發(fā)布時間：2018-09-17 18:19

【摘要】：背景房顫是臨床上最常見的心律失常之一。近年來,一類新的輔助性T細胞亞群-Th17細胞(T help cell 17,Th17)及其特異性效應(yīng)因子白介素-17(interleukin-17,IL17)備受關(guān)注,IL-17被證明是一種重要的炎癥反應(yīng)介質(zhì)。研究發(fā)現(xiàn),IL-17在多種心血管疾病的發(fā)生發(fā)展中發(fā)揮了重要的作用,包括動脈粥樣硬化、慢性心力衰竭、急性冠狀動脈綜合征和病毒性心肌炎。但是目前關(guān)于IL-17與房顫發(fā)生相關(guān)的研究證據(jù)尚較少。為探索IL-17是否是房顫的獨立危險因素和重要的炎癥標記物,本課題組前期通過配對的病例對照研究發(fā)現(xiàn),外周血漿中IL-17A與房顫發(fā)生相關(guān)聯(lián),且隨著因子水平增高,關(guān)聯(lián)強度越強,且可能通過影響心臟結(jié)構(gòu)重構(gòu)而影響房顫發(fā)生。但是IL-17A在房顫發(fā)生和維持中的具體作用和機制如何,外周血和心房組織中IL-17A水平是否存在一致性,仍有待進一步研究。目的本課題利用SD大鼠,通過食道快速心房起搏的方法來構(gòu)建房顫動物模型,進一步探討IL-17與房顫發(fā)生的關(guān)聯(lián),分析外周血中IL-17A水平與心房組織中是否存在一致性及二者與心房纖維化的相關(guān)性。同時,分析IL-17A與基質(zhì)金屬蛋白酶(matrix metalloproteinase,MMP)、基質(zhì)金屬蛋白酶抑制劑(tissue inhibitor metalloproteinase,TIMP)之間的相關(guān)性,進一步驗證IL-17A通過引起MMPs/TIMPs失衡誘導(dǎo)心房纖維化的機制。研究結(jié)果將為IL-17作為房顫早期診斷和監(jiān)測疾病進展的重要生物標記物提供依據(jù),也將為全面認識房顫發(fā)生和維持的機制以及尋找新的治療靶點提供參考依據(jù)。方法1.采用經(jīng)食道快速心房起搏的方法構(gòu)建大鼠房顫模型,比較分析房顫組與對照組心電數(shù)據(jù)的變化,包括QRS間期、QT間期等,以及兩組心房纖維化程度的改變。2.通過酶聯(lián)免疫吸附法測定房顫組與對照組血漿中IL-17A與C反應(yīng)蛋白(c-reactive protein,CRP)水平的變化,以及兩組血漿中MMP2、MMP9、TIMP2的水平。3.采用免疫組化法測定兩組心房組織中IL-17A、MMP2、MMP9及TIMP2的水平。4.采用Pearson相關(guān)分析的方法,分析血漿和心房組織中IL-17A的一致性及二者與心房纖維化的相關(guān)性,同時分析血漿和心房組織中MMP2、MMP9、TIMP2的一致性及三者與心房纖維化的相關(guān)性,血漿和心房組織中MMP2、MMP9、TIMP2與IL-17A的相關(guān)性。結(jié)果第一章大鼠房顫模型的構(gòu)建1.房顫組大鼠的心率顯著加快,正常時為(159.63±18.25)次/min,房顫發(fā)作時達(203.24±33.56)次/min,同時,QRS間期和QT間期明顯延長,分別由正常時的50.00±4.28 ms,103.00±1.85 ms增加至68.85±14.73 ms和134.80±22.05 ms,差異均有統(tǒng)計學意義(P0.05)。2.房顫組大鼠心房纖維化程度較對照組嚴重,膠原容積分數(shù)較對照組明顯升高[(18.15±9.45)%vs(6.51±4.49)%,P0.01]。第二章大鼠房顫模型中IL-17A的作用及其與心房纖維化的關(guān)聯(lián)研究1.大鼠外周血血漿中IL-17A和CRP的水平與房顫的發(fā)生密切相關(guān),且隨房顫發(fā)生次數(shù)的增多有上升趨勢。2.房顫組大鼠心房組織中IL-17A的蛋白表達水平較對照組明顯升高(0.17±0.05vs 0.04±0.01,P=0.003)。3.房顫組大鼠外周血血漿中IL-17A水平與心房組織中IL-17A水平存在高度相關(guān)性(r=0.721,P=0.019)。血漿中IL-17A水平與心房纖維化呈正相關(guān)(r=0.830,P=0.003),心房組織中IL-17A水平與心房纖維化也呈正相關(guān)(r=0.685,P=0.029)。第三章大鼠房顫模型中IL-17A與MMPs/TIMPs的關(guān)聯(lián)研究1.尚未發(fā)現(xiàn)房顫組與對照組大鼠外周血血漿中MMP2、MMP9、TIMP2水平存在統(tǒng)計學差異。2.與對照組相比,房顫組大鼠心房組織中MMP2、MMP9水平明顯升高(0.12±0.05vs 0.04±0.02,P=0.021;0.08±0.01 vs 0.03±0.01,P0.001),而TIMP2水平在房顫組明顯降低(0.02±0.00 vs 0.06±0.02,P=0.004)。3.房顫組大鼠外周血血漿和心房組織中MMP2、MMP9、TIMP2的水平存在高度相關(guān)性,心房組織中MMP9水平與心房纖維化呈正相關(guān)(r=0.809,P=0.005),TIMP2水平與心房纖維化呈負相關(guān)(r=-0.884,P=0.001),未發(fā)現(xiàn)心房組織中MMP2水平與心房纖維化之間的相關(guān)性(r=0.599,P=0.067)。4.房顫組大鼠外周血血漿中MMP9水平與IL-17A呈正相關(guān)(r=0.648,P=0.043),TIMP2水平與IL-17A呈負相關(guān)(r=-0.695,P=0.037),未發(fā)現(xiàn)血漿中MMP2與IL-17A水平之間的相關(guān)性(r=0.576,P=0.082)。5.房顫組大鼠心房組織中MMP2、MMP9與IL-17A水平均呈正相關(guān)(r=0.801,P=0.005;r=0.778,P=0.008),TIMP2與IL-17A水平呈負相關(guān)(r=-0.841,P=0.002)。結(jié)論通過大鼠房顫動物模型研究,發(fā)現(xiàn)IL-17A與房顫的發(fā)生發(fā)展密切相關(guān),IL-17A可能是通過影響MMPs/TIMPs平衡,引起心房結(jié)構(gòu)重構(gòu),從而導(dǎo)致房顫的發(fā)生與發(fā)展。
[Abstract]:Background Atrial fibrillation (AF) is one of the most common arrhythmias in clinical practice. In recent years, a new helper T cell subset-Th17 (Th17) and its specific effector interleukin-17 (IL-17) have attracted much attention. IL-17 has been proved to be an important inflammatory mediator in many cardiovascular diseases. Interleukin-17 (IL-17) plays an important role in the development of atrial fibrillation, including atherosclerosis, chronic heart failure, acute coronary syndrome and viral myocarditis. Phase I matched case-control study showed that IL-17A in peripheral plasma was associated with AF, and with the increase of factor levels, the stronger the association was, and it may affect AF by affecting cardiac structural remodeling. However, the specific role and mechanism of IL-17A in the occurrence and maintenance of AF, and the role of IL-17A in peripheral blood and atrial tissue were also discussed. Objective To explore the relationship between