本文選題：孕期炎癥　切入點：心室重構(gòu)　出處：《第三軍醫(yī)大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：近年來，炎癥在心血管疾病發(fā)病中重要作用的認(rèn)識是對高血壓的病因?qū)ふ抑凶钪匾倪M(jìn)展之一。本研究室將高血壓的病因追溯至母體孕期，選取脂多糖(Lipopolysaccharide, LPS)作為炎癥刺激劑，建立了孕期炎癥刺激導(dǎo)致子代大鼠血壓升高的動物模型；在此模型的基礎(chǔ)上，選取了炎癥通路上的關(guān)鍵靶點核轉(zhuǎn)錄因子kappa-B(Nuclear factor kappa-B, NF-κB)在母體孕期進(jìn)行干預(yù)，可以有效阻止子代出生后高血壓的發(fā)生，但關(guān)于其機(jī)制尚待深入闡明。心室重構(gòu)是高血壓、心力衰竭和心肌病等許多心血管疾病發(fā)生發(fā)展的共同病理生理過程，從病理生理學(xué)角度來說，一方面是心肌細(xì)胞發(fā)生代償性肥厚、細(xì)胞凋亡、壞死和增生；另一方面是心肌細(xì)胞外基質(zhì)膠原的沉積，膠原網(wǎng)的組成和含量的改變；最終引起心肌實質(zhì)和間質(zhì)比例的失衡，使心臟的結(jié)構(gòu)改變和功能失償。近年來研究認(rèn)為，神經(jīng)內(nèi)分泌因子的過度激活在心室重構(gòu)病程的發(fā)生、發(fā)展中極為重要，其中腎素-血管緊張素系統(tǒng)(renin angiotensin system, RAS)則發(fā)揮著尤為重要的作用。研究發(fā)現(xiàn)，RAS系統(tǒng)中除了ACE-AngⅡ-AT1R通路以外，還存在著與其作用相拮抗的另外一條通路即血管緊張素轉(zhuǎn)化酶-血管緊張素(1-7)-Mas受體軸(Angiotensin converting enzyme2-Angiotensin(1-7)-Mas axis)，ACE2-Ang(1-7)-MasR軸，此條通路通過ACE2降解AngⅡ，其在高血壓、心力衰竭的防治及改善心肌纖維化等方面均得到了證實。我室前期研究發(fā)現(xiàn)，與循環(huán)RAS系統(tǒng)變化不明顯相比，腎小球數(shù)量減少和腎功能的下降的同時存在腎臟局部RAS系統(tǒng)的明顯激活。在脂肪RAS系統(tǒng)中也得到了相同的結(jié)論，提示局部組織RAS系統(tǒng)的變化可能是高血壓發(fā)病的另一重要原因之一。在我室研究中還發(fā)現(xiàn)孕期炎癥刺激會引起子代大鼠心室肌發(fā)生凋亡和重構(gòu)，且這種重構(gòu)的發(fā)生早于高血壓的發(fā)生，提示子代的心室重構(gòu)是引起高血壓發(fā)生的機(jī)制之一。但子代大鼠哪些因子的變化引起了心室重構(gòu)的發(fā)生？以及孕期炎癥刺激對子代大鼠心臟局部RAS系統(tǒng)是否產(chǎn)生影響？其規(guī)律和特征是什么等尚待進(jìn)一步研究。 本課題擬通過觀察心肌組織膠原的變化，膠原代謝酶MMP-2、TIMP-2及促纖維化因子TGFβ-1、TGFβ-2的變化來研究心肌發(fā)生纖維化及心肌重構(gòu)。并通過觀察子代大鼠心臟局部RAS系統(tǒng)上兩條代謝通路即ACE-AngⅡ-AT1R通路和ACE2-Ang(1-7)-MasR通路的變化，進(jìn)一步了解孕期炎癥刺激后子代大鼠發(fā)生心肌纖維化及心肌間質(zhì)重構(gòu)的機(jī)制進(jìn)行研究。 方法： 1.孕鼠分組及給藥：將SD孕鼠采用完全隨機(jī)法分為3組。正常對照組：在孕第8~14天每天腹腔注射無菌生理鹽水0.5ml；LPS模型組：在孕第8、10、12天腹腔注射LPS0.79mg/kg，孕第9、11、13、14天給予生理鹽水；LPS+PDTC組：在孕第8、10、12天腹腔注射LPS0.79mg/kg，孕第8~14天每天腹腔注射PDTC100mg/kg。 2.稱重指標(biāo)的測定：子大鼠每周進(jìn)行體重(body weight,BW)的測定，三組實驗動物在6周齡和16周齡時麻醉，打開腹腔，快速取出心臟，置于冰凍無菌生理鹽水中洗凈后，濾紙吸干水分稱重，天平稱量心臟重量(heart weight,HW)。 3.用天狼星紅及Masson膠原染色觀察心肌膠原形態(tài)學(xué)及心肌膠原容積分?jǐn)?shù)的變化。 4.免疫組織熒光的方法檢測6周齡和16周齡子代大鼠心肌組織AngⅡ及Mas受體蛋白水平； 5.免疫組織化學(xué)方法檢測6周齡和16周齡心肌組織中AT1受體的蛋白表達(dá)。 6.采用Real-time PCR測定6周齡和16周齡子大鼠心臟組織中ACE、ACE2、AT1R、MasR、MMP-2、TIMP-2、TGFβ-1及TGFβ-2mRNA基因表達(dá)水平。 7.采用Western blotting方法測定6周齡和16周齡子代大鼠心肌組織ACE、ACE2、MMP-2、TIMP-2、TGFβ-1及TGFβ-2的蛋白含量； 結(jié)果 1.孕期炎癥刺激對子代大鼠發(fā)生心室重構(gòu)的影響 (1)孕期炎癥刺激對子代大鼠心臟指數(shù)的影響 與對照組相比，子代大鼠6周齡時，LPS組心臟重量、心臟質(zhì)量分?jǐn)?shù)明顯升高(P0.05)，PDTC干預(yù)后明顯下降(P0.05)；子代大鼠16周齡時，LPS組較對照組心臟重量、心臟質(zhì)量分?jǐn)?shù)明顯升高(P0.05)，PDTC干預(yù)后顯著性降低(P0.01)，LPS組體重明顯升高(P0.05)；差異有統(tǒng)計學(xué)意義。 (2)孕期炎癥刺激對子代大鼠膠原蛋白的影響 天狼星紅及Masson染色結(jié)果顯示，與正常對照組相比，6w、16w，LPS組沉積在心肌間質(zhì)的膠原表達(dá)明顯增多；L+P組，紅色膠原表達(dá)較LPS組減少。 (3)孕期炎癥刺激對子代大鼠心肌組織MMP-2、TIMP-2、TGFβ-1及TGFβ-2mRNA水平的影響 子代大鼠6周齡時，與正常對照組相比，LPS組TGFβ-1mRNA表達(dá)顯著性升高(P0.01)；TGFβ-2mRNA表達(dá)水平明顯升高(P0.05)，PDTC干預(yù)后顯著性降低(P0.01)；LPS組MMP-2mRNA表達(dá)明顯降低(P0.05)；TIMP-2mRNA表達(dá)顯著性升高(P0.01)。