【作者】 李海荣；

【导师】 景雅；

【作者基本信息】 山西医科大学 ， 生理学， 2008， 博士

【摘要】 胚胎心脏发育过程中,流出道经复杂的分隔和结构重建,参与心包内升主动脉和肺动脉干、左右心室流出道及动脉瓣膜结构的发育。流出道发育和分隔异常将导致永久性动脉干,右心室双流出道,室间隔缺损,主动脉和肺动脉易位,法洛氏四联症等多种先天性心脏病。由于转基因或基因敲除后对胚胎发育影响的复杂性和不同动物的种属差异,对心脏流出道的正常发育机理尚有许多争议。受精后前8周是人胚心脏流出道发育、分隔和重建的重要时期。对人胚心脏流出道发育和分隔机理的探讨大多局限于较大胚龄的胚胎,缺乏对早期人胚心脏流出道正常发育和分隔机理的系统研究。探讨胚胎早期心脏流出道的正常发育和分隔机理不但是重要的基础理论研究,也可为探讨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,对此类先天性心脏病的预防和治疗有重要意义。有研究认为静脉窦心肌来自后生心区的间充质,受独特的基因调控,也有著者否认哺乳类动物心脏发育过程中静脉窦结构的存在。以往对人胚心脏发育的研究多集中在C14期(Carnegie stage 14)后较大胎龄的胚胎,此时静脉窦已经并入右心房,因此这些研究并没有观察到静脉窦的早期发育。结蛋白(DES)基因突变导致结蛋白性心肌病,常伴有不同程度的心脏传导障碍和心律失常,严重时引起猝死,其原因不明。本实验第一章通过观察α-平滑肌肌动蛋白(α-SMA)、α-横纹肌肌动蛋白(α-SCA)、肌球蛋白重链(MHC)和Caspase-3(CAS-3)在C10期～C16期(排卵后22±1～37天)人胚心脏、咽中胚层的时空表达型及各期心脏和咽中胚层HE染色的形态学特征,探讨了人胚心脏流出道增长的机制、心内膜垫的发育及动脉囊的分隔。本实验第二章通过观察α-SMA、α-SCA和DES在C10～C16期人胚心脏的时空表达型,探讨了人胚心脏静脉窦的发生、分化及传导系统的发育。第三章通过观察α-SMA、α-SCA、MHC、GATA-4、CAS-3、增殖细胞核抗原(PCNA)在胚龄9～12天小鼠胚胎心脏的时空表达型探讨了小鼠胚胎心脏流出道的缩短机制。第一章人胚早期心脏流出道的发育取29例C10期～C16期(Carnegie stage 10～Carnegie stage 16,排卵后22±1～37天)人胚心脏连续切片,用抗α-平滑肌肌动蛋白(α-SMA)、抗α-横纹肌肌动蛋白(α-SCA)、抗肌球蛋白重链(MHC)和抗活性Caspase-3(CAS-3)抗体进行免疫组织化学染色,并用HE染色观察了各期心脏及咽中胚层的形态学特征,以探讨心包腔背侧脏壁中胚层上皮、咽前间充质及动脉囊与心肌性流出道发生的关系。结果显示:人胚发育C10期～C15期,由于流出道、动脉囊由颈部向胸部移位以及心包腔向胚胎背侧的逐渐扩展,使位于原始咽前方间充质中的动脉囊逐渐突向心包腔内,动脉囊表面的心包腔背侧脏壁中胚层上皮不断分化为α-SMA、α-SCA和MHC阳性的流出道心肌细胞。心包腔脏壁中胚层上皮向心肌细胞的分化在C16期人胚终止。人胚C10晚期～C12期,流出道远端的脏层心包和心包腔的背侧壁为复层上皮,向壁层心包内延伸逐渐变为单层扁平上皮。背侧心包于C13期～C15期变为单层上皮。人胚C10晚期-C13期,流出道远端α-SMA和α-SCA表达可延伸至脏层心包及咽部间充质细胞,流出道逐渐延长。人胚C14期～C15期,流出道明显延长,其远端的脏层心包细胞增生并迁至流出道,在近流出道心肌细胞处表达α-SMA和α-SCA。迁移至流出道动脉端前后壁的间充质在C15期人胚发生凋亡,可见流出道心肌细胞迁入间充质细胞团内,取代凋亡的间充质细胞。α-SMA阳性神经嵴细胞在C12期人胚心脏流出道心内膜垫中开始出现,随发育逐渐增多并在局部聚集形成两条纵形的螺旋状走形的嵴。人胚C15期～C16期,动脉囊后壁的α-SMA阳性神经嵴细胞向动脉囊内增生,形成主肺动脉隔,将动脉囊分隔为心包内升主动脉及肺动脉干。因此我们认为:心包腔背侧脏壁中胚层是人胚心脏第二生心区,可不断分化为心肌细胞,使胚胎心肌性流出道长度增加。细胞凋亡染色提示并非所有迁入流出道的咽前间充质细胞都可分化为心肌细胞。流出道远端心肌可诱导第二生心区或称前心区细胞增殖分化为心肌细胞。α-SMA阳性神经嵴细胞出现在流出道心内膜垫和主肺动脉隔的时间不同,提示神经嵴细胞经不同路线迁移至流出道嵴和主肺动脉隔。第二章人胚心脏静脉窦和传导系统的早期发育用抗α-平滑肌肌动蛋白(α-SMA)、抗α-横纹肌肌动蛋白(α-SCA)和抗结蛋白(DES)抗体对29例C10期～C16期(Carnegie stage 10～Carnegie stage 16,排卵后22±1～37天)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C12期～C13期,系统静脉汇集形成的静脉窦出现于心包腔尾端原始横膈间充质中,静脉窦壁间充质细胞逐渐分化为α-SCA阳性的静脉窦心肌细胞。人胚C14期,心包腔的扩张使静脉窦进入心包腔内,参与了右心房的形成。DES阳性传导系心肌的分化始于C10期人胚心脏房室管右侧壁,随发育逐渐向室间沟心肌扩展,发育为房室传导系统的希氏束、左右束支及心室腔面的小梁心肌。在心房,DES表达首先出现于C11期人胚心房背侧壁,在C13期人胚,可见静脉窦左背侧壁α-SCA、α-SMA、DES阳性心肌带与左心房底部、房室管背侧壁相延续,可能这条心肌带参与了人胚心脏静脉窦至房室管传导系统的发育。人胚发育C14～C16期,DES强阳性染色从窦房结经左、右静脉瓣及心房的背、腹侧壁延伸至房室管右侧壁,可能是原始的心房传导通路。因此我们认为,心包腔尾端原始横膈间充质是人胚静脉窦心肌发生区,原始横膈间充质细胞逐渐分化为心肌细胞,添加到人胚心管静脉端,形成心脏静脉窦心肌。人胚心脏传导系统心肌的分化始于房室管,随心管发育逐渐向动、静脉端扩展,在人胚发育C16期,已分化为形态学清晰可辨的DES阳性胚胎心脏传导系统。第三章小鼠胚胎心脏流出道早期并入右心室用抗α-横纹肌肌节肌动蛋白(α-SCA)、抗肌球蛋白重链(MHC)抗体标记心肌,用抗GATA-4抗体标记心肌及其前体细胞,用抗α-平滑肌肌动蛋白(α-SMA)抗体标记早期心肌细胞,抗增殖细胞核抗原(PCNA)抗体显示增殖细胞,用抗人/鼠活性Caspase-3(CAS-3)抗体检测凋亡早期细胞,对胚龄9～12天(胚龄12天分别取8时、12时、16时、20时胚胎)小鼠胚胎心脏连续切片进行了免疫组织化学PAP法染色。结果显示:①小鼠胚胎心脏流出道于胚龄12天8时～20时明显缩短。②在流出道缩短前及缩短过程中流出道全长均未检测到CAS-3阳性细胞。③胚龄11天至胚龄12天8时,动脉囊陷入心包腔,GATA-4、α-SCA、α-SMA、MHC染色示流出道远端心肌界向心室方向退缩至心包腔内,GATA-4染色示流出道远端心肌未转分化为大血管壁的细胞成分,可见第二生心区来源的间充质细胞不断分化为心肌细胞添加在心脏的动脉端使流出道延长。④胚龄11～12天,α-SMA阳性心神经嵴细胞逐渐从流出道远端迁入,GATA-4、α-SCA、α-SMA、MHC、PCNA染色表明流出道近段心肌不断增生形成小梁侵入邻近的流出道嵴内,使流出道嵴心肌化而并入右心室;胚龄12天,在近流出道心肌处,可见与流出道心肌相延续的α-SCA、α-SMA弱阳性间充质细胞流,高倍光镜下可见其细胞突起向心肌细胞延伸或靠近甚至接触;胚龄12天流出道嵴内可见散在的α-SCA、α-SMA、GATA-4阳性细胞,表明流出道嵴内的间充质细胞在不断转分化为心肌细胞。因此我们认为流出道嵴愈合形成间充质性流出道隔前,其近段即在通过心肌化、间充质细胞转分化为心肌细胞不断肌化,使流出道由近向远并入了右心室,心肌性流出道逐渐缩短,流出道远端界逐渐向心室方向退缩。流出道近段嵴内间充质细胞向心肌细胞的转分化受心肌细胞直接接触及释放的因子的调节。心肌细胞凋亡及流出道远端心肌转分化为大血管壁的细胞不是流出道缩短的机制。更多还原

