【作者】 杨艳萍；

【导师】 景雅；

【作者基本信息】 山西医科大学 ， 生理学， 2009， 博士

【摘要】 胚胎早期单管状心脏流出道连于动脉囊和原始心室之间。随发育,流出道经复杂的分隔和结构重建,参与心包内主、肺动脉与所属动脉瓣膜以及左右心室流出道的发育。有关不同种属动物胚胎心脏流出道发育和分隔机理已有大量研究,但目前尚有许多争议。探讨胚胎早期心脏流出道的正常发育和分隔机理可以为流出道发育和分隔异常导致的先天性心脏病的发病机理提供理论依据。心脏神经嵴是指从耳板中部到第3对体节之间的神经嵴细胞,迁移进入胚胎心脏,参与主肺动脉隔、流出道心内膜垫的形成与流出道的分隔。迁移前去除心脏神经嵴,可表现为永久性动脉干等畸形。因此,有关神经嵴细胞在流出道的分布与功能成为研究焦点之一,但由于种属差异和实验方法不同,对心脏神经嵴在流出道分隔中的作用仍存在分歧。流出道嵴融合形成间充质性流出道隔与其肌性化相伴随,间充质组织肌性化的机制仍存在争议。有学者认为流出道壁心肌细胞不断向隔内生长,同时募集相邻间充质细胞向心肌细胞分化共同完成流出道隔的肌性化。而本课题组观察到流出道嵴及其融合形成的流出道隔内存在间充质细胞的原位分化。因此,流出道心肌性隔的形成机制有待进一步研究。本实验通过观察α-平滑肌肌动蛋白(α-SMA)、α-横纹肌肌节肌动蛋白(α-SCA)、肌球蛋白重链(MHC)在C10期～C19期人胚心脏的表达规律,探讨流出道嵴、主肺动脉隔的形成与动脉囊、心肌性流出道的分隔过程;阐明α-SMA在流出道心肌与心内膜垫内的表达规律及其意义。通过观察胚龄10～14d小鼠胚胎心脏流出道内PlexinA2、α-SMA阳性细胞分布规律与流出道嵴内致密细胞团融合过程中间充质细胞超微结构变化,探讨PlexinA2、α-SMA时空表达特点及其对间充质细胞超微结构的影响。通过观察胚龄12～16d小鼠胚胎心脏流出道心肌性隔的形成过程,探讨流出道心肌性隔的形成机制。第一章人胚早期心脏流出道的分隔对32例C10期～C19期[Carnegie stage 10～Carnegie stage 19, Carnegie分期法,排卵后(22±1～47)d]人胚心脏连续切片,经抗α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、抗α-横纹肌肌节肌动蛋白(α-sarcomeric actin,α-SCA)、抗肌球蛋白重链(myosin heavy chain, MHC)抗体免疫组织化学染色,观察进入流出道嵴的α-SMA阳性细胞的迁移路线、动脉囊的演变及其与心肌性流出道的分隔过程。结果显示:人胚C10期～C13期,动脉囊位于心包腔外,与心肌性流出道的界限位于流出道与心包腔背侧壁反折处;人胚C14期～C15期,第4弓动脉以下的动脉囊逐渐降入心包腔内,动脉囊壁心肌化成为流出道的远端,使心肌性流出道增长;人胚C16期,第4弓动脉水平的动脉囊也降入心包腔内,但未发生心肌化,动脉囊与心肌性流出道界限移至心包腔内。人胚C12期～C15期,α-SMA阳性细胞逐渐在流出道心内膜垫聚集,参与形成螺旋状流出道嵴;C16期,α-SMA阳性主肺动脉隔形成,流出道嵴缩短。人胚C14期,心包腔外的动脉囊分隔。人胚C16期,进入心包内的动脉囊分隔为心包内升主动脉和肺动脉干,心肌性流出道远端可见两动脉瓣膜原基,流出道嵴尚未融合。人胚C19期,瓣膜水平流出道嵴开始融合形成流出道隔,分隔心肌性流出道,来自两侧嵴内α-SMA阳性细胞在隔中央聚集。由此我们认为:流出道嵴的形成早于主肺动脉隔的出现,流出道嵴的远端、近端界限与心肌性流出道的远端、近端界限始终保持一致。动脉囊首先在心包腔外分隔,进入心包腔后由主肺动脉隔分隔形成心包内升主动脉、肺动脉干,流出道嵴远端形成两动脉瓣膜。流出道嵴大部融合分隔瓣膜以下的心肌性流出道。第二章人胚早期心脏流出道内α-SMA阳性细胞的发育用抗α-平滑肌肌动蛋白(α-SMA)、抗α-横纹肌肌节肌动蛋白(α-SCA)、抗肌球蛋白重链(MHC)抗体对29例C10期～C16期[Carnegie stage 10～Carnegie stage 16,排卵后(22±1～37)d]人胚心脏连续切片进行免疫组织化学染色。结果显示:人胚发育C10期～C13期,与流出道反折处的心包腔背侧壁中胚层上皮细胞部分呈α-SMA阳性,而α-SCA和MHC表达较弱。提示心包腔背侧壁上皮不断分化为心肌细胞添加至流出道远端使其增长,这些心肌细胞α-SMA的表达早于α-SCA和MHC。人胚C14期～C15期,进入心包腔的动脉囊壁α-SMA染色为强阳性,而α-SCA和MHC仅在左侧壁表达较强。提示动脉囊壁正在心肌化。人胚C12期～C15期,α-SMA阳性细胞由咽部间充质逐渐迁入流出道心内膜垫内,同时可见流出道心内膜的部分内皮细胞由α-SMA阴性转为阳性,向间充质细胞分化。不同来源的间充质细胞在流出道心内膜垫内聚集,参与形成流出道嵴。随着α-SMA阳性细胞不断进入,流出道嵴体积逐渐增大。人胚C16期,流出道嵴缩短,嵴内大部分间充质细胞α-SMA表达减弱,而在流出道嵴近心肌处出现成行排列的α-SMA强阳性细胞,相邻的心肌细胞伸出突起与其相连。由此我们认为:α-SMA可作为心肌细胞早期分化的标志;流出道嵴表面内皮细胞向间充质细胞分化时表达α-SMA;主肺动脉隔与流出道嵴内的α-SMA阳性细胞参与动脉囊分隔。第三章小鼠胚胎心脏流出道嵴的发育用抗α-平滑肌肌动蛋白(α-SMA)、抗α-横纹肌肌节肌动蛋白(α-SCA)单克隆抗体对胚龄10～14d小鼠胚胎心脏连续切片进行免疫组织化学染色;用原位杂交方法显示PlexinA2阳性细胞向胚龄10～14d小鼠胚胎流出道的迁移与分布;透射电镜观察胚龄12.5d心脏流出道嵴融合过程。结果显示:胚龄10～11d,神经管及其周围、动脉囊和弓动脉壁可见PlexinA2阳性细胞,并沿动脉囊壁迁入流出道嵴内,部分细胞同时表达α-SMA。胚龄12d,PlexinA2阳性细胞分布在脊神经节、咽前间充质、主肺动脉隔以及主、肺动脉壁。主肺动脉隔显α-SMA强阳性,但动脉壁仅见少量α-SMA阳性细胞。胚龄12.5d,两侧流出道嵴内致密间充质细胞团形成,并在瓣膜水平开始融合,PlexinA2表达较弱,α-SMA表达呈强阳性。在流出道嵴融合开始后,嵴表面的内皮细胞带形成继而断裂消失,由含微丝少、排列稀疏的间充质细胞取代。两侧致密细胞团逐渐靠拢、融合。有的间充质细胞内含较多线粒体和微丝,细胞之间形成细胞连接点;有的间充质细胞含微丝少,细胞膜间断融合。还可见内皮细胞或间充质细胞融合形成双核细胞。由上述结果可得出以下结论:流出道心内膜垫内α-SMA阳性间充质细胞来自神经嵴;内皮细胞-间充质细胞转化参与流出道嵴融合;致密细胞团内间充质细胞富含微丝束和细胞连接点或发生细胞膜局部融合,增加了细胞之间连接的牢固性,有助于流出道嵴的融合;PlexinA2表达减弱可能有助于间充质细胞的粘附与聚集。第四章小鼠胚胎心脏流出道间充质隔的肌性化用抗α-平滑肌肌动蛋白(α-SMA)、抗结蛋白(Desmin)、抗α-横纹肌肌节肌动蛋白(α-SCA)单克隆抗体对胚龄12～16d小鼠胚胎心脏连续切片进行染色。用TUNEL染色对胚龄13～15d小鼠胚胎心脏切片进行染色。结果显示:胚龄12d,小鼠胚胎心脏流出道嵴尚未融合,一侧嵴内观察到少量α-SMA、Desmin、α-SCA阳性的心肌细胞,并可见流出道壁心肌细胞开始向嵴内延伸。胚龄12.5～14d,动脉瓣膜及其以下流出道嵴融合形成间充质性流出道隔。流出道壁α-SCA阳性的心肌细胞进一步向间充质隔内长入。隔中央可见α-SMA、α-SCA阳性细胞独立存在。胚龄13～15d,流出道隔中央可见α-SMA阳性细胞聚集形成漩涡状结构,隔内更多间充质细胞由心肌细胞取代,仅在其中央呈α-SCA阴性。胚龄16d流出道心肌性隔形成。胚龄13～15d,间充质隔中央凋亡细胞逐渐增多。因此我们认为:流出道心肌性隔形成的机制包括心肌化、募集与间充质细胞的原位分化。在此过程中,部分α-SMA阳性细胞凋亡;未凋亡的α-SMA阳性细胞可能原位转分化为心肌细胞。更多还原