子代大鼠16周齡時，LPS組較正常對照組TGFβ-1mRNA表達(dá)明顯升高(P0.05)，PDTC干預(yù)后明顯降低(P0.05)；TGFβ-2mRNA表達(dá)明顯升高(P0.05)；TIMP-2mRNA表達(dá)明顯升高(P0.05)PDTC干預(yù)后顯著性降低(P0.01)；MMP-2mRNA表達(dá)明顯降低(P0.01)。 (4)孕期炎癥刺激對子代大鼠心肌組織MMP-2和TIMP-2蛋白水平的影響 子代大鼠6w、16w，LPS模型組較正常對照組MMP-2蛋白水平顯著性降低(P0.05)；TIMP-2蛋白水平顯著性升高(P0.05)；LPS組TIMP2-/MMP-2的蛋白水平比值顯著升高(P0.05)，PDTC干預(yù)后蛋白水平明顯降低(P0.05)。 (5)孕期炎癥刺激對子代大鼠心肌組織TGFβ-1和TGFβ-2蛋白水平的影響 子代大鼠6w、16w，LPS模型組較正常對照組TGFβ-1的蛋白水平明顯升高(P0.05)，PDTC干預(yù)后明顯降低(P0.05)；TGFβ-2的蛋白水平明顯升高(P0.05)。 2.孕期炎癥刺激對子代大鼠心臟局部RAS系統(tǒng)的影響 (1)免疫組織熒光測定子代大鼠心肌組織中AngⅡ：LPS組較對照組熒光強(qiáng)度及熒光點明顯增強(qiáng)，L+P組較LPS組減少；MasR蛋白表達(dá)水平LPS組較對照組熒光強(qiáng)度明顯減弱，熒光點明顯減少，L+P組紅色熒光區(qū)較LPS組增多。 (2)免疫組織化學(xué)測定子代大鼠心肌組織中AT1R蛋白水平：對照組，陽性細(xì)胞數(shù)(細(xì)胞核著色)少，LPS組，陽性細(xì)胞數(shù)(細(xì)胞核著色)明顯增多，L+P組，，陽性細(xì)胞(細(xì)胞核著色)較LPS組減少。 (3)Real time PCR檢測子代大鼠心肌組織ACE、ACE2、MasR及AT1R mRNA水平 子代大鼠6周齡時，與正常對照組比，LPS組ACE mRNA表達(dá)有升高趨勢，PDTC干預(yù)后明顯降低(P0.05)；AT1R mRNA表達(dá)明顯升高(P0.05)； MasR mRNA表達(dá)顯著性降低(P0.01)，PDTC干預(yù)后顯著性升高(P0.01)。子代大鼠16周齡時，與正常對照組比，LPS組較ACE mRNA表達(dá)明顯升高(P0.05)，PDTC干預(yù)后顯著降低(P0.01)AT1R mRNA表達(dá)顯著性升高(P0.01)，PDTC干預(yù)后明顯降低(P0.05)；MasR、ACE2mRNA表達(dá)明顯降低(P0.05)。 (4)Western blotting檢測子代大鼠心肌組織ACE和ACE2蛋白水平 子代大鼠6w、16w，LPS模型組較正常對照組ACE蛋白水平顯著性升高(P0.05)，PDTC干預(yù)后明顯降低(P0.05)；LPS組ACE2蛋白水平顯著性降低(P0.05)；LPS組ACE/ACE2的蛋白水平比值顯著升高(P0.05)，PDTC干預(yù)后明顯降低(P0.05)。 結(jié)論 1.孕期炎癥刺激可導(dǎo)致子代大鼠發(fā)生心室重構(gòu)，NF-κB的抑制劑PDTC能有效逆轉(zhuǎn)心室重構(gòu)。 2.孕期炎癥刺激可引起子代大鼠發(fā)生基質(zhì)金屬蛋白酶及基質(zhì)金屬蛋白酶抑制酶表達(dá)失衡，轉(zhuǎn)化生長因子β1、轉(zhuǎn)化生長因子β2表達(dá)增加，膠原代謝異常，從而引起心肌纖維化，促進(jìn)了心室重構(gòu)的發(fā)生。 3.孕期炎癥刺激一方面可導(dǎo)致子代大鼠心肌組織ACE表達(dá)增加，AngⅡ生成增多，激活A(yù)CE-AngⅡ-AT1R通路；另一方面使ACE2表達(dá)降低減少了AngⅡ的降解和Ang(1-7)的生成，降低了ACE2-Ang(1-7)-MasR通路的活性，造成心臟局部ACE-AngⅡ-AT1R和ACE2-Ang(1-7)-MasR兩條通路失衡，引起心臟局部RAS系統(tǒng)的過度激活，而這可能是孕期炎癥刺激致子代大鼠發(fā)生心室重構(gòu)的重要分子基礎(chǔ)。
[Abstract]:In recent years, understanding of the important role of inflammation in the pathogenesis of cardiovascular diseases is one of the most important progress in the search for the causes of hypertension. This study room will be traced to maternal hypertension, lipopolysaccharide (Lipopolysaccharide, LPS) were selected as inflammatory stimuli, established the animal model of blood pressure during pregnancy leads to inflammation of rat offspring increased; on the basis of this model, the key target inflammatory pathways on nuclear transcription factor kappa-B (Nuclear factor kappa-B, NF- K B) to intervene in the mother during pregnancy, can effectively prevent the occurrence of hypertension sub generation after birth, but the mechanism needs to be further elucidated. Ventricular remodeling is a common hypertension. The pathophysiology of heart failure and