巨噬细胞极化在钙化性主动脉瓣中的表达及作用研究

发布时间：2018-02-25 02:20

本文关键词： 巨噬细胞极化钙化性主动脉瓣疾病瓣膜间质细胞　出处：《上海交通大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景和目的:钙化性主动脉瓣疾病是全世界范围内发病率最高的心脏瓣膜疾病之一。而随着老龄化社会的来临,这一数字仍将不断增加,造成极大的社会医疗负担。目前对于该病,除了手术治疗,尚无有效的药物可用于延缓疾病的进展。事实上大量的研究提示主动脉瓣钙化的病变过程类似于动脉粥样硬化,包括脂质沉积、炎症浸润、瓣膜血管新生和纤维化重塑,最终导致钙化。而在这些过程中,炎症浸润是其中关键因素之一,伴随主动脉瓣瓣膜钙化的整个过程。巨噬细胞作为瓣膜炎症浸润的主要炎症细胞无疑起着至关重要的作用。近年来发现巨噬细胞并非单一性状的炎症细胞,而是可以通过极化分化为不同亚型。大量研究认为巨噬细胞极化在冠状动脉粥样硬化发生发展过程中起着重要作用,而关于巨噬细胞极化在主动脉瓣钙化中的作用目前尚未明确。因此本研究旨在通过对巨噬细胞极化在钙化主动脉瓣中的表达以及其对瓣膜间质细胞的影响等相关研究,来探讨巨噬细胞极化在主动脉瓣钙化中的作用机制。方法:按一定纳入排除标准收集临床上行主动脉瓣置换手术的主动脉瓣瓣膜标本。首先通过比较硝酸、甲酸以及EDTA三种实验室常用脱钙液对钙化性主动脉瓣脱钙后HE和免疫组化染色效果,筛选出合适的钙化性主动脉瓣瓣膜脱钙液。对收集的钙化性主动脉瓣瓣膜进行脱钙处理后,采用免疫组化的方法来探测巨噬细胞极化后的不同亚型在钙化性主动脉瓣中的分布,得出其中主要的巨噬细胞分化后亚型。然后通过体外培养人主动脉瓣VICs,用不同亚型巨噬细胞来进行相应刺激,分别在蛋白水平、基因表达水平、形态学等方面来检测刺激后VICs的成骨钙化情况,以比较不同亚型巨噬细胞对VICs的成骨钙化作用。结果:在脱钙效果的比较上面,EDTA组尽管脱钙速度较慢,但其脱钙效果最好。通过EDTA对钙化主动脉瓣瓣膜脱钙后进行免疫组化发现:相比较正常主动脉瓣,钙化主动脉瓣中巨噬细胞呈大量表达,其中尤其以M1型巨噬细胞表达增加明显。而通过不同亚型巨噬细胞体外刺激VICs发现M1型巨噬细胞刺激后的VICs无论是在碱性磷酸酶活性水平,还是在成骨钙化相关基因ALP、BMP2、RUNX2的表达水平均增加最明显。而在后续VICs的茜素红染色中,更是发现相比于对照组和其他巨噬细胞亚型刺激组,M1型巨噬细胞刺激组阳性染色最明显。结论:本次研究结果提示,EDTA对于钙化性主动脉瓣的脱钙效果最好。相比其他亚型巨噬细胞,M1型巨噬细胞在钙化性主动脉瓣中呈高表达,并且能在体外诱导VICs向成骨钙化方向分化。本研究证实巨噬细胞极化后的M1型巨噬细胞在钙化性主动脉瓣的发生发展中起着重要的作用。而关于钙化性主动脉瓣中巨噬细胞如何极化以及极化后通过何种途径诱导VICs成骨钙化分化则是未来研究的重点。
[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.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.2

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