【Abstract】 The single tubular outflow tract of early embryonic heart connects primary ventricle with the aortic sac, which wall is composed of primary myocardium and endothelium with cardiac jelly in between. With development, the outflow tract undergoes complex septation and remodeling to form intrapericardial ascending aorta and pulmonary trunk and their valves and the outlets of the two ventricles. Many debates remain to be elucidated though there have been plenty of studies about the outflow tract development and its septation mechanism of embryonic heart of different species. The investigation about these questions could provide theory foundation for the congenital heart disease pathogenesis resulting from the abnormality of the outflow tract development and septation.The neural crest cells between the mid-otic placode and the third somite are called as cardiac neural crest, which can migrate into embryonic heart and play important role in the formation of the aorto-pulmonary septum and endocardial cushion and the outflow tract septation. Ablating the cardiac neural crest prior to its migration could lead to cardiac deficiency such as persistent truncus arteriosus. Thus the studies about cardiac neural crest distribution in embryonic heart and its function become one of the focuses. Because of the differences in the species and experimental methods, the debates still exist on the cardiac neural crest function during the septation of the outflow tract.The fusion of the outflow tract ridges to form mesenchymal septum is accompanied with its musculization. But the musculization mechanism remains to be elucidated. Some scholars reported that cardiomyocytes of the outflow tract wall progressively extended into the septum and recruited neighboring mesenchymal cells differentiating towards cardiomyocytes to complete the myocardiac septum formation. But at the same time, we also observed in situ differentiation of the mesenchymal cells in the outflow tract ridges and the septum except myocardialization and recruitment. So the formation mechanism of myocardiac septum is open to be explored.In this study, we observed the expression patterns ofα-SMA (α-smooth muscle actin),α-SCA (α-sarcomeric actin) and MHC (myosin heavy chain) in embryonic human heart from Carnegie stage 10 to Carnegie stage 19 to explore the formation of the outflow tract ridge and the aorto-pulmonary septum and the septation of the aortic sac and the outflow tract, and also to elucidateα-SMA expression significance in the myocardium and endocardial cushion of the outflow tract. We observed the distribution of PlexinA2 positive cells andα-SMA positive cells in the outflow tract of embryonic mouse heart from ED(embryonic day)10 to ED14 and the celluar ultrastructure change of the condensed mesenchymal cell masses during their fusion to explore the spacio-temporal exression patterns of PlexinA2 andα-SMA and their