【作者】 邓方阁；

【导师】 李玉林；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 心肌细胞(CMs)不具有再生修复能力,心肌损伤后的修复重建工作一直困扰着人类,如何将骨髓间充质干细胞(MSCs)有效的诱导成CMs并用于细胞治疗,是亟需解决的问题。目前,关于体外MSCs向CMs定向诱导分化研究中,主要局限于动物MSCs水平,而关于人MSCs(hMSCs)向CMs诱导分化研究及实验性治疗尚少。本实验以此为出发点,分离纯化、培养扩增细胞特性稳定的hMSCs,作为细胞源,分别在体外用药物5-氮胞苷(5-Aza)诱导体系、体外复制正常和损伤状态下的心肌微环境二维共培养体系中进行向CMs诱导分化的研究,并通过形态结构、蛋白水平、基因水平、电生理功能以及生化酶学等检测,多方位多角度证实了hMSCs具有向CMs分化的潜能,为利用hMSCs作为种子细胞,进行缺血性心血管疾病的细胞治疗提供了重要的理论基础。更多还原

【Abstract】 Ischemic cardiac disease, including myocardial infarction, is a threat to people’s life increasingly. As adult cardiomyocytes have limited ability to regenerate myocardial infarction-induced loss of cardiomyocytes, the necrotic cardiomyocytes are progressively replaced by fibroblasts to form scar tissues. So the work of repair and reconstitution to heart is a great problem perplexing the people, causing the people to try searching the newer and the more effective ways to repair necrotic cardiomyocytes and to improve cardiac function. The recent development of cardiac cellular transplantation and gene therapy comes into being and has the possibility to replace damaged cardiomyocytes and to restore cardio-function gradually, which is a ideal heal strategy nowadays.Mesenchymal stem cells (MSCs) are multi-potent stem cells derived from mesoderm, possessing powerfully proliferative capacity and self-renewal and multi-lineage differentiation. MSCs have been used in the research on cardiac regeneration tissue engineering and genetic engineering because of their particular advantages, including drawing the materials easily, no immune rejection after autologous implantation, large numbers of amplification easily in vitro, induced differentiation easily and inserting and expression of exogenous genes easily.MSCs have potentiality in multi-lineage differentiation and can differentiate into multi-type cells, of which the hardest is differentiation into cardiomyocytes. The most emergent peoblem to tackle at present is how to induce effectually MSCs into CMs and to be used in cell therapy. But the precondition is how to effectually isolate, culture and amplify of hMSCs. So our aim in this study is to isolate, purify, culture and amplify the hMSCs derived from human bone marrow on the base of the methods of isolation and culture of hMSCs improved by the laboratory. With the stabile characteristics, as seeding cells, hMSCs will be used in inducing differentiation in vitro in this research.There are two main ways in inducing hMSCs differentiation into CMs in vitro now, including the chemoattractant induction and the simulation inner microenvironment in vitro induction. Up to now, 5-Aza is only effective chemical in induction into CMs. Moreover, stem cells have high plasticity and environmental dependent differentiation. It is hard to manipulate the stem cells and their surrounding environment inner organism. So in order to get to know the effect of the normal cardiac microenvironment on hMSCs differentiating into CMs in an intuitional way, in this study, we simulated a normal cardiac microenvironment in vitro and co-cultured hMSCs with beating rat CMs. Our aim is to get to know the information of the differentiation in the 2D co-culture system. However, the main purpose of research on the differentiation from hMSCs into CMs is for clinical application in future. Namely, using cellular cardiomyoplasty or cellular transplant save tens of thousands patients’lives who are on the edge of death with impossibly recovered diseases such as myocardium damage, myocardial ischemia or necrosis. We also built up a model of injury patho-station of cardiac microenvironment in vitro to observe hMSCs trans-differentiation into CMs.Results were as following.1. Utilizing the improved methods of isolation and culture of hMSCs in my laboratory, hMSCs were selected from human bone marrow by gradient centrifugation and further selected and expanded by adhering peculiarity. Then carrying out a series of detections in aspects of morphology structure, protein expression, and electrophysiology which to confirm the hMSCs got in this study have stable biologic characteristics, status of keeping undifferentiation, easy getting and easy multiplication in vitro, can offer cells source to research on differentiation from hMSCs into CMs.2. 5-Aza can induce hMSCs differentiating into CMs. The aspects of morphology structure, protein expression, mRNA expression, electrophysiology and enzymology in biochemistry hint that the cells induced by 5-Aza have a certain of properties and relevant functions which are as same as those of CMs’.3. With culture technique of primary cardiomyocytes of 2 days neonatal rats setting up 2D co-culture system of beating CMs and hMSCs is to simulate the normal cardiac microenvironment in vitro, in which hMSCs were successfully differentiated into CMs. Otherwise, this co-culture system not only has nothing toxic action to hMSCs but also out-weights the 5-Aza inducing system.4. Utilizing H2O2 as a hazard factor to damage CMs to simulate and reproduce the pathologic processes of cardiomyocytes damaged by ROS at the moment of oxidative stress and ischemic reperfusion in vivo, we built up a 2D model of injury patho-station of cardiac microenvironment in vitro and co-culture hMSCs with CMs to study how hMSCs differentiate into CMs and to observe the potentiality of directional differentiation of hMSCs in this 2D model. So this style of research is a great innovation in my laboratory.5. The induced hMSCs were selected, purified, expanded and harvested by adhering peculiarity, which is called CMG (Cardiomyocytes group, CMG). At present, the serial passage of CMG has been to passage eight. It was confirmed that CMG has some morphology structures and functions of CMs’by detections.6. The condition medium made of by normal beating or damaged CMs’culture supernate induces hMSCs differentiation into CMs, which demonstrates primely that the main mechanism of hMSCs differentiation into CMs under simulating normal cardiac microenvironment in vitro is cell-cell contact, mechanical tension and traction stimulus of beating CMs, and that the minor mechanism is soluble molecular signal such as cytokine or protein secreted by CMs. Whereas the main mechanism of inducing hMSCs differentiation into CMs in damaged cardiac microenvironment in vitro is related to the effects of a great quantity of cytokines or proteins as molecular signal emitted and released by CMs after membranolysis when they come to death, degeneration or apoptosis.In short, this experiment systemically explained in vitro that hMSCs could differentiate into CMs. It is confirmed the possibility of transplant hMSCs to treat cardiovascular diseases. It also offer the important theoretic basis for utilizing hMSCs as seeds cells in cell therapy to treat ischemic heart diseases.更多还原