IL-17 and atrial fibrillation in SD rats by rapid esophageal atrial pacing, and to analyze the consistency of IL-17A level in peripheral blood with atrial tissue and the relationship between them and atrial fibrosis. The correlation between IL-17A and matrix metalloproteinase (MMP) and tissue inhibitor metalloproteinase (TIMP) was analyzed to further validate the mechanism of IL-17A inducing atrial fibrosis by inducing imbalance of MMPs/TIMPs. Methods 1. A rat model of atrial fibrillation was established by transesophageal rapid atrial pacing, and the changes of electrocardiogram data, including QRS, were compared and analyzed between atrial fibrillation group and control group. The changes of plasma levels of IL-17A and C-reactive protein (CRP) and plasma levels of MMP2, MMP9 and TIMP 2 in AF group and control group were measured by enzyme-linked immunosorbent assay. 3. The levels of IL-17A, MMP2 and MMP in atrial tissues were determined by immunohistochemistry. Pearson correlation analysis was used to analyze the consistency of IL-17A in plasma and atrial tissues and their correlation with atrial fibrosis. The consistency of MMP2, MMP9, TIMP2 in plasma and atrial tissues and their correlation with atrial fibrosis were also analyzed. Result The first chapter was the establishment of rat atrial fibrillation model. 1. The heart rate of rats in AF group increased significantly from (159.63 (+ 18.25) beats per minute in normal time to (203.24 (+ 33.56) beats per minute in AF attack. Meanwhile, the QRS interval and QT interval prolonged significantly from 50.00 (+ 4.28 ms), 103.00 (+ 1.85 ms) to 68.85 (+ 14.73 ms) and 134.80 (+ 22.05) beats in normal time, respectively. The degree of atrial fibrosis in AF group was more serious than that in control group. The volume fraction of collagen in AF group was significantly higher than that in control group [(18.15+9.45)% vs (6.51+4.49)%, P 0.01]. The level of IL-17A in atrial tissue of AF group was significantly higher than that of control group (0.17.05 vs 0.04.01, P = 0.003). 3. The level of IL-17A in plasma of AF group was significantly higher than that of control group (0.17.05 vs 0.04.01, P = 0.003). The level of IL-17A in plasma was positively correlated with atrial fibrosis (r = 0.830, P = 0.003), and the level of IL-17A in atrial tissue was positively correlated with atrial fibrosis (r = 0.685, P = 0.029). Compared with the control group, the levels of MMP 2 and MMP 9 in atrial tissue of AF group were significantly higher (0.12.05 vs 0.04.02, P = 0.021; 0.08.01 vs 0.03.01, P 0.001), while the levels of TIMP 2 were significantly lower in AF group (0.02.00 vs 0.06.02, P = 0.004). The levels of MMP 2, MMP 9 and TIMP 2 in atrial tissues were positively correlated with atrial fibrosis (r = 0.809, P = 0.005). The levels of TIMP 2 were negatively correlated with atrial fibrosis (r = - 0.884, P = 0.001). No correlation was found between the levels of MMP 2 in atrial tissues and atrial fibrosis (r = 0.599, P = 0.067). 4. The levels of MMP-9 in plasma were positively correlated with IL-17A (r = 0.648, P = 0.043), and TIMP-2 was negatively correlated with IL-17A (r =-0.695, P = 0.037). No correlation was found between the levels of MMP-2 and IL-17A (r = 0.576, P = 0.082). MMP-2, MMP-9 and IL-17A were positively correlated in atrial tissues of AF rats (r = 0.801, P = 0.005; r = 0.778, P = 0.008). Conclusion IL-17A is closely related to the occurrence and development of atrial fibrillation in the rat model of atrial fibrillation. IL-17A may induce atrial remodeling by affecting the balance of MMPs/TIMPs, leading to the occurrence and development of atrial fibrillation.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R541.75;R-332

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