【Abstract】 After complex septation and remodeling,the outflow tract of embryonic heart developed intrapericardial asending aorta and pulmonary trunk,outlets of ventricles and semilunar valves. Abnormality in outflow tract development and septation resulted in congenital heart defects, such as persistent truncus arteriosus,ventricular septal defects,double outlets of right ventricle, transition of great arteries and tetralogy of Fallot,etc.Due to effects of transgene and gene knock-out on embryonic development and differences in species,there were many debates on mechanism underling the outflow tract development.The outflow tract of human embryonic heart completed septation and remodeling before the eighth week.The investigation of the outflow tract development of human embryonic heart was mainly converged on the older embryos,lack of studies of the very younger embryos.Exploration of the early development of outflow tract was not only important to theoretical research,but also in favor of unraveling the mechanism of congenital heart defects resulting from disturbance of outflow tract development and septation.It was reported that myocardium of sinus venosus derived from mesenchyme in the posterior heart-forming field,controlled by distinct gene program.It was even argued that sinus venosus had never been appeared in mammals.There was no date about the early development of sinus venosus of human embryonic heart since studies were almost manipulated on old embryos after C14(Carnegie stage 14),when the sinus venosus had incorporated into right atrium.Mutation in DES gene caused DES myopathy,often accompanied with conduction blocks,arrhythmias and sudden death.The cause of DES myopathy was still unclear so far.In chapter I,we observed the spacio-temporal expression patterns ofα-SMA(α-smooth muscle actin),α-SCA(α-sarcomeric actin),myosin heavy chain(MHC) and active Caspase-3 (CAS-3) in cardiac outflow tract and pharyngeal mesoderm of human embryos from C10 to C16 (22±1～37 postovulatory day) and morphologic charicteristic of heart and pharyngeal mesoderm at each stage with HE staining to explore the mechanism underling the elongation of outflow tract,the development of the endocardial cushions and the division of the aortic sac.In chapterⅡ, the spacio-temporal expression patterns ofα-SMA,α-SCA and desmin(DES) in human embryonic heart through C10 to C16 were observed to investigate the early development of sinus venosus and cardiac conduction system.In chapterⅢ,the spacio-temporal expression patterns ofα-SMA,α-SCA,GATA-4,MHC,CAS-3 and PCNA(proliferating cell nuclear antigen) in mouse embryonic heart from E9(embryonic day 9) to E12 were detected to explore the