本文選題：肺動(dòng)脈高壓 + 肺血管重構(gòu)�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文

【摘要】：背景和目的 致病因素的作用下,肺循環(huán)調(diào)節(jié)功能的失衡啟動(dòng)一系列病理生理改變,包括內(nèi)皮功能障礙、炎癥激活等,引起肺血管異常收縮,進(jìn)而平滑肌細(xì)胞增殖、血管重構(gòu),最終導(dǎo)致肺動(dòng)脈高壓。腎素血管緊張素系統(tǒng)是心血管系統(tǒng)的重要調(diào)節(jié)系統(tǒng),它在肺動(dòng)脈高壓發(fā)病過程中發(fā)揮重要作用,但是干預(yù)經(jīng)典的腎素血管緊張素系統(tǒng)(ACE-Ang Ⅱ-AT1R軸)治療肺動(dòng)脈高壓的效果欠佳。血管緊張素轉(zhuǎn)化酶2(ACE2)及其參與的ACE2-Ang-(1-7)-Mas軸是腎素血管緊張素系統(tǒng)的新成員,起著拮抗經(jīng)典腎素血管緊張素軸的作用,而且在呼吸系統(tǒng)疾病中發(fā)揮著廣泛的作用。本研究應(yīng)用ACE2激動(dòng)劑Resorcinolnaphthalein(Rec)干預(yù)野百合堿聯(lián)合肺葉切除構(gòu)建重度肺動(dòng)脈高壓模型,探討ACE2激活在預(yù)防和逆轉(zhuǎn)重度肺動(dòng)脈高壓中的作用及其機(jī)制。 方法 1.實(shí)驗(yàn)動(dòng)物和分組：SD大鼠,隨機(jī)分為6組：空白對照組,ACE2對照組,肺動(dòng)脈高壓組,ACE2預(yù)防組,ACE2治療組,Mas受體拮抗組(Rec+A-779)。左肺切除后1周,皮下注射野百合堿誘導(dǎo)重度肺動(dòng)脈高壓。ACE2對照和ACE2預(yù)防組在野百合堿注射后第1天開始持續(xù)注射Rec, ACE2治療組第14天開始注射Rec, Mas受體拮抗組第1天開始同時(shí)注射Rec和Mas受體拮抗劑A-779。 2.干預(yù)2周后,除ACE2治療組外(干預(yù)42天),其余各組取8只,心導(dǎo)管測定平均肺動(dòng)脈壓和動(dòng)脈收縮壓,分離右心室測定右心室肥厚指數(shù),彈力纖維染色(Verhoeff-鐵蘇木素染色)后分析肺動(dòng)脈(100-200gm)中膜厚度和肺小動(dòng)脈(50-100gm)內(nèi)膜梗阻性增生程度。 3.內(nèi)皮功能檢測：肺動(dòng)脈內(nèi)注射不同濃度的乙酰膽堿和硝普鈉,檢測內(nèi)皮依賴的血管舒張反應(yīng)的變化。 4.活性熒光共振能量轉(zhuǎn)移法檢測肺組織ACE2酶活性。 5.生存分析：采用Kaplan-Meier生存分析圖分析。 6. ELISA檢測肺組織Ang Ⅱ和Ang-(1-7)濃度,Western blot檢測肺組織ACE, ACE2, AT1R, Mas表達(dá)水平。 7.免疫組化檢測直徑50-100μm肺動(dòng)脈管壁平滑肌抗體(a-SMA)和增殖細(xì)胞核抗原表達(dá),分析肺動(dòng)脈平滑肌細(xì)胞增殖情況。 8. RT-PCR檢測肺組織炎癥因子IL-6, MCP-1, TNF-α, IL-10。 結(jié)果 1.Rec對大鼠肺組織ACE2酶活性的影響ACE2對照組ACE2酶活性是正常的1.74倍；肺動(dòng)脈高壓組ACE2酶活性下降38.2%,ACE2預(yù)防和治療組ACE2酶活性則分別比肺動(dòng)脈高壓組升高1.87和1.78倍,提示Rec可以激活大鼠肺組織ACE2。 2.ACE2激活對PAH大鼠生存率的影響 實(shí)驗(yàn)終點(diǎn)時(shí),肺動(dòng)脈高壓大鼠和Mas受體拮抗組大鼠全部死亡,ACE2預(yù)防組未出現(xiàn)死亡,ACE2治療組生存率為80%。統(tǒng)計(jì)學(xué)分析,ACE2治療組和預(yù)防組與肺動(dòng)脈高壓組差異顯著。 3.ACE2激活對血液動(dòng)力學(xué)和右心室的影響 大鼠左肺切除聯(lián)合野百合堿注射2周后,肺動(dòng)脈壓力升高,右心室肥大；ACE2對照組肺動(dòng)脈壓力沒有明顯變化。ACE2激活可以防止肺動(dòng)脈高壓和右心室肥厚,同樣ACE2治療4周也可以降低肺動(dòng)脈壓力,但是無法逆轉(zhuǎn)肥厚的右心室。各處理組大鼠動(dòng)脈收縮壓沒有變化。 4.ACE2激活對肺動(dòng)脈內(nèi)皮細(xì)胞功能的影響 在相同的基礎(chǔ)肺動(dòng)脈壓力下,肺動(dòng)脈高壓大鼠乙酰膽堿誘導(dǎo)的內(nèi)皮依賴的舒張反應(yīng)減弱,ACE2不但可以抑制它的減弱,而且可以修復(fù)受損的舒張反應(yīng)。硝普鈉誘導(dǎo)的肺內(nèi)皮依賴的舒張反應(yīng)各組之間沒有明顯差異。 5.ACE2激活對肺動(dòng)脈中膜肥厚和內(nèi)膜增生的影響 肺動(dòng)脈高壓大鼠的直徑為100-200gm的肺小動(dòng)脈主要表現(xiàn)為中膜肥厚,ACE2激活可以抑制和逆轉(zhuǎn)中膜肥厚。直徑為50-100μm的肺小動(dòng)脈主要表現(xiàn)為內(nèi)膜增生,梗阻明顯,ACE2預(yù)防組大鼠則主要表現(xiàn)為中膜肥厚,ACE2治療組大鼠小動(dòng)脈梗阻程度也顯著下降。 6.Mas受體對ACE2效果的影響 同時(shí)應(yīng)用ACE2激活劑Rec和Mas受體阻斷劑A-779,肺動(dòng)脈高壓預(yù)防作用消失,包括改善生存率,降低肺動(dòng)脈壓力,抑制右心室肥厚,肺小動(dòng)脈中膜肥厚和內(nèi)膜增生。 7.ACE2激活對腎素血管緊張素系統(tǒng)各主要成份的影響 肺動(dòng)脈高壓形成以后,ACE和AT1R上升為正常的2.49和2.75倍,Ang IⅡ則由25.06±3.81pg/mg上升到91.00±6.70pg/mg,ACE2激活可抑制ACE(78.5％), AT1R(78.9%)和Ang Ⅱ(46.60±10.00pg/mg)的上升,但是肺動(dòng)脈形成以后再激活A(yù)CE2,ACE和AT1R下降不明顯,僅能降低Ang Ⅱ(52.01±7.61pg/mg,). 