cardiomyopathy of many cardiovascular diseases, from the perspective of pathophysiology, one is myocardial cells have compensatory hypertrophy, apoptosis Death, necrosis and hyperplasia; on the other hand is the deposition of myocardial extracellular matrix collagen, composition and content of collagen network changes; causing imbalance myocardial stroma ratio, the structural change and functional cardiac decompensation. Recent studies suggest that, the excessive activation of neuroendocrine factors in ventricular remodeling course happen, is very important in the development, of which the renin-angiotensin system (renin angiotensin system, RAS) plays a particularly important role. The study found that the RAS system in addition to the ACE-Ang II -AT1R pathway also exists and its role in antagonizing a pathway that angiotensin-converting enzyme - vascular (1-7) angiotensin -Mas receptor axis (Angiotensin converting enzyme2-Angiotensin -Mas axis (1-7)), ACE2-Ang (1-7) -MasR axis, this pathway by ACE2 degradation in Ang II, the prevention and improvement of hypertension, heart failure Myocardial fibrosis were confirmed. Our previous studies showed that, compared with the circulating RAS system did not change significantly, significantly reduce the number of glomeruli activated simultaneously and renal function in the presence of reduced renal local RAS system. Also obtained the same conclusion in fat RAS system, suggests that the changes in local tissue RAS system may be the incidence of hypertension is another important factor. In our study also found that during pregnancy will cause inflammation of offspring rat ventricular myocytes apoptosis and remodeling, and this remodeling occurs earlier than the occurrence of hypertension, suggesting that ventricular remodeling offspring is one of the mechanism of hypertension. But the change of rat offspring which the factor caused the occurrence of ventricular remodeling and inflammation during pregnancy? Offspring rats cardiac RAS system impact? The rules and characteristics of what is yet to be further research.
This paper through the changes of collagen of myocardial tissue was observed and collagen metabolism enzymes MMP-2, TIMP-2 and profibrotic factor TGF beta -1 and TGF beta -2 to study the changes of myocardial fibrosis and myocardial remodeling. And through the observation of offspring rat cardiac RAS system on the two metabolic pathways: -AT1R pathway and ACE2-Ang ACE-Ang II (1-7) changes of -MasR pathway, further understanding of prenatal inflammation after stimulation of rat offspring occurrence of myocardial fibrosis and myocardial interstitial remodeling mechanism were studied.
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