心肌梗死导致的慢性心肾综合征中免疫激活及相关机制的研究

发布时间：2018-07-05 17:57

本文选题：心肾综合征 + 心肌梗死　；参考：《上海交通大学》2014年博士论文

【摘要】：背景:慢性心力衰竭和慢性肾病往往共存于同一机体内互相影响,并导致了心力衰竭加重,晚期肾脏病变的进一步恶化和预后不良。临床研究证实了心肌梗死后的患者肾功能可发生慢性恶化,特别是那些已伴有肾功能损害的人群中,而糖尿病肾病则是这些人群的首要病因。这种慢性心力衰竭和慢性肾病共存且相互影响并导致病变加重的这一复杂的病理生理现象称之为心肾综合征。既往已有许多证据提示免疫激活参与了多种病因导致的肾损害进程,然而在心肌梗死后是否同样参与和诱导了肾脏功能的损害而导致预后不良,即慢性心肾综合征,及其相关机制仍无深入研究。目的:本研究旨在:1.通过建立心肌梗死合并切单肾(UNX+MI)和心肌梗死合并I型糖尿病两个联合损伤大鼠模型(即出现心肾综合征病理生理改变),观察其心、肾结构与功能的改变,并比较其与单纯心肌梗死大鼠相关指标的差异;2.观察心肌梗死12周后各组模型大鼠肾内细胞免疫和体液免疫等指标改变的同时,观察肾脏血流动力学/血管反应性,肾脏内质网应激等改变;3.观察Rho激酶抑制剂是否能够改善联合损伤模型大鼠中上述免疫机制等异常并改善心、肾功能;以进一步探讨肾脏免疫等机制是否参与了心梗后心、肾功能的进一步损害,以及内质网应激在慢性心肾综合征病理生理过程中的分子作用机制和价值。方法:实验分组:1):假手术对照组(control,Ctr.n=12);2)冠脉结扎诱导的心肌梗死组(MI,n=12);3)切单肾组(UNX,n=11);4)切单肾合并心肌梗死组(UNX+MI,n=11);5)切单肾合并心肌梗死给予Fasudil治疗组(UNX+MI+Fas,n=11);6)单纯STZ诱导I型糖尿病组(DB,n=11);7)STZ诱导糖尿病合并心肌梗死组(DB+MI,n=11)和8)糖尿病合并心肌梗死组给予Fasudil治疗组(DB+MI+Fas,n=11)。分别比较Ctr和MI(protocol 1);UNX+MI和UNX,UNX+MI+Fas(protocol 2);DB+MI和DB,DB+MI+Fas(protocol 3);以及联合损伤模型——UNX+MI,DB+MI与单纯MI组之间的心肾结构、功能和相关病生机制的差异。以对照研究:1)切单侧肾后心肌梗死对心脏和肾脏功能的进一步损害;2)伴有糖尿病肾病是否加重心肌梗死对心肾功能的影响;3)通过电子显微镜,流式细胞仪,免疫组化,免疫荧光以及多导myograph系统等检测手段观察各组大鼠的心、肾结构和肾局部免疫、血流动力学和内质网应激等机制的异常;4)观察Fasudil治疗是否通过改善免疫细胞的侵润和免疫复合物的沉积以及肾脏内质网应激而改善CRS中的肾脏功能。结果:第一部分:心梗12周后各组大鼠心、肾结构和功能的改变1)心功能:心梗12周后,心超(LVEDD,LVESD,FS)及导管(dp/dt max,dp/dt min,LVEDP)检测的各项心脏结构和功能指标以及血浆BNP等均显示各模型大鼠心功能发生了一定的恶化。但联合模型组(UNX+MI及DB+MI)较单纯心梗组的改变并不一致:一方面UNX+MI及DB+MI组的血浆BNP值显著高于单纯MI组,导管检测也显示DB+MI组较单纯MI组的心功能更差。但另一方面,切单肾合并心梗组较MI组的导管和心超参数并未显示出显著的差异。2)Rho信号通路的改变:联合模型组(UNX+MI及DB+MI组)的Rho信号途径显著上调——p-ERM/t-ERM的比值上调,fasudil则改善了其p-ERM/t-ERM的比值。3)肾功能:肾结构和功能的检测显示:心梗12周后不同程度的加重了肾组织纤维化(MASSON,TGF-β),肾小球硬化(PAS),足细胞损伤(nephrin,desmin),微量白蛋白尿,且联合模型组的肾微结构和功能的恶化较单纯MI组更显著,Fasudil改善了UNX+MI组及DB+MI组的肾微结构和功能的异常。第二部分:心梗12周后各组大鼠血流动力学和血管活性的改变心梗12周后联合模型组——UNX+MI及DB+MI组的肾灌注压,肾叶间小动脉收缩反应性,肾静脉压,肾重/体重比等均发生显著异常,显示该两个模型同时存在肾缺血和淤血,且较单纯MI组更为显著,而MI组则未显示出与Ctr组的差异。Fasudil改善了联合模型组的肾静脉压、肾重/体重比及肾叶间小动脉的收缩反应性。第三部分:心梗12周后各组大鼠肾脏内质网应激的激活心梗12周后各种模型大鼠肾内质网应激标志物CHOP,GRP78的表达均有所增加,且联合模型组——UNX+MI组及DB+MI组较单纯MI组更为显著。免疫组化显示内质网应激发生的主要部位在肾小球,而Fasudil改善了联合模型组的肾小球内质网应激。第四部分:心梗12周后各组大鼠肾脏免疫应答的改变1)肾脏免疫细胞的浸润:流式细胞仪检测显示心梗加重了各模型大鼠肾脏单核细胞的浸润(KMNCs)。联合损伤模型组——UNX+MI及DB+MI组较单纯MI组的免疫细胞浸润更为显著,并且肾CD3+/KMNCs,CD4+/CD3+,也分别较其假手术组和单纯心梗组更为显著。此外免疫组化显示上述免疫细胞浸润的部位主要在肾小球。2)肾小球免疫复合物沉积:电镜显示联合模型组肾小球系膜区出现了高电子密度物质的沉积,免疫荧光检测显示其主要成分为免疫复合物Ig G和补体C4。同时,肾浸润的CD4+T细胞的比重和Ig G沉积量与微量白蛋白尿及血浆BNP呈一定的正相关性。3)免疫应答与炎症反应:流式细胞仪检测显示心梗12周后各组模型肾内浸润的产炎症因子IFN-γ,IL-4的CD4+T细胞的比例显著增加,此外,在UNX+MI组及DB+MI组产炎症因子IL-17的CD4+T细胞,Treg细胞进一步上调,而Treg/Th17比例下调提示免疫相关性炎症参与了该疾病的进程。此外,Fasudil改善了联合模型组中免疫细胞的浸润和免疫复合物的沉积。结论:心肌梗死诱导的慢性心力衰竭加重了肾脏结构和功能损伤,特别是在独肾及有糖尿病肾损害的大鼠模型中,其主要的机制可能是肾细胞和体液免疫的激活,而伴随的血流动力学/血管反应性和内质网应激可能也参与了其中的进程,Rho激酶抑制剂改善了上述机制和心、肾功能的异常。
[Abstract]:Background: chronic heart failure and chronic kidney disease often coexist with each other in the same body, resulting in exacerbation of heart failure, further deterioration of advanced renal disease and poor prognosis. Clinical studies have confirmed chronic deterioration of renal function in patients with myocardial infarction, especially among those with renal impairment, Diabetic nephropathy is the leading cause of these people. This complex pathophysiological phenomenon, which coexists with chronic renal failure and chronic kidney disease and causes aggravation of the disease, is called cardio renal syndrome. There is a lot of evidence that immune activation is involved in the process of renal damage caused by multiple causes, but in myocardial infarction The purpose of this study is: 1. the purpose of this study was to establish two combined injured rat models of myocardial infarction combined with single kidney (UNX+MI) and myocardial infarction combined with I type glycan disease (that is, the synthesis of heart and kidney syndrome) The changes of the heart, the structure and function of the kidney were observed, and the differences in the related indexes of the rats with simple myocardial infarction were compared. 