shortening mechanism of outflow tract during its remodeling.ChapterⅠThe development of the outflow tract in the early human embryonic heartSerial sections of twenty-nine human embryonic hearts from Carnegie stage 10 to Carnegie stage 16(C10～C16) were stained immunohistochemically with antibodies againstα-SMA (α-smooth muscle actin),α-SCA(α-sarcomeric actin),MHC(myosin heavy chain) and CAS-3 (active Caspase-3) to investigate the relationship of splanchnic epithelium lining the dorsal wall of the pericardial cavity,the prepharyngeal mesenchyme and the aortic sac with the embryogenesis of the outflow tract myocardium.HE staining was carried out to observe morphologic charicteristic of heart and pharyngeal mesoderm at each stage.We found that with the caudal translocation of the aortic sac and outflow tract relative to the pharyngeal arches during C10 to C15 and the dorsal expansion of the pericardial cavity on both lateral sides of the outflow tract,the aortic sac originally embedded in the prepharyngeal mesenchyme gradually protruded into the pericardial cavity.The progressive differentiation of the pericardial splanchnic epithelium covering the mesenchymal wall of the aortic sac intoα-SCA and MHC positive cardiomyocytes resulted in the elongation of the myocardial outflow tract.The ability of the dorsal pericardial splanchnic epithelium to differentiate into cardiomyocyte was terminated at C16.From C10 to C12,splanchnic pericardium distal to the outflow tract and dorsal pericardium were stratified epithelium,when extending into parietal mesoderm pericardium gradually transformed into simple squamous epithelium.During the following three stages, dorsal pericardium gradually changed into simple epithelium.During C10 to C13,expression ofα-SMA andα-SCA reached splanchnic pericardium and pharyngeal mesenchymal cells distal to the outflow tract,which caused the progressively elongation of the outflow tract.From C14 to C15,the outflow tract gradually elongated.Over these stages,splanchnic pericardial cells distal to the outflow tract proliferated and progressively migrated into the outflow tract, expresssingα-SMA andα-SCA when closed to cardiomyocytes.The prepharyngeal mesenchyme migrated to the dorsal and ventral walls of the arterial pole of the outflow tract was seen being apoptosed at C15,the outflow tract cardiomyocytes were detected to proliferate, migrate into and replace the apoptosised outflow tract mesenchymal masses.α-SMA positive neural crest cells began to appear in the endocardium of the outflow tract at C12 and gradually aggregated to form two opposite spiral ridges during the following stages.During C15 and C16, α-SMA positive neural crest cells in the posterior wall of the aortic sac proliferated and grew into the aortic sac to form the aorto-pulmonary septum that divided the aortic sac into the intrapericardial ascending aorta and pulmonary trunk.We suggest that the splanchnic mesodermal epithelium of the pericardial cavity is the secondary heart field of the human embryonic heart,the continuous