【作者】 陈康；

【导师】 沈卫峰；

【作者基本信息】 上海交通大学 ， 内科学（心血管专业）， 2013， 博士

【摘要】 【背景与目的】在缺血性心脏疾病中,细胞移植治疗具有广阔的前景,而具有多项分化潜能的胚胎干细胞作为一种具有治疗作用的组织,拥有巨大的临床应用潜力。研究表明,胚胎干细胞来源的内皮细胞对缺血性心脏疾病可能有治疗作用。目前对多能干细胞向心肌细胞分化的细胞分子和微环境调节机制,仍不完全明确。内皮细胞和心肌细胞都来源于相同的生心中胚层前体细胞。但迄今为止,没有直接证据表明,两者在胚胎干细胞分化过程中有着功能性的联系。研究发现,EphB4基因敲除胚胎干细胞向心肌细胞分化能力明显减弱,因而推论心肌细胞分化过程可能与分别表达在静脉、动脉内皮上的EphB4及其配体ephrin B2有关。本研究以胚胎干细胞向心肌细胞定向分化体系为研究基础,利用EphB4基因敲除胚胎干细胞或者使用外源性物质抑制EphB4信号通路或内皮细胞的方法,研究内皮细胞功能变化对胚胎干细胞向心肌细胞分化的影响及EphB4/ephrin B2信号通路在其中所发挥的作用,揭示内皮细胞和心肌细胞在胚胎干细胞分化过程中存在的功能性联系,为缺血性心脏疾病的治疗中基于内皮细胞的移植替代疗法提供明确理论基础。【研究方法】1、采用针对心肌细胞定向分化的常规悬滴培养分化形成拟胚体的方法,利用EphB4基因敲除的胚胎干细胞,观察当EphB4基因缺失,或者在普通胚胎干细胞向心肌细胞定向分化体系中应用抑制EphB4基因功能的多肽,观察上述两种情况下胚胎干细胞向心肌细胞定向分化的变化。同时,使用维生素C观察外源性促进定向分化的物质对于该反应体系的影响。2、采用CGR8-GFP小鼠胚胎干细胞系作为普通胚胎干细胞进行观察,采用共聚焦显微镜分析其定向分化过程中EphB4基因的表达情况。如果EphB4在该反应体系中直接表达在心肌细胞上,则说明该基因直接调控心肌细胞。如果与成体一样,表达在内皮细胞上,则在后续试验分析可能的机制。3、采用添加外源性内皮细胞以及使用血管生成抑制剂,特异性抑制内源性内皮细胞的方法,观察当内皮细胞功能发生变化,心肌细胞定向分化的情况变化。4、在EphB4基因敲除的胚胎干细胞向心肌细胞定向分化体系中,添加表达EphB4基因的内皮细胞,观察其是否能挽救心肌细胞定向分化明显减少的情况。采用条件培养液以及跨膜培养方法,明确内皮细胞以及EphB4信号通路调节心肌细胞定向分化的机制。【实验结果】1、使用针对心肌细胞定向分化的常规悬滴培养分化形成拟胚体的方法,EphB4基因敲除的胚胎干细胞系(EphB4-/-ESCs)向心肌细胞定向分化能力明显障碍。采用外源性多肽限制EphB4基因功能同样使得普通胚胎干细胞向心肌细胞分化减少。表现为具有自主收缩功能的心肌细胞数量明显减少,心肌细胞代表性基因,包括心肌特异性α-重链肌球蛋白(α-MHC)和心肌特异性轻链肌球蛋白-2V(MLC-2V)表达明显降低。提示EphB4基因可能参与心肌细胞的定向分化过程。而添加维生素C促进EphB4-/-胚胎干细胞向心肌细胞的定向分化,说明EphB4基因和维生素C在心肌细胞的定向分化中可能有共同的下游信号通路。2、在上述胚胎干细胞向心肌细胞定向分化体系中,共聚焦显微镜结果提示在绿色区域(代表心肌细胞)富集区域,红色区域也非常集中(代表EphB4阳性细胞)。同时,红色区域与CD31染色的蓝色阳性区域(代表内皮细胞)重叠,免疫组化染色提示为紫色,说明EphB4阳性细胞为内皮细胞。因此EphB4基因是表达在具有自主收缩能力心肌细胞下部的内皮细胞中,而不是心肌细胞内。内皮细胞可能借助EphB4信号通路介导心肌细胞的定向分化。3、在胚胎干细胞向心肌细胞定向分化体系中,当胚胎干细胞与人类内皮细胞共培养时,具有自主收缩能力的心肌细胞定向分化明显增多,且心肌细胞特异性基因表达明显上调,表明外源性内皮细胞能够促进胚胎干细胞向心肌细胞的定向分化。4、采用针对内皮细胞的特异性血管生成抑制剂---Angiostatin,发现其在抑制胚胎干细胞向内皮细胞分化的同时,也明显抑制胚胎干细胞向心肌细胞定向分化,表明当内源性内皮细胞功能缺失时,心肌细胞定向分化受到明显影响。5、在EphB4基因敲除的胚胎干细胞向心肌细胞定向分化体系中,添加表达EphB4基因的人类内皮细胞,能够部分挽救由于EphB4基因功能缺失所导致的心肌细胞定向分化明显减少的情况。6、内皮细胞跨膜培养可以促进心肌细胞定向分化,表明内皮细胞是通过旁分泌细胞因子参与胚胎干细胞向心肌细胞的分化过程。【研究结论】1、内皮细胞在胚胎干细胞向心肌细胞分化过程中采用旁分泌的方式促进心肌细胞的定向分化。2、内皮细胞可能通过EphB4信号通路介导调节胚胎干细胞向心肌细胞分化过程。更多还原