肺動(dòng)脈高壓大鼠肺組織ACE2降低為正常的41.67%,而預(yù)防和治療組ACE2則達(dá)到肺動(dòng)脈高壓大鼠的2.52和2.35倍。肺動(dòng)脈高壓對Mas和Ang-(1-7)的影響不大,ACE2激活后,Mas則分別升高了的2.43和2.51倍,Ang-(1-7)也相應(yīng)升高。 肺動(dòng)脈高壓大鼠肺組織ACE/ACE2和AT1R/Mas比值明顯升高,而預(yù)防組和治療組這兩個(gè)比值則降低。 8.ACE2激活對肺動(dòng)脈平滑肌細(xì)胞增殖的影響 各組直徑為50-100gm肺小動(dòng)脈壁均廣泛表達(dá)平滑肌細(xì)胞抗體,提示平滑肌細(xì)胞在其病變中占重要地位。肺動(dòng)脈高壓大鼠肺動(dòng)脈增殖細(xì)胞核抗原陽性率顯著升高,ACE2激活后可以降低平滑肌細(xì)胞的增殖細(xì)胞核抗原陽性率,抑制增殖。 9.ACE2激活對肺組織炎癥因子的影響 大鼠肺動(dòng)脈高壓形成后,IL-6上升47倍,MCP-1上升31倍,TNF-a上升16倍,IL-10下降79%；而預(yù)防組IL-6降低85%,MCP-1降低86%, TNF-α降低82%,IL-10上升11倍,治療組則IL-6降低82%,MCP-1降低80%, TNF-α降低78%,IL-10升高9倍。 結(jié)論 1.Rec持續(xù)注射可以在大鼠體內(nèi)有效的激活肺組織ACE2 2.ACE2激活可預(yù)防左肺切除聯(lián)合野百合堿注射誘導(dǎo)的大鼠重度肺動(dòng)脈高壓形成 3.ACE2激活一定程度上逆轉(zhuǎn)大鼠重度肺動(dòng)脈高壓的血液動(dòng)力學(xué)和病理學(xué)改變 4.ACE2激活通過調(diào)控腎素血管緊張素系統(tǒng)內(nèi)縮血管／促增殖軸(ACE-AngⅡ-AT1R軸)和血管保護(hù)軸(ACE2-Ang-(1-7)-Mas軸)之間的平衡起作用。 5.在大鼠肺動(dòng)脈高壓的形成和進(jìn)展過程中激活A(yù)CE2,可修復(fù)內(nèi)皮依賴的血管舒張反應(yīng),改善內(nèi)皮功能；可減少新生內(nèi)膜中平滑肌細(xì)胞PCNA的表達(dá),抑制平滑肌細(xì)胞增生；可降低肺組織促炎因子IL-6, MCP-1, TNF-α,升高抗炎因子IL-10,從而減輕炎癥反應(yīng)。
[Abstract]:Background and purpose
Under the action of pathogenic factors, the imbalance of pulmonary circulation regulating function starts a series of pathophysiological changes, including endothelial dysfunction, inflammation activation and so on, causing abnormal contraction of pulmonary vessels, proliferation of smooth muscle cells, vascular remodeling, and pulmonary arterial hypertension. Renin angiotensin system is an important regulatory system of the cardiovascular system. It plays an important role in the pathogenesis of pulmonary hypertension, but the intervention of the classical renin angiotensin system (ACE-Ang II -AT1R axis) is not good for the treatment of pulmonary hypertension. Angiotensin converting enzyme 2 (ACE2) and the ACE2-Ang- (1-7) -Mas axis involved in the renin angiotensin system are new members of the renin angiotensin system and play an antagonism to the classical renin blood vessels. In this study, the ACE2 agonist Resorcinolnaphthalein (Rec) was used in this study to construct a model of severe pulmonary hypertension with monocrotaline combined with lobectomy, and to explore the role and mechanism of ACE2 activation in the prevention and reversal of severe pulmonary vein pressure.
Method
1. experimental animals and groups: SD rats were randomly divided into 6 groups: blank control group, ACE2 control group, pulmonary hypertension group, ACE2 prevention group, ACE2 treatment group, Mas receptor antagonist group (Rec+A-779). 1 weeks after left pneumonectomy, the subcutaneous injection of monocrotaline induced severe pulmonary arterial hypertension,.ACE2 control and ACE2 prevention group began to hold on first days after the injection of lilocinine. Continuous injection of Rec, ACE2 treatment group began Rec injection on the fourteenth day, while Mas receptor antagonist group started injecting Rec and Mas receptor antagonist A-779. on the first day.
