【作者】 历志；

【导师】 易定华； 韩骅； 魏旭峰；

【作者基本信息】 第四军医大学 ， 外科学， 2010， 硕士

【摘要】 在成体,心脏瓣膜稳态的失衡会引起获得性瓣膜退行性病变。获得性瓣膜退行性病变是第三大心血管疾病,严重威胁的人们的身体健康。然而,关于成体瓣膜的稳态是如何维持,获得性瓣膜退行性病变是如何启动,却少有报道。有研究表明,Notch信号通路在维持瓣膜的稳态中起重要的作用。因此,Notch信号通路可能成为寻找治疗和预防获得性瓣膜病变的新的靶点。但是,Notch信号通路对成体瓣膜稳态的调节机制尚有待阐明。近年来,许多研究小组通过转基因技术建立了小鼠瓣膜疾病模型,从而揭开了对人类瓣膜病变发生的机制进行探索。RBP-JК是Notch信号通路下游的关键的转录调控因子,如果剔除RBP-JК,将导致Notch信号通路阻断。因此,RBP-JК基因条件性剔除小鼠成为寻找瓣膜稳态失衡机制的理想模型。然而,正常小鼠主动脉瓣的组织形态学结构却没有被系统的观察和分析过,导致难以对基因剔除小鼠主动脉瓣病变模型进行正确的研究和评价。本实验首次对正常小鼠主动脉瓣的结构进行了全面的组织形态学观察和分析,同时将其与Notch基因剔除小鼠的瓣膜进行比较,从而探讨Notch信号通路在瓣膜稳态中的调节作用及可能机制,为进一步寻找瓣膜病变的治疗和预防的新靶点提供依据。目的1.观察正常小鼠主动脉瓣膜组织形态学结构及超微结构。2.探讨Notch信号通路在瓣膜稳态维持的作用及可能机制。方法1.采用3月龄C57成年小鼠20只,分离小鼠心脏称重后固定,通过HE染色、免疫荧光、激光共聚焦和Massoon染色后经光镜及电镜观察,分别对主动脉瓣膜的细胞数量、分布、胶原含量及瓣膜超微结构进行组织形态学分析。2.采用成体条件性剔除RBP-JК小鼠,分离小鼠心脏称重后固定,通过HE染色、免疫荧光、激光共聚焦和Massoon染色后经光镜及电镜观察,分别将其主动脉瓣膜的细胞数量、分布、胶原含量及瓣膜超微结构与正常小鼠主动脉瓣膜的组织与形态学进行比较分析。结果1.正常小鼠的主动脉瓣膜呈半透明状的纤维膜,其根部固定于心肌纤维环上,游离缘位于管腔。主动脉瓣的左瓣及无冠瓣与二尖瓣前瓣的根部相连。通过对瓣膜切片的白片采集和编号观察到瓣膜发现瓣膜切片平面的变化呈对称性分布,且中间大于两边。采用3D-doctor软件对瓣膜进行三维重建,同时对瓣膜的局部体积进行比较,发现游离缘>根部>中间部。正常小鼠主动脉瓣的面积为(0.1978±0.003) mm2,周长为(9.18×102±0.06×102)μm,平均细胞数为(9.75×102±0.04×102)个/mm2,其中内皮细胞数为(3.23×102±0.1×102)个/mm2 ,间质细胞数为(5.05×102±0.07×102)个/mm2,胶原含量为(23.77%±8.38)%。激光共聚焦显微镜观察显示,成体内皮下层存在一些共表达CD31及α-SMA的细胞。电镜观察发现,瓣膜的心室面内皮与主动脉面内皮在形态上存在有很大的差异:心室面内皮呈扁平形与血流方向一致,主动脉内皮呈方形与血流方向垂直。间质中成纤维细胞处于静止状态,其细胞胞体较小,呈长梭形,粗面内质网和高尔基复合体均不发达。细胞外基质胶原的含量丰富,主要集中于流出道。2.成体条件敲除RBP-JК缺陷导致瓣膜稳态失衡发生重塑异常。阻断内皮细胞上的RBP-JК,内皮细胞上的VEGFR2表达上调,同时瓣膜内皮细胞发生增殖。瓣膜超微结构显示RBP-JК剔除导致内皮细胞功能紊乱、基底膜紊乱和与瓣膜重塑相关的双征黑色素细胞增多。干扰RBP-JК引起主动脉瓣膜上发生上皮-间质转化,同时瓣膜间质细胞增多,胶原分泌量增多。结论1.首次对了小鼠主动脉瓣组织形态学进行了详细的观察,通过体式显微镜对正常小鼠主动脉瓣膜大体标本观察发现其形态与正常人的瓣膜相似,但其透光度却异于人的瓣膜呈半透明状。通过对瓣膜进行HE染色、免疫荧光、激光共聚焦和Massoon染色后经光镜及电镜观察并进行分析,建立了正常小鼠主动脉瓣组织形态学研究的方法,为今后小鼠主动脉瓣疾病模型的研究提供重要的参考依据。2.首次对瓣膜切片的白片采集和编号,成功的对瓣膜切片平面进行了立体的定位。首次采用3D-doctor对小鼠主动脉瓣膜进行三维重建,使对瓣膜的局部体积进行比较成为可能。3.激光共聚焦显微镜观察显示,成体内皮下层存在一些共表达CD31及α-SMA的细胞,表明成体小鼠主动脉瓣膜仍保留具有上皮-间质转化潜能的细胞。4.首次对Notch基因成体条件性剔除小鼠主动脉瓣膜进行组织形态学进行比较和分析,发现瓣膜内皮细胞上敲除RBP- JК引起瓣膜异常重塑,提示RBP- JК很可能为治疗和预防瓣膜病变的新的靶点。5.瓣膜内皮细胞上RBP-JК缺陷引起VEGFR2表达上调促使内皮细胞增殖,同时导致瓣膜内皮细胞及基底膜紊乱,提示正常情况下,Notch信号通路是通过RBP-JК抑制VEGFR2表达来维持瓣膜内皮细胞的稳态的。6.瓣膜内皮细胞上阻断RBP-JК引起主动脉瓣膜上发生上皮-间质转化,引起瓣膜间质细胞增多,胶原分泌量增多。同时,发现与瓣膜重塑可能相关的双征黑色素细胞在敲除小鼠中增多。更多还原

【Abstract】 In adults , perturbations of heart valve homeostasis will lead to diseased from acquired,‘degenerative valve disease’. Degenerative valvular disease(DAVD) is the third most common cause of cardiovascular disease.It is unclear, however, concerning how the normal valve homeostasis is maintained and how valve disease is initiated in normal adults. So,notch singnal will be the key to the new therapy and prevention of heart valve diseases.But, the mechanism is still not very clear.Recently, many research groups have succeded in the establishment of transgenic mouse model of valve disease, which open a new way for human to explore the mechanisms of valve disease. RBP-JК, the transcription factor downstream of Notch receptors is essential for notch