基于心肌干细胞3D力学环境的工程化微心肌组织构建研究
发布时间：2018-07-31 12:40
【摘要】：心肌梗塞以及充血性心力衰竭等心脏类疾病一直有很高的死亡率,此类疾病都伴随着大量的心肌细胞死亡以及心脏功能的减退,其他心脏疾病如心率不齐等症状、心脏药物筛选等,都需要一个理想的体外组织模型,来了解细胞移植后心肌功能的改善和心机细胞肥大的减缓,以及细胞移植对干细胞心肌向分化、心肌成纤维细胞增殖分化的影响或者药物的有效性等。心脏修复中应用较广泛的是细胞移植疗法,此方法旨在使用细胞改善心脏功能或者是修复受损的心肌层。多种类型的细胞可以应用于心脏修复,并且都取得一定效果,但是这些方法都有不同的缺点,比如细胞在宿主体内的存活、分化以及与原组织的交联。心肌干细胞(Cardiac Stem Cells,CSCs)具有成体干细胞特性,并且自体移植不会发生免疫排斥反应,因此,心肌干细胞是构建工程化心肌组织的理想细胞来源。利用心肌干细胞、心肌细胞及心肌成纤维细胞作为种子细胞,发展一种体外的心肌组织模型,为完善心肌损伤的细胞移植疗法、认识组织力学环境对种子细胞的增殖分化的影响、药物干预心脏的功能提供了可能的体外模型。Matrigel包含多种ECM蛋白以及生长因子,可以作为构建3D心肌组织工程的理想材料。本文主要探究在力学环境下,体外构建3D工程化心肌组织。将Matrigel与DMEM培养基按照不同比例稀释,将心肌干细胞,心肌细胞以及心肌成纤维细胞按照按一定的比例与Matrigel工作液混匀。使用FlexCell力学加载设备,首先将六孔组织培养版形成凹槽,然后将混合均匀的细胞加到凹槽中,待静置40分钟后,3D组织构建成功,然后静置培养2-3天,再使用FlexCell力学加载设备进行拉力加载,体外构建3D工程化心肌组织。通过合适的力学加载、细胞比例及数量、3D凝胶的稀释比例,构建3D微心肌组织,发展了在体外构建3D心肌组织的方法,为构建心肌组织用于体外研究心肌损伤等疾病的研究,奠定可用的组织模型。本文共分为四章进行介绍,第一章为绪论,综合介绍了心肌梗死以及当前最主要的治疗方案;第二章为种子细胞的分离纯化培养及鉴定工作;第三章使用FlexCell力学加载系统,构建3D工程化微心肌组织,并对微心肌组织进行心肌细胞检测;第四章为全文总结以及工作展望。
[Abstract]:Heart diseases such as myocardial infarction and congestive heart failure have a high mortality rate. These diseases are accompanied by a large number of cardiac cell death and cardiac dysfunction, other heart diseases such as heart rate disorders such as symptoms, The screening of cardiac drugs requires an ideal tissue model in vitro to understand the improvement of myocardial function and the slowdown of cardiomyocyte hypertrophy after cell transplantation, as well as the differentiation of stem cell myocardium by cell transplantation. Effects of myocardial fibroblasts on proliferation and differentiation or the availability of drugs. Cell transplantation is widely used in cardiac repair, which aims to improve heart function or repair damaged myocardial layer. Many types of cells can be applied to heart repair, and all of these methods have different disadvantages, such as cell survival, differentiation and cross-linking with original tissue. Myocardial stem cells (Cardiac Stem cells) have the characteristics of adult stem cells, and there is no immune rejection in autologous transplantation. Therefore, myocardial stem cells are the ideal cell source for the construction of engineering myocardial tissue. Myocardial stem cells, cardiomyocytes and myocardial fibroblasts were used as seed cells to develop a myocardial tissue model in vitro to improve cell transplantation therapy for myocardial injury. In order to understand the effect of tissue mechanics environment on the proliferation and differentiation of seed cells, drug intervention provides a possible in vitro model. Matrigel contains a variety of ECM proteins and growth factors, which can be used as an ideal material for the construction of 3D myocardial tissue engineering. The aim of this study was to construct 3 D engineered myocardial tissue in vitro under mechanical environment. The Matrigel and DMEM media were diluted in different proportions, and the myocardial stem cells, cardiomyocytes and myocardial fibroblasts were mixed with the working solution of Matrigel in a certain proportion. Using the FlexCell mechanical loading equipment, the six-hole tissue culture plate was first formed into a groove, and then the mixed cells were added to the grooves. After 40 minutes of statics, the 3D tissue was successfully constructed, and then the cells were statically cultured for 2-3 days. Then FlexCell mechanical loading equipment was used to construct 3D engineered myocardial tissue in vitro. Through proper mechanical loading, cell proportion and dilution ratio of 3D gel, 3D micromyocardial tissue was constructed, and the method of constructing 3D myocardial tissue in vitro was developed, which was used to study myocardial injury and other diseases in vitro. Establish an organization model that is available. This paper is divided into four chapters, the first chapter is the introduction, the second chapter is the isolation, purification, culture and identification of seed cells, the third chapter is the use of FlexCell mechanical loading system. The 3D engineered micromyocardial tissue was constructed and the cardiac myocytes were detected. Chapter 4 was the summary of the whole paper and the prospect of the work.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R54
