| Background and Objective:Valvular heart disease accounts for a large proportion of the adult heart disease, is one of the main cause of heart failure. Chronic aortic valve stenosis is a common clinical left ventricular overload diseases, through the replacement of aortic valve to relieve outflow obstruction is a typical process of unloading. In theory, valve replacement can remove left ventricular pressure overload and clinical symptoms should be improved thoroughly. But the cardiac function cannot be recovered effectivelly for some patients. All of them caused the clinic symptoms can not alleviate, or even be worse, it does not benefit to patients. The specific reasons are not clear, whether or not be related to the degree of myocardial fibrosis?myocardial remodeling determined recovery of heart function after pressure over load / unload. Myocardial remodeling is the pathophysiological processes including myocardial hypertrophy, fibrosis, extracellular matrix components increase and cell ultrastructure features change under various mechanical impact force, metabolic and genetic factors. The necessary adaptive response to myocardial hypertrophy in pressure overload is generated under the action, it occurs mainly in the long-term pressure overload. Myocardial content increased and contraction enhanced, are an effective compensation method. However, the compensatory ability is limited, when myocardial oxygen demand can not be met, myocardial interstitial fibrosis, eventually leading to myocardial systolic dysfunction.KLFs family is a large class of basal transcription factor in eukaryotic organisms(BTEBP), which is involved in early embryonic development, cell differentiation, organ formation, proto oncogene mutations and blood vessel occurrence of physiological and pathological processes. They are the largest, one of the most important basic regulatory factors in eukaryotes. The KLFs are widely expressed in many cell types in organisms, play an important role in different. Many factors in this family have been widely studied, such as the KLF5 gene knockout studies in rats showed that it is the essential factor in the reaction process of continuous injection of angiotensin II induced cardiac hypertrophy, fibrosis. But, cultured cardiac fibroblasts in vitro, KLF5 genes directly regulated transcription in the platelet derived growth factor PDGF-A related to tissue remodeling and wound healing. In addition, myocardial fibroblast specific knockout of KLF5 gene reduced cardiac hypertrophy induced by the moderate pressure overload. It is an important negative regulatory factor of cardiovascular remodeling, in cardiac fibrosis caused by pressure overload. KLF15 is a member of the KLFs family have been found 17 basic regulation factor, it plays an important role in all aspects of cell growth, differentiation, apoptosis, etc.Above the research and found, we think that the changes of ultrastructure induced by myocardial remodeling influencing the recovery of heart function are the main molecular event, KLF15 is the most critical factor for that. So, the thesis intends to establish an animal and cell model effectively, in this two levels, we can explore the regulation mechanism of KLF15 in cardiac hypertrophy and fibrosis.Methods:This paper based on the clinical problems, combined with the related research, by using the method of in vitro and in vivo experiments to carry on. It is divided into the following four parts:Part I The wild type and 3-4 weeks old male Sprague-Dawley(SD) rats, in the non artificial ventilation conditions, at the aorta ascendens above aortic valve, aortic banding was performed to establish an animal model of cardiac hypertrophy and fibrosis caused by pressure overload, and at different time making debanding model; Using color Doppler echocardiography to detect the corresponding indexes on this animal model in order to clear the molding effect.Part II Utilizing the ascending aortic banding and debanding rat models without artificial ventilation, to explore myocardial pathologic changes and outcome of myocardial fibrosis in different time points after pressure overload or unload. To study the effect of KLF15 gene in this process, and the mutual relationships between TGF- β, CTGF, MRTF-A, clarifying the specific mechanisms of KLF15 gene in cardiac fibrosis induced by pressure overload.Part III The rat cardiac fibroblasts cultured in vitro were made to observe the identification, and the cell proliferation, apoptosis of cultured primary cells, and on this basis, we established cultured cardiac fibroblast hypertrophy model in vitro by TGF- β and provide a cell experimental platform to study the concrete molecular mechanism of myocardial hypertrophy, fibrosis occurrence, development and outcome.Part IV on the basis of the rat cardiac fibroblasts cultured in vitro, to the TGF- β cell hypertrophy model as a platform, the interference and overexpression of KLF15 adenovirus vector and transfection, we explore the specific molecular mechanism