2. the changes in the renal cell immunity and humoral immunity of each model rats after 12 weeks of myocardial infarction were observed, and the renal hemodynamic / vascular reactivity, and the renal endoplasmic reticulum stress were observed. 3. to observe whether the Rho kinase inhibitor can improve the abnormality of the above-mentioned immune mechanism in the combined injury model rats and improve the heart and kidney function, to further explore whether the mechanism of renal immunity is involved in the further damage of the post infarction heart, the renal function, and the molecules of endoplasmic reticulum stress in the pathophysiological process of chronic heart and kidney syndrome. Effect mechanism and value. Methods: experimental group: 1): control (Ctr.n=12); 2) coronary artery ligation induced myocardial infarction (MI, n=12); 3) single kidney group (UNX, n=11); 4) single kidney combined with myocardial infarction (UNX+MI, n=11); 5) single kidney combined with myocardial infarction (UNX+MI+Fas, n=11); 6) simple STZ induced diabetic diabetes mellitus The disease group (DB, n=11); 7) STZ induced diabetes combined with myocardial infarction (DB+MI, n=11) and 8) diabetes combined with myocardial infarction group was given Fasudil treatment group (DB+MI+Fas, n=11). Differences in cardiac and renal structure, function and related pathogenesis between groups. Control study: 1) further impairment of unilateral post renal infarction on cardiac and renal function; 2) whether diabetic nephropathy aggravates myocardial infarction on cardiac and renal function; 3) through electronic microscopes, flow cytometry, immunohistochemistry, immunofluorescence, and multi conductance Myograph system and other detection methods observe the heart, renal structure and local immune, hemodynamics and endoplasmic reticulum stress in rats. 4) observe whether Fasudil treatment improves the renal function in CRS by improving the deposition of immune cells and the deposition of immune complex and renal endoplasmic reticulum. Results: the first part: Heart, renal structure and function change 1) cardiac function after 12 weeks of myocardial infarction. After 12 weeks of myocardial infarction, Cardiac Ultrastructure and function indexes, such as dp/dt max, dp/dt min, LVEDP and dp/dt max, dp/dt min, LVEDP, and plasma BNP all showed that the cardiac function of each model rats had been deteriorated, but the combined model group (UNX+MI and DB+MI) was compared. The changes in the simple myocardial infarction group were not consistent: on the one hand, the plasma BNP value of the UNX+MI and DB+MI groups was significantly higher than that in the simple MI group. The catheter detection also showed that the cardiac function of the DB+MI group was worse than that in the simple MI group. On the other hand, the single renal combined myocardial infarction group had no significant difference in the Rho signaling pathway from the catheter and cardiac parameters of the MI group: the change of the Rho signaling pathway. The Rho signal pathway in the combined model group (UNX+MI and DB+MI) was significantly up-regulated, the ratio of p-ERM/t-ERM was up, and fasudil improved the p-ERM/t-ERM ratio.3) renal function. The renal structure and function test showed that renal tissue fibrosis (MASSON, TGF- beta), glomerulosclerosis (PAS), and foot cell injury (nephrin) were aggravated after 12 weeks of myocardial infarction. Desmin), microalbuminuria, and the deterioration of renal microstructures and functions in the combined model group was more significant than that in the simple MI group. Fasudil improved