神經(jīng)系統(tǒng)激活、氧化應(yīng)激、炎癥反應(yīng)等。目前CRS動(dòng)物模型多采用心腎手術(shù)構(gòu)建、以急性變化為主,沒有很好的模擬病理生理機(jī)制的慢性CRS動(dòng)物模型,限制了對(duì)其病生理機(jī)制的探究。高醛固酮高鹽飲食(DOCA-Salt)小鼠模型模擬了鹽敏感高血壓的病生理狀態(tài),前期我們觀察到腎臟結(jié)構(gòu)和功能的損害。本研究試圖探索該模型是否同時(shí)存在心臟損害,從而為探究CRS機(jī)制提供平臺(tái)。腺苷1型受體(Adenosine A1 Receptor, A1AR)通過調(diào)節(jié)腎臟管球反饋、促進(jìn)心臟釋放心房利鈉肽(Atrial Natriuretic Peptide, ANP)調(diào)控水鹽負(fù)荷；在非血流動(dòng)力學(xué)機(jī)制方面,A1AR對(duì)RAAS起到直接抑制作用、對(duì)炎癥和凋亡起到調(diào)節(jié)作用。但目前沒有研究系統(tǒng)地探索在DOCA-Salt小鼠模型上A1AR對(duì)于心腎損害的意義。前期我們觀察到A1AR主要通過血流動(dòng)力學(xué)因素保護(hù)DOCA-Salt小鼠腎臟結(jié)構(gòu)和功能,本研究試圖通過血流動(dòng)力學(xué)機(jī)制和非血流動(dòng)力學(xué)機(jī)制兩方面探索A1AR對(duì)DOCA-Salt小鼠心臟損害是否同樣具有保護(hù)作用。二、研究目的：1.觀察DOCA-Salt鼠心臟損害,探索DOCA-Salt模型作為慢性CRS模型的可能性；2.探索A1AR在DOCA-Salt小鼠水鈉潴留中的作用；3.探索A1AR在DOCA-Salt小鼠心肌肥大、心肌組織微血管密度、炎癥和纖維化中的作用。三、研究方法及結(jié)果：1. DOCA-Salt小鼠心臟損害在C57BL/6J野生型小鼠上,行左腎切除術(shù),術(shù)中于左腎窩埋置醋酸脫氧皮質(zhì)酮(Deoxycorticosterone Acetate, DOCA)緩釋藥片,術(shù)后予高鹽飲食,建立DOCA-Salt小鼠模型。采用尾套法測(cè)量血壓、心率,觀察到DOCA-Salt小鼠心率隨實(shí)驗(yàn)周數(shù)顯著下降(0周685.8±±25.4 vs.8周526.6±103.9次/分,P0.01),平均動(dòng)脈壓顯著上升(0周74.6±4.4 vs.8周88.5±6.5 mmHg, P0.01)。分別于第4周和第8周處死小鼠,并留取心臟標(biāo)本,稱量全心重量,觀察到第8周時(shí)心臟重量指數(shù)顯著增加(7.05±0.83 vs.5.16±0.20mg/g, P=0.007)。常規(guī)HE染色觀察心肌組織鏡下改變并測(cè)量左心室厚度無顯著變化。Van Gieson膠原染色觀察心肌組織纖維化程度,發(fā)現(xiàn)從第4周起出現(xiàn)局灶性間質(zhì)纖維化；運(yùn)用Realtime PCR方法分析轉(zhuǎn)化生長(zhǎng)因子-β1 (Transforming Growth Factor-β1, TGF-β1)、膠原纖維1 (Collagenl)在實(shí)驗(yàn)組和對(duì)照組間無差異。CD34免疫組化染色觀察并分析心肌微血管密度,發(fā)現(xiàn)第4周時(shí)微血管密度顯著增加([1.01±0.003]%vs.[0.59±0.001]%,P=0.030),第8周時(shí)微血管密度較對(duì)照組無顯著變化。CD68免疫組化染色觀察并分析巨噬細(xì)胞浸潤(rùn)程度,發(fā)現(xiàn)DOCA-Salt小鼠巨噬細(xì)胞浸潤(rùn)程度與對(duì)照組無差異；運(yùn)用Realtime PCR方法分析趨化因子2(Chemokine C-C motif ligand 2, CCL2) mRNA表達(dá)水平也無差異。2. DOCA-Salt小鼠水鹽負(fù)荷在第0、4、8周用代謝籠留24h尿,觀察到DOCA-Salt小鼠24h尿量(0周1173±464 vs.4周3606±2359 vs.8周7635±3017μl,兩兩比較P0.05)及24h尿鈉排出量(0周133.3±39.5 vs.4周798.4±719.4 vs.8周1788±854.0μmol/d,兩兩比較P0.05)隨實(shí)驗(yàn)周數(shù)顯著增加,尿滲透壓(0周1801±642 vs.4周910±213mOsm/kg H2O,P0.05;0周1801±642 vs.8周860±233mOsm/kg H2O, P0.05)顯著下降。Realtime PCR方法觀察到心肌組織Corin和ANP mRNA表達(dá)在第4周顯著增加,分別為31.9倍(P=0.005)和180.7倍(P=0.030),第8周時(shí)與對(duì)照組無差異。Realtime PCR方法觀察到DOCA-Salt小鼠心肌組織腺苷合成酶CD73 mRNA表達(dá)從第4周起顯著上調(diào)(4周18.79倍,P=0.001；8周8.28倍,P=0.036), A1AR mRNA第8周開始出現(xiàn)顯著上調(diào)(12.12倍,P=0.013),但Western Blot方法沒有觀察到A1AR蛋白表達(dá)水平的顯著變化。3.A1AR在DOCA-Salt小鼠心臟損害中的作用建立A1AR-/- DOCA-Salt小鼠模型,觀察到A1AR-/- DOCA-Salt小鼠心率隨實(shí)驗(yàn)周數(shù)下降(4周636±64.2 vs.0周696.7±35.2,P0.05),平均動(dòng)脈壓無顯著變化。24h尿量相對(duì)于野生型實(shí)驗(yàn)組顯著增加(7003±3742 vs.3606±2359μl, P0.05),24h尿鈉排出量較對(duì)照增加(:1246.5±859.8 vs.146.6±71.2μmol/d, P0.05),尿滲透壓較對(duì)照下降(702±165 vs.1394±397 mOsm/kg, P0.05)。心肌組織ANPmRNA表達(dá)顯著上調(diào)(12.65倍,P=0.026),Corin mRNA表達(dá)在實(shí)驗(yàn)組與對(duì)照組間無差異。A1AR-/- DOCA-Salt小鼠第4周心臟重量指數(shù)及左心室厚度無顯著變化；心肌組織微血管密度無顯著升高；心肌巨噬細(xì)胞浸潤(rùn)程度、CCL2 mRNA表達(dá)水平在實(shí)驗(yàn)組與對(duì)照組間無差異；僅1只(共6只)小鼠心臟出現(xiàn)局灶性間質(zhì)纖維化,TGF-β1和Collagenl mRNA表達(dá)水平在實(shí)驗(yàn)組與對(duì)照組間無差異。四、研究結(jié)論：本實(shí)驗(yàn)條件下1. DOCA-Salt小鼠血壓升高,心率下降,早期(4周)心臟微血管密度顯著增加、出現(xiàn)局灶性間質(zhì)纖維化,晚期(8周)心臟重量指數(shù)顯著增加。結(jié)合前期研究觀察到腎臟損害,DOCA-Salt小鼠存在心腎共同損害,可作為慢性CRS模型。2. DOCA-Salt小鼠血壓升高,提示存在水鈉潴留,其水鹽負(fù)荷受到相關(guān)因素調(diào)節(jié)：心臟CD73-A1AR和Corin-ANP通路激活,ANP限制水和鈉的重吸收；24h尿量和24h尿鈉排出量顯著增加；前期研究觀察到腎臟CD73-A1AR通路激活,提示管球反饋持續(xù)激活。A1AR-/- DOCA-Salt小鼠管球反饋喪失,24h尿量顯著增加,血壓較基線和對(duì)照組均無升高,提示A1AR加重水鈉潴留。3.A1AR-/- DOCA-Salt小鼠早期(4周)出現(xiàn)局灶性間質(zhì)纖維化,心臟重量指數(shù)和微血管密度無顯著變化。未觀察到A1AR在DOCA-Salt小鼠模型中對(duì)心肌肥大、炎癥和纖維化的明確保護(hù)作用。
