本文關(guān)鍵詞： 巨噬細(xì)胞極化 鈣化性主動脈瓣疾病 瓣膜間質(zhì)細(xì)胞　出處：《上海交通大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的:鈣化性主動脈瓣疾病是全世界范圍內(nèi)發(fā)病率最高的心臟瓣膜疾病之一。而隨著老齡化社會的來臨,這一數(shù)字仍將不斷增加,造成極大的社會醫(yī)療負(fù)擔(dān)。目前對于該病,除了手術(shù)治療,尚無有效的藥物可用于延緩疾病的進(jìn)展。事實上大量的研究提示主動脈瓣鈣化的病變過程類似于動脈粥樣硬化,包括脂質(zhì)沉積、炎癥浸潤、瓣膜血管新生和纖維化重塑,最終導(dǎo)致鈣化。而在這些過程中,炎癥浸潤是其中關(guān)鍵因素之一,伴隨主動脈瓣瓣膜鈣化的整個過程。巨噬細(xì)胞作為瓣膜炎癥浸潤的主要炎癥細(xì)胞無疑起著至關(guān)重要的作用。近年來發(fā)現(xiàn)巨噬細(xì)胞并非單一性狀的炎癥細(xì)胞,而是可以通過極化分化為不同亞型。大量研究認(rèn)為巨噬細(xì)胞極化在冠狀動脈粥樣硬化發(fā)生發(fā)展過程中起著重要作用,而關(guān)于巨噬細(xì)胞極化在主動脈瓣鈣化中的作用目前尚未明確。因此本研究旨在通過對巨噬細(xì)胞極化在鈣化主動脈瓣中的表達(dá)以及其對瓣膜間質(zhì)細(xì)胞的影響等相關(guān)研究,來探討巨噬細(xì)胞極化在主動脈瓣鈣化中的作用機(jī)制。方法:按一定納入排除標(biāo)準(zhǔn)收集臨床上行主動脈瓣置換手術(shù)的主動脈瓣瓣膜標(biāo)本。首先通過比較硝酸、甲酸以及EDTA三種實驗室常用脫鈣液對鈣化性主動脈瓣脫鈣后HE和免疫組化染色效果,篩選出合適的鈣化性主動脈瓣瓣膜脫鈣液。對收集的鈣化性主動脈瓣瓣膜進(jìn)行脫鈣處理后,采用免疫組化的方法來探測巨噬細(xì)胞極化后的不同亞型在鈣化性主動脈瓣中的分布,得出其中主要的巨噬細(xì)胞分化后亞型。然后通過體外培養(yǎng)人主動脈瓣VICs,用不同亞型巨噬細(xì)胞來進(jìn)行相應(yīng)刺激,分別在蛋白水平、基因表達(dá)水平、形態(tài)學(xué)等方面來檢測刺激后VICs的成骨鈣化情況,以比較不同亞型巨噬細(xì)胞對VICs的成骨鈣化作用。結(jié)果:在脫鈣效果的比較上面,EDTA組盡管脫鈣速度較慢,但其脫鈣效果最好。通過EDTA對鈣化主動脈瓣瓣膜脫鈣后進(jìn)行免疫組化發(fā)現(xiàn):相比較正常主動脈瓣,鈣化主動脈瓣中巨噬細(xì)胞呈大量表達(dá),其中尤其以M1型巨噬細(xì)胞表達(dá)增加明顯。而通過不同亞型巨噬細(xì)胞體外刺激VICs發(fā)現(xiàn)M1型巨噬細(xì)胞刺激后的VICs無論是在堿性磷酸酶活性水平,還是在成骨鈣化相關(guān)基因ALP、BMP2、RUNX2的表達(dá)水平均增加最明顯。而在后續(xù)VICs的茜素紅染色中,更是發(fā)現(xiàn)相比于對照組和其他巨噬細(xì)胞亞型刺激組,M1型巨噬細(xì)胞刺激組陽性染色最明顯。結(jié)論:本次研究結(jié)果提示,EDTA對于鈣化性主動脈瓣的脫鈣效果最好。相比其他亞型巨噬細(xì)胞,M1型巨噬細(xì)胞在鈣化性主動脈瓣中呈高表達(dá),并且能在體外誘導(dǎo)VICs向成骨鈣化方向分化。本研究證實巨噬細(xì)胞極化后的M1型巨噬細(xì)胞在鈣化性主動脈瓣的發(fā)生發(fā)展中起著重要的作用。而關(guān)于鈣化性主動脈瓣中巨噬細(xì)胞如何極化以及極化后通過何種途徑誘導(dǎo)VICs成骨鈣化分化則是未來研究的重點(diǎn)。
[Abstract]:Background and objective: calcified aortic valve disease is one of the most common heart valve diseases in the world. There is no effective drug available to delay the progression of the disease other than surgical treatment. In fact, a large number of studies have shown that aortic valve calcification processes in a similar way to atherosclerosis. These include lipid deposition, inflammatory infiltration, valve angiogenesis and fibrosis remodeling, which ultimately lead to calcification. Inflammatory infiltration is one of the key factors in these processes. Along with the whole process of aortic valve calcification, macrophages play an important role as the main inflammatory cells in valvular inflammatory infiltration. In recent years, it has been found that macrophages are not monomorphic inflammatory cells. Macrophage polarization is thought to play an important role in the development of coronary atherosclerosis. However, the role of macrophage polarization in aortic valve calcification has not been clarified, so this study aims to investigate the expression of macrophage polarization in calcified aortic valve and its effect on the interstitial cells of aortic valve. Methods: to investigate the mechanism of macrophage polarization in aortic valve calcification. Methods: the aortic valve specimens undergoing aortic valve replacement were collected according to certain exclusion criteria. Three kinds of decalcification solution, formic acid and EDTA, were used to decalcify calcified aortic valve after decalcification, and HE and immunohistochemical staining were used to screen suitable decalcification solution for calcified aortic valve. Immunohistochemical method was used to detect the distribution of different subtypes of macrophages in calcified aortic valve after polarization. The major subtypes of macrophages were obtained after differentiation, and then cultured in vitro with different subtypes of macrophages to stimulate them, respectively, at the protein level and gene expression level. In order to compare the osteogenic calcification effect of different subtypes of macrophages on the osteogenic calcification of VICs, the osteogenic calcification of VICs was detected by morphology. Results: compared with the decalcification effect, the decalcification rate of the VICs group was slower than that of the control group. After decalcification of calcified aortic valve by EDTA, it was found that macrophages expressed a lot of macrophages in calcified aortic valve compared with normal aortic valve. In particular, the expression of M1 macrophages increased significantly, and the VICs activity of M1 macrophages stimulated by different subtypes of macrophages was found to be at the level of alkaline phosphatase (ALP) regardless of the activity of alkaline phosphatase (ALP) after stimulation of VICs by different subtypes of macrophages in vitro. The expression level of osteoblastic calcification related gene ALPMP2rUNX2 was also significantly increased, but in the subsequent VICs staining with alizarin red, the expression of RUNX2 was significantly higher than that of BMP2RUNX2. It was also found that the positive staining of M 1 macrophage stimulation group was the most obvious compared with the control group and other macrophage subtype stimulation groups. Conclusion: the results of this study suggest that EDTA has the best decalcification effect on calcified aortic valve. Other subtypes of macrophages, M 1 macrophages, were highly expressed in calcified aortic valve. This study confirmed that M1 type macrophages after macrophage polarization play an important role in the development of calcified aortic valve. How to polarization and how to induce osteoblastic calcification differentiation of VICs after polarization is the focus of future research.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R654.2

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