influence on the mesenchymal cell ultrastructure. We observed the myocardial septum formation of the outflow tract in ED12 to ED16 embryonic mouse heart to investigate the formation mechanism of the myocardial septum.Chapter I The septation of the outflow tract in the early embryonic human heartSerial sections of thirty-two human embryonic hearts from Carnegie stage 10 to Carnegie stage 19 (C10～C19, 22±1～47 postovulatory day) were stained immunohistochemically with antibodies againstα-SMA (α-smooth muscle actin),α-SCA (α-sarcomeric actin) and MHC (myosin heavy chain) to observe the migrating route of theα-SMA positive cells to the outflow tract ridge, the development of the aortic sac and the septation mechanism of the aortic sac and the outflow tract. The results showed that from C10 to C13, the aortic sac was situated outside of the pericardial cavity and the demarcation between the aortic sac and the outflow tract was located at the reflection of the dorsal wall of pericardial cavity to the outflow tract. Between C14 and C15, the proximal part of the aortic sac began to progressively protrude into pericardial cavity and its wall gradully myocardialized to become the distal pole of the myocardial outflow tract leading to lengthening of the latter. At C16, the aortic sac at the fourth aortic arch level invaginated into the pericardial cavity, but its wall was not myocardialized. So the demarcation between the aortic sac and the myocardial outflow tract became to be located in the pericardial cavity. From C12 to C15,α-SMA positive cells aggregated in the endocardial cushion of the outflow tract and took part in forming the two opposite spiral ridges. At C16,α-SMA positive aorto-pulmonary septum could be observed and the outflow tract ridge shortened. The aortic sac was septated before protruding into the pericardial cavity at C14. In the pericardial cavity, although the outflow tract ridges were not fused, the aortic sac had been divided into the ascending aorta and pulmonary trunk by the aorto-pulmonary septum at C16. The valve anlagen could be seen at the distal pole of the myocardial outflow tract. At C19, the outflow tract ridges began to fuse with each other at the valve level to form the outlet septum and septate the outflow tract. Theα-SMA positive cells from the two ridges aggregated in the centre of the septum. The results suggest that the formation of the outflow tract ridge is earlier than the aorto-pulmonary septum. The distal and proximal ends of the ridge keep at the same level with the two poles of the myocardial outflow tract. The aortic sac has been septated when it is embeded in the pharyngeal mesenchyme. After descending into the pericardial cavity, the aortic sac is septated into the ascending aorta and pulmonary trunk by the aorto-pulmonary septum. The distal poles of the outflow tract ridges form the valve anlagen of the two arteries. The main part of the ridges fuses to septate the outflow tract under the valves.Chapter II The development of theα-SMA positive cells in the outflow tract of the early embryonic human heartSerial sections of twenty-nine human embryonic hearts from Carnegie stage 10 