the renal microstructures and function abnormalities in group UNX+MI and DB+MI group. The second part: the changes of hemodynamics and vasoactivity in each group after 12 weeks of myocardial infarction, the combined model group after 12 weeks of myocardial infarction - UNX+MI and DB+M The renal perfusion pressure in the I group, the contractile responsiveness of the interlobular arteriole, the kidney static pulse pressure, the kidney weight / weight ratio, and so on, showed that the two models had renal ischemia and congestion at the same time, which was more significant than that in the simple MI group, while the MI group did not show the difference from the Ctr group. The renal vein pressure, the kidney weight / weight ratio and the ratio of the renal weight / weight ratio in the combined model group were improved by.Fasudil. The contractile responsiveness of the interlobular arteriole of the renal interlobular. Third: after 12 weeks of myocardial infarction, the activation of the renal endoplasmic reticulum stress in each group was 12 weeks after 12 weeks. The expression of CHOP and GRP78 in the renal endoplasmic reticulum was increased, and the combined model group - the group UNX+MI and the DB+MI group were more significant than the simple MI group. Immunohistochemistry showed the endoplasmic reticulum The main part of stress occurred in the glomerulus, and Fasudil improved the glomerular endoplasmic reticulum stress in the combined model group. The fourth part: changes in renal immune response in each group after 12 weeks of myocardial infarction 1) the infiltration of renal immune cells: flow cytometry showed that the myocardial infarction aggravated the infiltration of mononuclear cells in the kidney of each model rat (KMNCs). In the injury model group, the immune cell infiltration in the UNX+MI and DB+MI group was more significant than that in the simple MI group, and the renal CD3+/KMNCs and CD4+/CD3+ were more significant than those in the sham operation group and the simple myocardial infarction group. In addition, the immunohistochemical staining showed that the site of the immune cell infiltration was mainly in the glomerular.2) glomerular immune complex deposition: the electron microscopy showed the combination of the immune cells. In the glomerular mesangial region of the model group, high electron density materials were deposited. Immunofluorescence detection showed that the main components were immune complex Ig G and complement C4., the proportion of CD4+T cells in the renal infiltration and the Ig G deposition and the positive correlation.3 in microalbuminuria and plasma BNP: the immune response and inflammatory response: flow cytometry After 12 weeks of myocardial infarction, the proportion of inflammatory factors IFN- gamma and IL-4 CD4+T cells increased significantly in each group of intrarenal infiltration. In addition, Treg cells were further up-regulated in group UNX+MI and DB+MI group of CD4+T cells producing inflammatory factor IL-17, and the downregulation of Treg/Th17 suggested that immune phase inflammation was involved in the process of the disease. In addition, Fasudil, Fasudil. Conclusion: chronic heart failure induced by myocardial infarction aggravates renal structural and functional damage, especially in the rat model of kidney and diabetic kidney damage, the main mechanism may be the activation of renal cell and humoral immunity, accompanied by blood flow. Kinetics / vascular reactivity and endoplasmic reticulum stress may also be involved in the process. Rho kinase inhibitors have improved the above mechanisms and cardiac and renal dysfunction.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R542.22;R692

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