【作者】 何勇；

【导师】 郑强荪；

【作者基本信息】 第四军医大学 ， 内科学， 2010， 博士

【摘要】 胚胎干细胞(embryonic stem cells,ESC)是源于胚胎囊胚内细胞群的一类具有自我复制和无限增殖潜能的细胞。它在增殖的过程中保持着强大的分化强力,能形成各种机体组织细胞。已经证实,在特定条件下,ESC能够分化为具有收缩和兴奋功能的心肌细胞,其形态和功能也接近于体内CM。然而,由于促使ESC有效分化的分子机制和信号途径尚未完全明确,分化的过程难以控制。目前的研究显示,尽管很多诱导剂已经在ESC分化实验中使用,ESC的分化效率仍然很低,分化过程中只有少量EB能形成搏动的CM。由于某些受体系统能够在ESC中检测出来,激素作为新型的诱导剂备受关注,近年来发现催产素及受体系统在胚胎心脏有表达。催产素的添加有利于对ESC向CM的分化。然而,催产素仅对ESC的早期分化有促进作用,无助于分化细胞的超微结构改善。为了进一步探讨ESC的分化机制以及研究微环境对ESC分化的影响,我们在国外已成功地用催产素诱导出搏动CM的基础上,重点观察细胞-细胞及细胞-基质的相互作用对于ESC向ESCM分化的影响,特别是对ESC后期分化的影响,阐明促进ESC定向分化为CM的机制。本研究分成两步,第一步研究催产素对CGR8系细胞的分化作用。采用经典的ESC诱导方法,即ESC经悬滴培养法形成拟胚体(embryoid bodies, EB)进行诱导分化,然后往培养液10-7M催产素,设立对照组,观察并分析两组分化细胞(ESC-derived CM, ESCM)的结构和功能的变化。我们发现,采用催产素为诱导剂,在诱导过程中可见EB形成,诱导第8天出现可自发搏动的细胞。在催产素的作用下,MLC-2V和GATA4表达有所增加;分化第10天的免疫组化结果显示,心肌特异性蛋白α-sarcomeric actin、cTnT均在实验组心肌样细胞中表达,但是在催产素作用下,细胞中α-sarcomeric actin、cTnT的表达分布更接近于天然CM。第二步在第一步的实验的基础上,研究3D和共培养对促进催产素诱导下的ESC向CM分化的影响。设立普通分化组、3D培养组、共培养组、3D共培养组,观察并分析各组分化细胞的结构和功能的变化。实验发现,催产素处理ESC后确能增强其分化,但分化水平仍不令人满意。跳动EBs的数目平均只占17.76±5.99%,并且兴奋收缩的自律性不能长时间维持:在分化18天后,跳动EBs的数量开始明显减少。在3D的培养环境中,跳动EBs的数目平均占22.53±3.75%,兴奋收缩的自律性维持优于普通2D的环境。令人惊奇的是,在体外模拟的心脏微环境中,跳动EBs的数目明显增加,兴奋收缩的自律性能维持更长时间。进一步分析ESCM的超微结构,提示在体外模拟的心脏微环境中,ESCM显示出典型的肌原纤维节结构和胞间的闰盘结构(粘合膜fascia adherens、桥粒desmosomes、间隙连接gap junctions)。我们推测,天然CM群的存在促使ESCM形成更加成熟的肌原纤维节结构,这些结构也为ESCM具有真正CM的功能提供了结构基础。本实验的结果表明,催产素诱导对于ESC的早期分化有帮助;心脏微环境是促进ESC心源性分化的关键因素; 3D共培养可以解决催产素诱导下ESC后期分化的问题,有助于ESC向CM的定向分化并有利于维持ESCM的兴奋收缩的自律性。这些因素可能与细胞-细胞间相互作用和天然CM分泌的细胞因子等有关。总之,我们的研究显示3D共培养诱导策略能够显著地促进ESC分化为有兴奋和收缩功能CM,为体外控制ESC特异性分化提供了一条新思路。此方法对于ESC在组织工程中的应用及其它医学再生领域的应用有重要意义。更多还原