of myocardial fibrosis and prognosis on the cell level.Results:1. We complete aortic banding and debanding operation by using SD rats without artificial ventilation conditions, Through color Doppler echocardiography detection, from images and relevant data measured results to know: the effect of constriction is definite, coarctation of ascending aorta diameter reduced after about 50%, cross-sectional area is reduced about 75%, narrow distal has obvious expansion, and posterior wall of left ventricle, interventricular septal thickness increased significantly, while the cardiac ejection fraction, left ventricular shortening fraction, left ventricular systolic pressure was significantly lower, left ventricular end diastolic pressure was significantly higher, and after neurolysis, ascending aorta diameter, ejection fraction, left ventricular shortening, left ventricular systolic pressure was restored, left ventricular wall and interventricular septum thickness, diastolic pressure decreased.2. In the non artificial ventilation aortic coarctation and loosening rat model, 1 weeks after the aortic coarctation, it is starting to appear hypertrophy and myocardial fibrosis of left ventricle, with narrow time prolonged, the hypertrophy and fibrosis increased, m RNA and protein expression of the cytokines of TGF- β、CTGF and MRTF-A gradually increased, and the KLF15 factor level is gradually reduced; After releasing coarctation of ascending aorta, the indicators of heart(including the degree of myocardial fibrosis, ventricular hypertrophy etc.) have improved, but the degree of recovery solution in early(1W), medium(3W) and late(6W) after coarctation loose, there are some differences, the sooner to narrow, the indexes had significantly greater improvements. The degree of recovery of cardiac structure and function are related to the duration of coarctation. While the m RNA and protein expression of TGF- β, CTGF, MRTF-A after neurolysis has gradually decreased, the variation of the KLF15 factor is the opposite, the sooner to narrow the better to the recovery to normal.3. Through observing these biological activities, such as cell morphology, immunofluorescence identification, proliferation and apoptosis in the cultured rat myocardial fibroblasts, we found that the culture method is reliable, simple operation, better cell growth, high purity, and can be stably transfered; Through the detection of type I collagen m RNA levels analysis of the results, TGF- β has an obvious effect on inducing cardiac fibroblast hypertrophy and fibrosis.4. There are a higher rate of transfection efficiency in the KLF15 over expression adenovirus and its interference fragment in cultured rat myocardial cells transfected. The transfection efficiency and time、concentration were positively correlated in a certain range. After over expression of adenovirus KLF15 transfected the cardiac fibroblasts, the expression level of KLF15 m RNA and protein was significantly increased, the expression level of m RNA and protein of TGF- β, CTGF, MRTF-A significantly decreased. The cell hypertrophy and fibrosis are reduced, Ⅰ, Ⅲ collagen fiber contents are decreased too. But after the KLF15 interference fragment transfected cell, change of the above indicators are on the contrary.Conclusions:1. We use SD rats successfully complete coarctation and loosening operation of ascending aorta in the non artificial ventilation conditions. Establishing an animal model of consistency, reproducibility, simplicity and economy. It provides a good experimental platform for the future research on the load of cardiac pressure overload;2. Based on culturing rat cardiac fibroblasts in vitro, we successful constructed the cell hypertrophy model by using TGF- β, for the further study on molecular mechanism of myocardial hypertrophy, fibrosis, development and outcome provides a good technical means and cell experiment platform;3. By feedback regulation TGF-β, KLF15 inhibited the effect of CTGF and MRTF-A, reducing myocardial interstitial fibrosis and cardiac fiber cell phenotype transformation. It improved cardiac function, plays a key role in promoting the negative regulatory factor of myocardial hypertrophy and fibrosis.4. The possible mechanism of KLF15 on myocardial fibrosis: Stimulating cardiac fibroblasts under mechanical or chemical stress when pressure overloading, the expression of TGF-β increased, it caused the activation of P38 MAPK and inhibition of KLF15 expression by the self(side)-secreted form effecting on fibroblasts. On the one hand, low expression of KLF15 losed the inhibition to the expression of CTGF and promoted the development of myocardial fibrosis; On the other hand, the low expression of KLF15 also losed competitive binding capacity on the myocardin related transcription factor A(MRTF-A), and then through the serum response factor(SRF), MRTF-A could promote myocardial fibroblasts differentiate into myofibroblasts, leading to interstitial fibrosisfibroblasts eventually. |
PDF Full Text Request