[Abstract]:One, study background: Cardiorenal Syndrome (CRS) is a general name for a large group of diseases involving the heart and kidney, including acute or chronic dysfunction of the heart (or kidney), causing acute or chronic dysfunction of the kidney (or heart), or co factors that cause structural and functional impairment of the heart and kidney simultaneously. There is a close relationship between function and renal function, and the heart and kidney are simultaneously involved, which greatly increases the difficulty and mortality of the diagnosis and treatment of the disease 1,2. according to the consensus of the 2010 ADKI (Acute Dialysis Quality Initiative). The pathophysiological mechanism of the heart and kidney syndrome divided into 5 types of 3.CRS has not been fully explained, and the various types of CRS have common characteristics in the physiological mechanism of the disease, The hemodynamic mechanism and non hemodynamic mechanism 4., the core of the hemodynamic mechanism is sodium retention and increased volume load, while non hemodynamic mechanisms include the renin angiotensin aldosterone system (Renin-Angiotensin-Aldosterone System, RAAS) and sympathetic nervous system activation, oxidative stress, and inflammatory response At present, most of the CRS animal models are constructed by cardiac and renal surgery. The chronic CRS animal model with acute changes and no good simulation of pathophysiology has limited the physiological mechanism of its disease. The model of high aldosterone high salt diet (DOCA-Salt) mice model simulated the physiological state of salt sensitive hypertension, and we observed the kidney in the early stage. Damage of structure and function. This study attempts to explore whether the model has cardiac damage at the same time, and thus provides a platform for exploring the CRS mechanism. Adenosine A1 Receptor (A1AR) regulates the cardiac release of natriuretic peptide (Atrial Natriuretic Peptide, ANP) by regulating the feedback of the renal tubular bulb (A1AR) to regulate the water and salt load in the heart, and in non blood flow. In the dynamic mechanism, A1AR plays a direct inhibitory effect on RAAS and regulates inflammation and apoptosis. However, there is no systematic exploration of the significance of A1AR on the damage to the heart and kidney in the DOCA-Salt mouse model. We observed that A1AR mainly protects the structure and function of the kidney of DOCA-Salt mice by hemodynamic factors. The study tries to explore whether A1AR has the same protective effect on the heart damage of DOCA-Salt mice through two aspects of hemodynamic mechanism and non hemodynamic mechanism. Two, the