骨髓间充质干细胞定向肝细胞分化及不同细胞外基质对分化率的影响

发布时间：2018-08-15 12:05

【摘要】：背景肝脏疾病危害人类健康,肝功能衰竭、重型肝炎和肝癌等疾病的死亡率高达 65%-85%,虽然原位肝脏移植在一定程度上可以缓解病情,帮助病人度过危险期,但是由于肝脏器官来源匮乏,术后免疫排斥反应和巨额的手术治疗费用等因素,都使得这一治疗措施不能广泛的应用。肝脏组织工程( Engineering Liver Regeneration)是近年来正在兴起的,由生物学、医学、材料科学、生物医学工程学等学科交叉产生的一门高新技术学科,最终的目标是永久性的修复和取代人类病损的肝脏组织,从而彻底的治愈肝脏疾病,这为肝脏疾病的治疗开辟了全新的思路。肝脏组织工程研究包括种子细胞的研究、细胞外基质的研究和组织器官的构建等三部分。其中,种子细胞选取,是一切研究的基础。随着人们对干细胞研究的逐步深入,选取干细胞作为种子细胞越来越为人们所关注,尤其是自体干细胞。已经有研究表明骨髓来源的干细胞具有多向分化潜能,在体外可以分化为骨、软骨、肌肉、神经、脂肪细胞和肝细胞等。在骨髓中存在两种主要的干细胞:造血干细胞(Hematopoietic stem cell HSCs)和间充质干细胞 (Mesenchymal stem cells MSCs),已经有研究表明:间充质干细胞在体外不同条件下可以定向的被诱导分化为成骨细胞[1]、成软骨细胞[2]、脂肪细胞[3]、肌肉细胞[4]、神经元细胞[5]等不同细胞,因其具有高度的自我更新能力和多向分化潜能以及取材方便、自体植入后不良反映较弱等优点,将成为肝脏组织工程的理想种子细胞。目前,MSCs 在骨、软骨、和肌腱损伤的修复方面研究比较深入,但在体外向肝细胞诱导分化的条件和机制方面刚刚起步,而且面临一系列的困难:如何模拟体内微环境使骨髓间充质干细胞定向肝细胞分化;如何提高诱导分化效率;建立高效完整的扩增体系等。研究目的探讨 MSCs 在体外是否具有定向分化为功能性的肝细胞的潜能。方法与结果本实验第一部分首先分离、培养并鉴定 MSCs。利用骨髓间充质干细胞的密度特性,用一定密度的 Percoll 对提取出的骨髓进行密度梯度分离。1.073g/mL 密度梯度离心法结合贴壁法分离小鼠骨髓间充质干细胞,经传代培养后,取第 3 代的 MSCs,观察不同的血清浓度、种植密度对细胞体外生长的影响,观察细胞生长曲线,并应用流式细胞仪检测细胞表面抗原。第 3 代的 MSCs 经过流式细胞仪鉴定发现:培养的第 3 代 MSCs,干细胞表面标志 CD90 阳性,基质细胞表面标志 CD44 阳性,不表达内皮细胞表面标志 CD31 不表达白细胞表面标志 D45;不表达造血干细胞表面标志 CD34。说明在第 3 代的 MSCs中不含有造血干细胞,其他杂质细胞含量少,已经成为比较纯的MSCs.,为下一步的实验奠定基础。本实验第二部分探讨 MSCs 是否具有定向肝细胞分化的潜能。取第 3 代骨髓间充质干细胞,0.25%胰酶消化,台盼兰拒染试验显示细胞活率大于 95%,以 1×105/mm 的密度种植在 24 孔和 6 孔培养板中。约 7-12 天,细胞铺满板底。单独应用 HGF、EGF 及 HGF 联合 EGF对 MSCs 进行诱导,各组加入诱导因子后,MSCs 分裂增殖逐渐停止,开始有少量细胞发生形态学的改变,梭形细胞的两极开始回缩,细胞开始由梭形变圆。其中 HGF 和 EGF 联合诱导组的 MSCs 在 48h内就部分细胞变圆;而单独应用 HGF 组和单独应用 EGF 组在 48h 内细胞无明显改变,而在 72h 后可以发现有部分细胞变圆;诱导 10 天后,各组变圆细胞均有“聚集”现象出现。诱导分化 20 天,各组细
[Abstract]:background
Liver disease is harmful to human health. The mortality rate of liver failure, severe hepatitis and liver cancer is 65%-85%. Although orthotopic liver transplantation can alleviate the disease to a certain extent and help patients through the dangerous period, due to the lack of liver organs, immune rejection after surgery and huge surgical costs, etc. This treatment can not be widely applied.
Engineering Liver Regeneration (ELR) is a new and high-tech subject emerging in recent years. It is produced by the interdisciplinary of biology, medicine, materials science, biomedical engineering and so on. The ultimate goal is to repair and replace the damaged liver tissue permanently, thus completely curing the liver disease. This is the liver. Liver tissue engineering research includes three parts: seed cell research, extracellular matrix research and tissue organ construction. Seed cell selection is the basis of all research. With the development of stem cell research, stem cells as seed cells become more and more important. There are two main types of stem cells in bone marrow: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Mesenchymal stem cells MSCs have been shown to differentiate into osteoblasts [1], chondroblasts [2], adipocytes [3], muscle cells [4], neurons [5] under different conditions in vitro, because of their high self-renewal capacity.
It will be an ideal seed cell for liver tissue engineering because of its strength, multidirectional differentiation potential, convenience of sampling and poor response after autologous implantation.
At present, MSCs have been extensively studied in repairing bone, cartilage, and tendon injuries. However, the conditions and mechanisms of inducing differentiation into hepatocytes in vitro are just beginning, and a series of difficulties are confronted: how to simulate the microenvironment in vivo to make bone marrow mesenchymal stem cells differentiate into hepatocytes; how to improve the efficiency of inducing differentiation; Complete amplification system.
research objective
To investigate whether MSCs has potential to differentiate into functional hepatocytes in vitro.
Methods and results
In the first part of this experiment, MSCs were isolated, cultured and identified. MSCs of the third generation were obtained by density gradient separation of the extracted bone marrow with a certain density of Percoll. 1.073g/ml density gradient centrifugation combined with adherence method. The effect of different serum concentration and planting density on the cell growth in vitro was observed. The growth curve was observed and the cell surface antigen was detected by flow cytometry. CD31, the endothelial cell surface marker, did not express the leukocyte surface marker D45; CD34, the hematopoietic stem cell surface marker, did not express the hematopoietic stem cell surface marker CD34.
The second part of this study was to investigate whether MSCs have the potential to differentiate into hepatocytes. The third generation bone marrow mesenchymal stem cells were digested with 0.25% trypsin. Trypan blue exclusion test showed that the cell viability was more than 95%. The cells were planted in 24-hole and 6-hole plates at a density of 1 105/mm for about 7-12 days. The cells were covered with the bottom of the plate. MSCs were induced by HGF combined with EGF. After the induction factors were added to each group, the division and proliferation of MSCs stopped gradually, and a few cells began to undergo morphological changes. The polarity of spindle cells began to shrink, and the cells began to circle by spindle. There was no obvious change in the cells of the two groups within 48 hours, but some cells became round after 72 hours. After 10 days of induction, the cells in each group showed "aggregation" phenomenon.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

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