to Carnegie stage 16 (C10～C16, 22±1～37 postovulatory day) were stained immunohistochemically with antibodies againstα-SMA (α-smooth muscle actin),α-SCA (α-sarcomeric actin) and MHC (myosin heavy chain). It was observed that during C10 to C13, a few of the pericardial splanchnic epithelium cells showedα-SMA positive and expressedα-SCA, MHC weakly, which meant that the pericardial splanchnic epithelium progressively differentiated into myocardial cells and these new cardiomyocytes were added to the distal pole of the outflow tract to make it lengthen. The expression ofα-SMA of these cells was earlier than the expression ofα-SCA and MHC. At C14 and C15, the aortic sac wall was stainedα-SMA strongly when protruding into the pericardial cavity. But the expression ofα-SCA and MHC was strong only on the left wall, which suggested that the aortic sac was myocardializing. From C12 to C15,α-SMA positive cells gradually migrated into the endocardial cushion from the pharyngeal mesenchyme. At the same time, the endocardial cells of the outflow tract began to expressα-SMA and differentiate towards mesenchymal cells. Mesenchymal cells of different origins aggregated in the endocardial cushion to form the outflow tract ridge. Withα-SMA positive cells progressively migrating into the ridge, the ridge became larger. At C16, the outflow tract was shortened and most of the mesenchymal cells in the ridge expressedα-SMA weakly. But a few of positive cells expressedα-SMA strongly aligning with the myocardial cells of the outflow tract wall. The cardiomyocytes linked with theseα-SMA positive cells by their protrusion. We conclude thatα-SMA could be regarded as an early marker of cardiomyocytes differentiation. The endocardial cells of the outflow tract ridge expressα-SMA when differentiating towards mesenchymal cells.α-SMA positive cells in the ridge and the aorto-pulmonary septum take part in the septation of the aortic sac. Chapter III The development of the outflow tract ridge in the embryonic mouse heartSerial sections of ED(embryonic day)10 to ED14 mouse embryonic hearts were stained by monoclonal antibodies againstα-SMA (α-smooth muscle actin) andα-SCA (α-sarcomeric actin). In situ hybridization was performed on the sections of ED10 to ED14 embryos to visualize the migration and distribution pattern of PlexinA2 positive cells in the outflow tract. The outflow tract ridges fusion was observed by transmission electron microscope at ED12.5. The results showed that from ED10 to ED11, PlexinA2 positive cells were seen in the neural tube, the mesenchyme around it, the aortic sac and aortic arch. These cells also migrated into the outflow tract ridge along the aortic sac wall. Some of them expressedα-SMA. At ED12, PlexinA2 was expressed in the dorsal ganglia, the pharyngeal mesenchyme, the aorto-pulmonary septum and the ascending aorta and pulmonary trunk. The septum was shownα-SMA strongly positive. But only a few ofα-SMA positive cells were observed in the ascending aorta and pulmonary trunk. At ED12.5, two clusters of condensed mesenchymal cells in the outflow tract ridges formed and began to merge at the semilunar level expressing