purpose of this study is: 1. to observe the heart damage of DOCA-Salt rats and to explore the possibility of DOCA-Salt model as a chronic CRS model; 2. explore A1AR in the retention of water and sodium retention in DOCA-Salt mice. The role of A1AR to explore the role of A1AR in myocardial hypertrophy, microvascular density, inflammation and fibrosis in DOCA-Salt mice. Three, research methods and results: 1. DOCA-Salt mouse heart damage in C57BL/6J wild type mice, left nephrectomy, implantation of Deoxycorticosterone Acetate, DOC in the left renal fossa (DOC). A) sustained release tablets were given high salt diet and established DOCA-Salt mice model. The blood pressure and heart rate were measured by tail sleeve method. The heart rate of DOCA-Salt mice decreased significantly with the number of weeks (0 weeks 685.8 + + 25.4 vs.8 weeks, 526.6 + 103.9 / min, P0.01), and the average arterial pressure increased significantly (0 weeks 74.6 + 4.4 vs.8 weeks, 88.5 + 6.5 mmHg, P0.01), respectively. Respectively, at week fourth weeks, respectively. Eighth weeks and eighth weeks, the mice were killed and heart specimens were left. The cardiac weight was weighed and the heart weight index increased significantly at eighth weeks (7.05 + 0.83 vs.5.16 + 0.20mg/g, P=0.007). Routine HE staining was used to observe the changes of the myocardial tissue under the microscope and the left ventricular thickness was not significantly changed by.Van Gieson collagen staining to observe the degree of fibrosis of the myocardium. The focal interstitial fibrosis was observed from fourth weeks, and the Realtime PCR method was used to analyze the TGF - beta 1 (Transforming Growth Factor- beta 1, TGF- beta 1), and the collagen fiber 1 (Collagenl) was observed and analyzed between the experimental group and the control group. The microvascular density of the cardiac muscle was observed and the microvascular density was significant at fourth weeks. Increased ([1.01 + 0.003]%vs.[0.59 + 0.001]%, P=0.030), microvascular density in eighth weeks had no significant changes compared with the control group..CD68 immunohistochemical staining was observed and the infiltration degree of macrophages was analyzed. The infiltration degree of macrophage in DOCA-Salt mice was not different from that of the control group; the Realtime PCR method was used to analyze chemokine factor 2 (Chemokine C-C motif lig). The expression level of and 2, CCL2) was also no difference in the water and salt load of.2. DOCA-Salt mice. 24h urine was kept in the metabolic cage at week 0,4,8. The 24h urine volume was observed in DOCA-Salt mice (1173 + 464 vs.4 weeks, 1173 + 464 vs.4 weeks, 3606 + 2359 vs.8, 7635 + 3017 Mu L, 22 comparison) and sodium excretion (0 weeks 133.3 + 39.5). 