，
本文编号：2155611
[Abstract]:Heart diseases such as myocardial infarction and congestive heart failure have a high mortality rate. These diseases are accompanied by a large number of cardiac cell death and cardiac dysfunction, other heart diseases such as heart rate disorders such as symptoms, The screening of cardiac drugs requires an ideal tissue model in vitro to understand the improvement of myocardial function and the slowdown of cardiomyocyte hypertrophy after cell transplantation, as well as the differentiation of stem cell myocardium by cell transplantation. Effects of myocardial fibroblasts on proliferation and differentiation or the availability of drugs. Cell transplantation is widely used in cardiac repair, which aims to improve heart function or repair damaged myocardial layer. Many types of cells can be applied to heart repair, and all of these methods have different disadvantages, such as cell survival, differentiation and cross-linking with original tissue. Myocardial stem cells (Cardiac Stem cells) have the characteristics of adult stem cells, and there is no immune rejection in autologous transplantation. Therefore, myocardial stem cells are the ideal cell source for the construction of engineering myocardial tissue. Myocardial stem cells, cardiomyocytes and myocardial fibroblasts were used as seed cells to develop a myocardial tissue model in vitro to improve cell transplantation therapy for myocardial injury. In order to understand the effect of tissue mechanics environment on the proliferation and differentiation of seed cells, drug intervention provides a possible in vitro model. Matrigel contains a variety of ECM proteins and growth factors, which can be used as an ideal material for the construction of 3D myocardial tissue engineering. The aim of this study was to construct 3 D engineered myocardial tissue in vitro under mechanical environment. The Matrigel and DMEM media were diluted in different proportions, and the myocardial stem cells, cardiomyocytes and myocardial fibroblasts were mixed with the working solution of Matrigel in a certain proportion. Using the FlexCell mechanical loading equipment, the six-hole tissue culture plate was first formed into a groove, and then the mixed cells were added to the grooves. After 40 minutes of statics, the 3D tissue was successfully constructed, and then the cells were statically cultured for 2-3 days. Then FlexCell mechanical loading equipment was used to construct 3D engineered myocardial tissue in vitro. Through proper mechanical loading, cell proportion and dilution ratio of 3D gel, 3D micromyocardial tissue was constructed, and the method of constructing 3D myocardial tissue in vitro was developed, which was used to study myocardial injury and other diseases in vitro. Establish an organization model that is available. This paper is divided into four chapters, the first chapter is the introduction, the second chapter is the isolation, purification, culture and identification of seed cells, the third chapter is the use of FlexCell mechanical loading system. The 3D engineered micromyocardial tissue was constructed and the cardiac myocytes were detected. Chapter 4 was the summary of the whole paper and the prospect of the work.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R54
，
本文编号：2155611
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