pathways and knockout RBP-JКwill block nocth signaling.Therefore,RBP-JКdeficient mouses will be the ideal modle for the research on perturbations of heart valve homeostasis.Howerer, the normal structure of mouse aortic valve have not been systemistic studied,which make it difficult for gene knockout mouse model of aortic valve disease to do proper research and evaluation .In this study, it is the the first time to do comprhensive assessment on the structure of the aortic valve in normal mice ,while the heart valve of notch gene knockout mice were compared and analyzed with the normal one,to find the function of the notch signaling pathway in the regulation of homeostasis in the valve effect and possible mechanism. It may elucidate novel treatment and prevention strategies of heart valve disease..AIMS1. To observe normal mice aortic valve morphology and ultrastructure.2. to find the function of the notch signaling pathway in the regulation of homeostasis in the valve effect and possible mechanism ..METHODS1. Adult mouse (postnatal 3 months) hearts were harvested and histological and valve morphometric analyses of the valve cells population, distribution and collagen content as well as the valve microstructure were conducted by light microscope (HE stain, immunofluorescence, Laser scanning confocal microscope and Masson’s trichrome stain) and transmission electron microscope.2. Using the Cre-LoxP-mediated conditional gene deletion mouse , hearts were harvested and histological and valve morphometric analyses of the valve cells population, distribution and collagen content as well as the valve microstructure were conducted by light microscope (HE stain, immunofluorescence, Laser scanning confocal microscope and Masson’s trichrome stain) and transmission electron microscope.RESULTS1. Normal aortic valve mice showed semi- transparent fiber membranes, its roots fixed in the cardiac annulus and the free edge in the lumen. The aortic valve is connected with mitral valve. The white slices of aortic valve showed changes in symmetry distribution and the middle part longer than the two ends. By 3D-doctor software, we got the three-dimensional reconstruction of the valve, sothat we can found different among the differernt local volume of the valve and found that free edge> root> the middle. The aortic valve area of normal mice was (0.1978±0.003 mm2), with a perimeter of (9.18×102±0.06×102um), the average cell number (9.75×102±0.04×102/mm2), the endothelial cell number (3.23×102±0.1×102/mm2), the intersititial cell number (5.05×102±0.07×102 /mm2), and the collagen content (21.62%±9.33%). Confocal laser scanning displayed some co-expressed CD31 andα-SMA cells under endothelium.The electron microscope showed some morphological difference between ventricular face of valve and aortic face of valve endothelium. The ventricular face of valve endothelium was flat while the aortic face of valve endothelium was square