2. after 2 weeks of intervention, 8 were taken out of the other groups except the ACE2 treatment group (42 days of intervention). The mean pulmonary artery pressure and arterial systolic pressure were measured by cardiac catheterization. Right ventricular hypertrophy index was measured by the separation of right ventricle. The middle membrane thickness of pulmonary artery (100-200gm) and the intimal obstruction hyperplasia of pulmonary artery (50-100gm) were analyzed after elastin staining (Verhoeff- ferulin staining). Degree.
3. endothelial function test: different concentrations of acetylcholine and sodium nitroprusside were injected into the pulmonary artery to detect the changes of endothelium-dependent vasodilatation.
4. activated fluorescence resonance energy transfer assay was used to detect the activity of ACE2 in lung tissue.
5. survival analysis: Kaplan-Meier survival analysis.
6. ELISA was used to detect the concentration of Ang II and Ang- (1-7) in lung tissue. Western blot was used to detect the expression levels of ACE, ACE2, AT1R and Mas in lung tissue.
7. immunohistochemistry was used to detect the expression of smooth muscle antibody (a-SMA) and proliferating cell nuclear antigen (PCNA) of pulmonary artery smooth muscle cells in diameter of 50-100 m, and to analyze the proliferation of pulmonary artery smooth muscle cells.
8. RT-PCR was used to detect inflammatory cytokines IL-6, MCP-1, TNF- alpha, IL-10. in lung tissue.
Result
The effect of 1.Rec on the activity of ACE2 enzyme in the lung tissue of rats was 1.74 times as high as that of the normal ACE2 control group; the activity of ACE2 enzyme in the pulmonary hypertension group decreased by 38.2%, and the activity of ACE2 enzyme in the ACE2 prevention and treatment group was 1.87 and 1.78 times higher than that in the pulmonary hypertension group, suggesting that Rec could activate the lung tissue ACE2. in rats.
The effect of 2.ACE2 activation on the survival rate of PAH rats
At the end of the experiment, all the rats of the pulmonary hypertension rats and the Mas receptor antagonist group died, the ACE2 prevention group did not die, the survival rate of the ACE2 treatment group was 80%. statistical analysis, and the difference between the ACE2 treatment group and the prevention group was significantly different from the pulmonary hypertension group.