PlexinA2 weakly andα-SMA strongly. When the ridges began to fuse, the endothelial cells formed a cellular seam, which rapidly broke into pieces and disappeared and were replaced by the sparsed mesenchymal cells containing a few of microfilaments. Two clusters of condensed mesenchymal cells gradully moved to merge. It was noted that some mesenchymal cells contained plenty of microfilament bundles, mitochondria and unmatured focal contacts between them. Some mesenchymal cells only had a few of microfilaments and plasma membrane fusion was seen between them. Cell fusion of endothelial cells or mesenchymal cells was also observed to form double nuclus cells. From the results we conclude thatα-SMA positive cells in the outflow tract cushion may be derived from cardiac neural crest. The endothelial cells participate in the fusion of the outflow tract ridges by endothelial-mesenchymal transformation. Mesenchymal cells of the condensed cell mass contain plenty of microfilament bundles and focal contacts or membrane fusion, which increase connection firmness between cells and contribute to the ridges fusion. PlexinA2 down-regulation may facilitate the mesenchymal cell adhesion and aggregation.Chapter IV Musculization of the mensenchymal septum of the outflow tract in embryonic mouse heartSerial sections of mouse embryonic hearts from ED(embryonic day)12 to ED16 were stained by monoclonal antibodies againstα-SMA (α-smooth muscle actin), Desmin andα-SCA (α-sarcomeric actin) to investigate the mechanism of the myocardial septum formation in the outflow tract of embryonic mouse heart. Apoptosis was determined by TUNEL assay. We found that at ED12, the outflow tract ridges were not fused yet and a few ofα-SCA positive cells had been observed in one of the ridges. The cardiomyocytes of the outflow tract wall began to extend into the ridge. From ED12.5 to ED14, the outflow tract ridges fused to form the mensenchymal septum at and under the semilunar valve level.α-SCA positive cardiomyocytes could be observed progressively migrating from the wall of the outflow tract into the mesenchymal septum. It needed to be stressed that a few ofα-SMA andα-SCA positive cardiomyocytes independently existed in the centre of the septum. During ED13 to ED15,α-SMA positive cells aggregated to form the whorl in the centre of the septum. More mesenchymal cells in the septum were replaced by cardiomyocytes. Soα-SCA negative area only lied in the centre of the septum. At ED16, the septum was filled with cardiomyocytes to form the myocardial septum. From ED13 to ED15, the apoptosed cells in the centre of the septum gradually increased. The conclusions could be drawed that myocardialization is not the single mechanism to explain the transforming of the mesenchymal septum to the myocardial septum. Transdifferentiation and recruitment of the mesenchymal cells in the septum may also be reasons for it. The formation of the myocardial septum is companied by apoptosis of part of theα-SMA positive cells in the septum. Theα-SMA positive cells without being apoptosed may transdifferentiate into cardiomyocytes and take part in the formation of the myocardial septum.更多还原