【Abstract】 As a proliferative cell line, embryonic stem cells (ESCs), capable of propagating in undifferentiated state, have the potential to form various kinds of adult tissue cells. It is verified that ESCs can differentiate into cardiomyocytes (CMs) with excitability and spontaneous contractions under appropriate conditions. These cells have the similar structure and function to in vivo CMs, and even the same ion channels to control pacing. These features of ESCs suggest it is possible to apply cellular transplant and tissue repair to the therapy of heart diseases. However, since the molecular mechanisms and signal pathway of ESC differentiation are still unclear, it is hard to control the process of its differentiation. The recent studies demonstrate the low differentiation rate of ESC, that is, only few embryoid bodies (EB) can differentiate into the beating CM. As some receptors can be detected in the ESC differentiation, and the expressions of oxytocin and receptors in cardiomyocytes, oxytocin arouses great interests as a new inductor. However, although oxytocin does increase the beating efficiency of EB in ESC differentiation, it causes the increased differentiation only at early stage and no improvement in ultrastructure of differentiated cells. To further study the ESC differentiation mechanism and the effect of microenvironment on the differentiation, we studied the effect of the interaction between cell and cell, and cell and matrix on the differentiation of ESC into ESCM, especially at the later stage. Our study is to clarify the mechanism of the promoted differentiation of ESC into CM.Our study can be divided into two parts:Part 1. The effect of oxytocin on the differentiation of CGR8 cells. In this part, the classical induction of ESC was conducted, that is, EB from the hanging-drop culture of ESC was induced to differentiate, and then cells were treated with 10-7M oxytocin. The structures and functions of differentiated cells (ESC-derived CM, ESCM) in two groups (experimental group and control) were observed and analyzed. The results showed the formation of EB in the induction with the treatment of oxytocin and the appearance of spontaneous beating cells on the 8th day. The expressions of MLC-2V and GATA4 increased, suggesting the promoting effect of oxytocin on the differentiation of ESC into CM at the early stage. The immunohistochemistrical results on the 10th day showed thatα-sarcomeric actin and cTnT were expressed in cardiomyocytes in the experimental group, and with the treatment of oxytocin, the distribution of the expression ofα-sarcomeric actin and cTnT was more similar to that of primary CM. The results demonstrate that oxytocin can promote the early differentiation of ESC into CM in mouse.Part 2. The effects of 3-D culture and co-culture on the differentiation of ESC into CM induced by oxytocin. The structures and functions of the differentiated cells in each group (common group, 3D group, co-culture group and 3D plus co-culture group) were observed and analyzed. It was observed oxytocin could promote the differentiation of ESC with the unsatisfactory results (the average of beating EBs was 17.76±5.99% and the autorhythmicity of excitement contraction could not maintain for a long period of time). On the 18th day, the number of beating EBs started to decrease significantly. In the 3-D culture, the average of beating EBs was 22.53±3.75% and the autorhythmicity of excitement contraction maintained for longer period than that in the 2-D culture. Surprisingly enough, in vitro simulative microenvironment of heart, the number of beating EBs was increased significantly and the autorhythmicity of excitement contraction maintained for longer period. The ultrastructure of ESCM showed the typical sarcomere and intercellular intercalary disc (fascia adherens, demonsomes and gap junctions) in vitro simulative microenvironment of heart. We suppose that the primary CM may promote ESCM to form more full-grown sarcomere, which provides the base for ESCM to have the function of CM.The results of our study show that the induction of oxytocin contributes to the early differentiation of ESC; the cardiac microenvironment is the one of the key factors to promote the differentiation of ESC; 3-D culture plus co-culture can improve the differentiation of oxytocin-induced ESC at the later stage, contributing to the oriented differentiation of ESC into CM and the longer period of autorhythmicity of excitement contraction. All these factors may be associated with the interaction of cell and cell or cytokines secreted by the primary CM.In short, our study shows that 3-D culture plus co-culture can significantly promote the differentiation of ESC into CM with excitability and spontaneous contraction, which provides a new method to control specific differentiation of ESC in vitro and plays an important role in the application of ESC in tissue engineering and medical regeneration.更多还原