22 compared with P0.05), the urine osmotic pressure (0 weeks, 1801 + 642 vs.4 weeks 910 + 213mOsm/kg H2O, P0.05, 0 weeks 1801 + 642 vs.8 weeks, 860 + 233mOsm/kg H2O, P0.05) decreased significantly by.Realtime PCR method and observed that the myocardial tissue Corin and expressions were increased in fourth weeks, respectively, and 31.9 times and 180.7 times respectively, At eighth weeks, there was no difference between the control group and the control group.Realtime PCR method to observe the expression of adenosine synthetase CD73 mRNA in DOCA-Salt mice from fourth weeks (4 weeks, 18.79 times, P=0.001, 8 weeks 8.28 times, P=0.036), and A1AR mRNA eighth weeks began to rise significantly (12.12 times, P= 0.013), but Western Blot method did not observe the expression of protein The effect of.3.A1AR on the heart damage of DOCA-Salt mice was established by.3.A1AR. The heart rate of A1AR-/- DOCA-Salt mice decreased with the number of weeks (4 weeks 636 + 64.2 vs.0 weeks 696.7 + 35.2, P0.05), and the average arterial pressure had no significant change in the mean arterial pressure, which was significantly increased (7003 + 3742 VS) compared with the wild type experimental group. .3606 + 2359 L, P0.05), 24h urine sodium excretion increased (1246.5 + 859.8 vs.146.6 + 71.2 u mol/d, P0.05), and urinary osmotic pressure decreased (702 + 165 vs.1394 + 397 mOsm/kg, P0.05). There was no significant change in cardiac weight index and left ventricular thickness in fourth weeks, no significant increase in myocardial microvascular density, and no difference in the level of macrophage infiltration and CCL2 mRNA expression between the experimental group and the control group; only 1 (a total of 6) mice had focal interstitial fibrosis, and the expression level of TGF- beta 1 and Collagenl mRNA was in the heart. There was no difference between the experimental group and the control group. Four, the study concluded that the blood pressure of 1. DOCA-Salt mice increased, the heart rate decreased, the cardiac microvascular density increased significantly at the early (4 weeks), the focal interstitial fibrosis appeared, and the heart weight index of the late (8 weeks) increased significantly. The kidney damage was observed in the early stage of the study, and the heart of the DOCA-Salt mice existed. Common renal damage can be used as a chronic CRS model in.2. DOCA-Salt mice, suggesting the presence of sodium retention. The water and salt load is regulated by related factors: the activation of the heart CD73-A1AR and Corin-ANP pathway, the restriction of the reabsorption of water and sodium by ANP; the increase of 24h urine volume and the excretion of 24h urine sodium; the early study observed the CD73-A1AR pathway in the kidney. Activation, suggesting that tube feedback continued to activate.A1AR-/- DOCA-Salt mice feedback loss, 24h urine volume increased significantly, blood pressure was not higher than the baseline and control group, suggesting that A1AR increased sodium retention of.3.A1AR-/- DOCA-Salt mice early (4 weeks) focal interstitial fibrosis, cardiac weight index and microvascular density did not change significantly. The protective effect of A1AR on myocardial hypertrophy, inflammation and fibrosis in DOCA-Salt mice was observed.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R541.6;R692

【共引文獻(xiàn)】

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