in shape. Interstitial fibroblasts were in a quiescent state, in which the cell bodies were small and long spindle-shaped, and endoplasmic reticulum and Golgi complexes were undeveloped. Extracellular matrix was collagen-rich, which was mainly concentrated in the outflow tract.2. Maladaptive Valve Remodeling in RBP-J mutation mice.Disruptionof RBP-JКinduces up-regulation of VEGFR2 and VEC proliferation.Mice lacking RBP-JКlead to VEC dysfuction and basement membrane disruption in ultrastructural. Dual-character melanocytes increased in knockout mice.Interrupt of RBP-J cause EMT in aortic valve.Mesenchymal cell proliferation and fibrosis secretion increasing.CONCLUSIONS1. In this study, the mouse aortic valve morphology were first observed in detail.Through the gross microscope ,the gross structure of mice normal aortic was similar with the human normal valve, but more tansparant than the human one. Histological and valve morphometric analyses of the valve cells population, distribution and collagen content as well as the valve microstructure were conducted by light microscope (HE stain, immunofluorescence, Laser scanning confocal microscope and Masson’s trichrome stain) and transmission electron microscope. The results of this study provide some histological and morphometric data for normal mouse aortic valve structure and are useful as reference standards for future studies of mouse models of progressive aorta valve diseases.2. It was first time we used the white slice of aortic valve to locate the three-dimensional positioning of aortic valve .It was first used 3D-doctor on the three-dimensional reconstruction of mouse aortic valve, so that we can compare the local volume of the valve with the others.3. Confocal laser scanning displayed some co-expressed CD31 andα-SMA cells under endothelium, indicating the existence of epidermis-mesenchymal transformation potential cells in adult aorta valves.4. It was first that the aortic morphology of valve in adult conditional knockout Notch gene mice was compared and analyzed.We found that knockout RBP-JКin valve endothelial cells caused abnormal valve remodeling, which suggested that RBP-JКmay be the new drug therapy targets for treatment and prevention of valvular disease. These findings suggest that in adults RBP-J-mediated Notch signaling may play an essential role in the maintenance of valve homeostasis5. Knockout RBP-JКin valve endothelial cells upregulated VEGFR2 and promoted endothelial cell proliferation, which leads to heart valve endothelial cells dysfunction and basement membrane disorder in ultrastructura.So we believed that VEGFR2 is inhibited by RBP-JКto maintain the valve endothelial cell homeostasis.6. Interrupt of RBP-JКcause EMT in aortic valve,which caused mesenchymal cell proliferation and fibrosis secretion increasing.Meanwhile ,knockout RBP-JКlead to dual-character melanocytes increased which maybe fuction in valve remolding更多还原