differentiation of which into cardiomyocytes brings about the increase in the length of the myocardial outflow tract.CAS-3 positive staining suggests that not all of the mesenchymal cells migrated to the arterial pole of the outflow tract can differentiate into cardiomyocytes.Myocardium of distal outflow tract can induce cells derived from the secondary heart field(or the anterior heart field) to differentiate into cardiomyocytes. Appearance ofα-SMA positive neural crest cells in the endocardium of the outflow tract and aorto-pulmonary septum at different developmental stages indicates that migration of the neural crest cells towards the outflow tract ridges and the aorto-pulmonary septum is along the different routes.ChapterⅡThe early development of the sinus venosus and the cardiac conduction system in human embryonic heartSerial transverse sections of 29 human embryonic hearts from Carnegie stage 10 to Carnegie stage 16(C10-C16) were stained immunohistochemically with antibodies againstα-SMA (α-smooth muscle actin),α-SCA(α-sarcomeric actin) and DES(desmin).We found that during C12 and C13,the sinus venosus formed by confluence of systematic veins at the caudal end of the pericardial cavity could be recognized in the mesenchyme of primitive transverse septum. The mesenchymal cells of the sinus venosus gradually differentiated intoα-SCA positive cardiocyocytes.At C14,the sinus venosus was within the pericardial cavity due to expansion of the pericardial cavity and incorporated into the right atrium.Differentiation of DES positive conductive cardiomyocyte was initiated in the right wall of atrio-ventricular canal of C10 embryonic heart and with the development,extended towards the myocardium of the interventricular sulcus to form His bundle,left and right bundle branches as well as the ventricular trabecular myocardium.In the atium,the strong expression of DES was first detected in the dorsal wall of C11 atrium.At C13,unique myocardial band showingα-SCA,α-SMA and DES expression in the left dorsal wall of the sinus venosus were found to be continuous with the basal wall of left atium and the dorsal wall of the atrio-ventricular canal,this band might be related to the development of conduction system from sinoatrial node to atrio-ventricular canal. During C14 to C16,primary conduction pathway of atria with strong DES expression was formed that extended from sinoatrial node along venous valves,DES positive myocardium in the dorsal and ventral walls of the atria to the right atrio-ventricular canal,respectively.We suggest that the mesenchyme of the primitive transverse septum is heart forming field of human embryos responsible for formation of sinus venosus myocardium,and cardiomyocytes differentiated from mesenchymal cells in the primitive transverse septum progressively add to the venous pole of the heart tube to form myocardial sinus venosus.The differentiation of CCS of the early human embryo initiates in the atrio-ventricular canal and develops gradually towards the arterial and venous poles of the heart tube.By