【Abstract】 【Background and objective】Pluripotent embryonic stem(ES)cells have enormous potential as a therapeutic source of cell-based transplantation for ischemic heart disease.Currently,many studies indicate that endothelial cells derived from ES cells have potential therapeutic effects,but the molecules and environment that direct pluripotent stem cell differentiation into cardiomyocytes remain largely unknown.Cardiomyocytes and endothelial cells are both derived from the cardiogenic mesoderm at about the same time,but there is no direct evidence for the functional relationship between cardiomyocytes and endothelial cells during ES cell differentiation.In the studies of mouse ES cells,the number of functional beating cardiomyocytes was significantly decreased in EphB4-/-ES cells,suggesting that EphB4 signal may play an important role in regulating embryonic endothelial and cardiac development.In this study,we hypothesized that cardimyocytes’ differentiation from ES cells may have strong relationships with EphB4/ephrin B2 signal which is expressed in the endothelium of vein and artery,respectively.We used exogenous chemical compounds to inhibit EphB4 signal pathway/endothelial cells or EphB4-/-ES cells,and to investigate the differentiation of cardiomyocytes from ESCs in the absence of endothelial cells.We also studied the possible mechanism by which EphB4/ephrin B2 signal regulates cardiomyocyte differentiation.This study could provide theoretical evidence supporting cell replaced therapy in ischemic cardiomyopathy using endothelial cells.【Method】 1.With normal differentiation media,ES cells differentiate using “hanging drop” method which has been proved efficiency for cardiomyocyte differentiation.This study Investigated the cardiogenesis from ES cells when EphB4 signal pathway was inhibited by exogenous TNYL-RAW peptide--an antagonist of ephrin B2 binding to EphB4 or EphB4-/-ES cell line.Meanwhile,the effect of exogenous chemical compounds(such as ascorbic acid)which could promote cardiogenesis was also assessed in this differentiation system.2.Confocal analysis of ES cell differentiation could identify the expression of EphB4 when use CGR8-GFP ES cells as normal control compared to EphB4-null ES cells.EphB4 may directly regulate cardiogenesis during differentiation if confocal analysis indicates EphB4 is expressed in cardiomyocytes.Otherwise,the further investigation would be extended if EphB4 is expressed in endothelial cells like normal condition.3.The study evaluated the commitment of cardiomyocytes when function of endothelial cells varied within addition of exogenous endothelial cells or angiogenic inhibitors during ES cell differentiation.4.To further examine whether endothelial cells compensate EphB4 signal to rescue cardiomyocyte differentiation-defects in EphB4-/-ES cells,we co-cultured endothelial cells with EphB4-/-ES cells,and induced cardiomyocyte differentiation in hanging-drop EB formation.The mechanism of endothelial cell and its EphB4 signal pathway in regulating cardiomyocyte development from embryonic stem cell was studied using condition media and trans-well culture methods.【Results】 1.The commitment of cardiomyocytes in EphB4-null ES cells was retarded sharply.Same results were obtained when using exogenous TNYL-RAW peptide,an antagonist of ephrin B2 binding to EphB4.The number of spontaneous contracting cardiomyocytes and expression of cardiac-specific genes(including α-MHC and MLC-2V)were significantly reduced,indicating EphB4 signal pathway may be involved in cardiogenesis.Addition of ascorbic acid promoted cardiogenesis in EphB4-/-ES cells,demonstrating EphB4 and ascorbic acid may share downstream signal pathway in cardiac differentiation.2.Within a beating EB,EphB4 was expressed in cell aggregates underneath the cardiomyocytes,but not in GFP+ cardiomyocytes.These results indicate that EphB4+ cells are co-localized in regions of beating cardiomyocytes within a beating EB,whereas GFP,EphB4,and CD31 were expressed in the beating EB under a single fluorescence wavelength.Analyses of the beating EB with multi-wave lengths indicated that EphB4+ cells also expressed endothelial cells marker,CD31.However,neither EphB4 nor CD31 was expressed in GFP+ cardiomyocytes,suggesting that endothelial cells may regulate the cardiomyocytes’ commitment from ES cells via EphB4/ephrin B2 signal pathway.3.Both generation of functional cardiomyocytes and expression of cardiac-specific genes were significantly enhanced by co-culture of ES cells with exogenous human endothelial cells,indicating exogenous endothelial cells may advance the commitment of cardiomyocytes.4.Angiogenic inhibitors,including endostatin and angiostatin,inhibited endothelial cell differentiation and diminished cardiomyogenesis in ES cells.This finding indicates that commitment of cardiomyocytes could be influenced dramatically by an impairment of endogenous endothelial cells.5.The defects of cardiomyocyte differentiation in EphB4-null ES cells were rescued by exogenous human endothelial cells with expressing EphB4.6.Beating cardiomyocytes were significantly increased when using transwell co-culture of endothelial cells and ES cells,indicating that endothelial cells were involved in the commitment of cardiomyocytes through paracrine effect.【Conclusions】 Endothelial cells may regulate the cardiomyocytes’ commitment from ES cells through paracrine effect and via EphB4/ephrin B2 signal pathway.更多还原