Effects of 3.ACE2 activation on hemodynamics and right ventricle
After 2 weeks of left lung resection combined with monocrotaline injection, pulmonary arterial pressure increased and right ventricular hypertrophy was increased. Pulmonary arterial pressure in ACE2 control group was not significantly changed by.ACE2 activation to prevent pulmonary hypertension and right ventricular hypertrophy. Similarly, the pulmonary artery pressure could be reduced by the treatment of ACE2 for 4 weeks, but the right ventricle could not be reversed by the hypertrophy of the right ventricle. Rats in each group were treated. The systolic pressure of the arteries did not change.
Effect of 4.ACE2 activation on the function of pulmonary artery endothelial cells
Under the same basic pulmonary arterial pressure, the endothelium dependent relaxation response induced by acetylcholine in the pulmonary hypertension rats weakened, and ACE2 not only inhibited its weakening, but also repaired the damaged diastolic response. There was no significant difference in the endothelium dependent diastolic response induced by sodium nitroprusside.
Effect of 5.ACE2 activation on pulmonary artery intima thickening and intimal hyperplasia
The pulmonary arterioles with the diameter of 100-200gm in the pulmonary hypertension rats were mainly the middle membrane hypertrophy, and the ACE2 activation could inhibit and reverse the middle membrane hypertrophy. The pulmonary arterioles with the diameter of 50-100 u m were mainly intima hyperplasia, and the obstruction was obvious. The rats in the ACE2 prevention group were mainly the middle membrane fat, and the degree of the small artery obstruction in the ACE2 treatment group was also obvious. It's falling.
The effect of 6.Mas receptor on the effect of ACE2
At the same time, ACE2 activator Rec and Mas receptor blocker A-779 were used, and the prevention of pulmonary hypertension disappeared, including improving survival rate, reducing pulmonary artery pressure, inhibiting right ventricular hypertrophy, pulmonary arterioles hypertrophy and intima hyperplasia.
Effects of 7.ACE2 activation on the main components of renin angiotensin system
After the formation of pulmonary hypertension, ACE and AT1R increased to 2.49 and 2.75 times as normal, Ang I II increased from 25.06 + 3.81pg/mg to 91 + 6.70pg/mg, ACE2 activation inhibited ACE (78.5%), AT1R (78.9%) and Ang II (46.60 + 10.00pg/mg), but pulmonary artery formation reactivated ACE2. .01 + 7.61pg/mg,).
ACE2 in pulmonary tissue of pulmonary hypertension rats decreased to 41.67% of normal, while ACE2 in prevention and treatment group reached 2.52 and 2.35 times that of pulmonary hypertension rats. Pulmonary hypertension had little effect on Mas and Ang- (1-7). After ACE2 activation, Mas increased by 2.43 and 2.51 times respectively, and Ang- (1-7) increased correspondingly.
The ratio of ACE/ACE2 to AT1R/Mas in lung tissue of rats with pulmonary hypertension increased significantly, while the two ratios in the prevention group and the treatment group decreased.
Effect of 8.ACE2 activation on proliferation of pulmonary artery smooth muscle cells
The expression of smooth muscle cell antibody was widely expressed in the wall of 50-100gm pulmonary arterioles in each group, suggesting that smooth muscle cells play an important role in its pathological changes. The positive rate of proliferating cell nuclear antigen (PCNA) in pulmonary artery of pulmonary hypertension rats was significantly increased. ACE2 activation could reduce the positive rate of proliferating cell nuclear antigen (PCNA) of smooth muscle cells and inhibit the proliferation.
Effect of 9.ACE2 activation on inflammatory factors in lung tissue
After the formation of pulmonary hypertension, IL-6 increased 47 times, MCP-1 increased 31 times, TNF-a increased 16 times, IL-10 decreased by 79%, IL-6 decreased by 85%, MCP-1 decreased by 86%, TNF- alpha was 82%, IL-10 increased 11 times, IL-6 decreased by 82%, MCP-1 decreased 80%, TNF- alpha decreased 78%, IL-10 increased 9 times.
conclusion
Continuous injection of 1.Rec can effectively activate lung tissue ACE2 in rats.
2.ACE2 activation prevents the formation of severe pulmonary hypertension induced by left lung resection combined with monocrotaline injection in rats
3.ACE2 activation can reverse the hemodynamic and pathological changes of severe pulmonary hypertension in rats to some extent.
4.ACE2 activates the balance between the regulation of the renin angiotensin system (ACE-Ang II -AT1R axis) and the vascular protective axis (ACE2-Ang- (1-7) -Mas axis) by regulating the renin angiotensin system.
5. the activation of ACE2 in the formation and progression of pulmonary hypertension in rats can repair endothelium dependent vasodilatation, improve endothelial function, reduce the expression of PCNA in the smooth muscle cells in neointima, inhibit the proliferation of smooth muscle cells, and reduce the IL-6, MCP-1, TNF- alpha, and anti-inflammatory factors IL-10 of lung tissue, thus reducing the anti-inflammatory factor IL-10. Inflammatory reaction.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R363

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