C16,DES positive embryonic CCS can be clearly recognized morphologically.ChapterⅢCardiac outflow tract in the early development incorporating into right ventricle in mouse embryoSections of embryonic mouse heart from E9(embryonic day9)-E12(harvested respectively at eight,twelve,sixteen,twenty o’clock at E12) were stained with mouse monoclonal antibody respectively againstα-SCA(α-sarcomeric actin),α-SMA(α-smooth muscle actin),GATA-4, MHC(myosin heavy chain),PCNA(proliferating cell nuclear antigen) and rabbit polyclonal antibody against active CAS-3(Caspase-3).We found that the length of outflow tract from embryonic mouse heart became shortened during eight to twenty o’clock at E12.Before and during its shortening,no CAS-3 positive cell was detected in the whole outflow tract.Through E11 to 8 o’clock at E12,with the translocation of the aortic sac into the pericardial cavity,the expression ofα-SCA,α-SMA,GATA-4 and MHC at the distal part of the outflow tract retracted in the pericardial cavity towards the ventricle.GATA-4 staining suggested at the distal border of the outflow tract no cardiomyocytes transdifferentiated into cell components of the great arteries, and mesenchymal cells derived from the secondary heart field continued differentiating into cardiomyocytes to add to the arterial pole of the heart,which still resulted in the elongation of the outflow tract.Through E11 to E12,α-SMA positive cardiac neural crest-derived cells migrated into the distal outflow tract,α-SCA,α-SMA,GATA-4,MHC and PCNA expression showed that in the proximal outflow tract,cardiomyocytes proliferated into trabecula invading into adjacent ridges,resulting in the myocardilization of it and the outflow tract incorporating into the right ventricle.At E12,α-SCA andα-SMA weak positive mesenchymal cell confluents continuous with the myocardium of the outflow tract were detected.With higher magnification, mesenchymal cell processes were observed to extend towards myocardiocytes,some of which even contacted with myocardiocyte.Independent cells withα-SCA,α-SMA,GATA-4 coexisting were observed in the outflow tract ridges during E12.These results suggested that mesenchymal cells in the outflow tract ridges transdifferentiated into cardiomyocytes.We suggest that just before the fusion of outflow tract ridges into the outflow tract septum,their proximity musculizes through myocardilization and mesenchymal cell transdifferention into cardiomyocytes,with the result of the outflow tract being absorbed into the right ventricle,the shortening of the outflow tract and retraction of the distal end of the outflow tract toward the ventricle.In the proximal outflow tract ridges,mesenchymal cell transdifferention into cardiomyocytes is induced by contact with cardiomyocytes and substance released from cardiomyocytes.Neither apoptosis of cardiomyocytes nor transdifferention of cardiomyocytes into cell components of the great arteries at the distal border of the outflow tract is the mechanism underlying the outflow tract shortening during remodeling.更多还原