骨髓微环境对间充质干细胞特性的影响

发布时间：2018-03-12 20:19

本文选题：间充质干细胞　切入点：骨髓微环境　出处：《中国人民解放军医学院》2014年硕士论文　论文类型：学位论文

【摘要】：间充质干细胞（Mesenchymal stem cells，MSCs）是骨髓中除造血干细胞外另一重要的干细胞。MSCs具有自我更新和多向分化能力的特点，它可以分化为多种骨髓基质细胞如脂肪细胞、成骨细胞和内皮细胞等；在一定的诱导体系中还能被诱导分化为软骨细胞、肌细胞、心肌细胞和神经元等。因此，MSCs对于受损组织的修复具有重要意义。另外MSCs还具有免疫调控和支持造血的能力，而且来源广泛，容易在体外培养和扩增，正是因为这些特点，MSCs目前已经应用于临床并显示了良好的治疗效果。但是，许多关键问题如免疫调控的具体机制、骨髓微环境对MSCs特性的影响等问题并未解决，所以，建立一种方便、快捷、有效地体外分离培养扩增MSCs的方法能为后续的研究奠定良好的基础。本实验应用一种新的方法——骨髓加骨片法分离培养小鼠MSCs。通过将骨髓加骨片法与现有的全骨髓贴壁法和骨片消化法对比，发现这种新方法出现的MSCs集落时间最早，集落数最多，获得的细胞数也最多；细胞呈梭形贴壁生长，流式细胞术检测结果显示细胞高表达干细胞标志之一Sca-1，高表达CD44、CD29，不表达白细胞表面抗原CD45和内皮细胞标志CD31；得到的MSCs在成骨诱导体系和成脂诱导体系中能分别向成骨细胞和脂肪细胞分化，具有典型的MSCs特性。由于MSCs上述多种优越的特性，尤其是对许多组织的损伤修复能力，使得MSCs各方面功能的研究成为热点，，而MSCs又是骨髓微环境中的一种重要的干细胞，虽然它对骨髓微环境中其它细胞有重要作用如支持造血等，但其本身特性也会受到骨髓微环境的影响，当机体年老时，损伤修复过程中MSCs的动员和迁移就比年轻机体要慢，所以本研究着重探讨年轻骨髓和年老骨髓对MSCs迁移和增殖的影响。通过年轻骨髓和年老骨髓与骨片共培养，发现年老骨髓有抑制MSCs集落形成的作用。为了分析骨髓细胞中何种成分发挥作用，流式细胞术检测年轻骨髓和年老骨髓细胞结果显示B220+细胞在年老骨髓中所占比例明显低于在年轻骨髓中的比例，而CD11b+、CD3+、Gr-1+和F4/80+细胞则是在年老骨髓中所占比例明显高于在年轻骨髓中的比例。用磁珠分选年老骨髓中CD11b+、B220+、Ter119+细胞并分别与骨片共培养，发现这三个亚群并不能抑制MSCs集落形成。在增殖实验中，年老骨髓也并没有起到明显的抑制细胞增殖的作用，但是在划痕损伤修复实验和迁移实验中，年老骨髓作用的MSCs的迁移能力要弱于年轻骨髓作用的MSCs，进一步的QPCR结果显示年轻骨髓刺激后的MSCs表达SDF-1水平显著高于年老骨髓刺激后。另外western blot结果显示在年轻骨髓或是年老骨髓刺激下，MSCs的MAPK通路均被激活，但是p-38和ERK的磷酸化水平基本没有差异，而JNK磷酸化水平则是年轻骨髓刺激后的MSCs表达高。综上所述，骨髓加骨片法是一种方便、快捷、有效的小鼠骨髓MSCs分离方法；年老骨髓微环境具有抑制MSCs从干细胞龛中迁出的作用，但是具体是骨髓中哪部分细胞、细胞外基质或是细胞因子起作用还有待进一步研究。
[Abstract]:Mesenchymal stem cells (Mesenchymal stem cells, MSCs) bone marrow mesenchymal stem cells and other important.MSCs stem cell self-renewal and multilineage differentiation capability, it can differentiate into a variety of bone marrow stromal cells such as adipocytes, osteoblasts and endothelial cells; in certain induction system can were induced to differentiate into cartilage cells, muscle cells, myocardial cells and neurons. Therefore, MSCs has an important significance for the repair of damaged tissue. MSCs also has the ability to support hematopoiesis and immune regulation, and a wide range of sources, easy amplification in vitro, it is because of these characteristics, MSCs has been used in clinical application and showed good therapeutic effect. However, the specific mechanisms such as immune regulation of many key problems, the problem of bone marrow microenvironment influence on the character of MSCs has not been resolved, therefore, to build a convenient, fast Czechoslovakia, effective isolation and culture of MSCs in vitro, can lay a good foundation for subsequent research.
The experimental application of a new method, bone marrow and bone slices were isolated and cultured in MSCs. mice by bone marrow and bone graft method and whole bone marrow adherent method and the existing bone digestion method comparison, found the new method of MSCs colony in the earliest time, colony number, the most number of cells obtained; the cells were spindle shaped adherent growth, flow cytometry showed that the expression of stem cell markers of Sca-1, high expression of CD44, CD29, the expression of leukocyte surface antigen CD45 and endothelial cell marker CD31; the MSCs in osteogenic induction system and adipogenic induction system respectively into osteoblasts and fat cell differentiation, MSCs has typical characteristics.
Because of the variety of MSCs superior characteristics, especially the damage repair capacity for many organizations, the study of MSCs function in all aspects has become a hot spot, and MSCs is a kind of important stem cells in bone marrow microenvironment, although there are other cells of the bone marrow microenvironment plays an important role as supporting hematopoiesis, but itself the characteristics will be affected by the bone marrow microenvironment, when the body is old, mobilization and MSCs damage repair process in migration than the young body to slow, so this study focuses on the influence of young and old bone marrow on the migration and proliferation of MSCs.
The young and old bone marrow and bone marrow were cultured, found that elderly bone marrow suppression of MSCs colony formation. In order to analyze what elements play a role in bone marrow cells, flow cytometry in young and old bone marrow cells showed that B220+ cells accounted for in old bone marrow ratio was significantly lower in the proportion of young bone marrow CD11b+, CD3+, Gr-1+, and F4/80+ cells are occupied in the old bone marrow was significantly higher than in young bone marrow ratio. Using CD11b+, B220+ multisort old bone marrow, Ter119+ cells were co cultured with bone slices, found that these three subgroups did not inhibit MSCs colony formation in proliferation. In the experiment, the old bone marrow also did not play a significant role in the inhibition of cell proliferation, but the scratch damage repair assay and migration experiment, the migration ability of old bone marrow effect of MSCs is much weaker than the young bone Curative effects of MSCs, further QPCR results showed that young bone marrow stimulated MSCs SDF-1 expression level was significantly higher than that of old bone marrow stimulation. In addition Western blot results showed that in young or old bone marrow bone marrow stimulation, MAPK MSCs pathway is activated, but the phosphorylation level of P-38 and ERK basically no difference, and JNK phosphate the level is high expression of young bone marrow stimulation after MSCs.
In summary, bone marrow and bone slices method is a convenient, fast and effective method for separating MSCs old mouse bone marrow; bone marrow microenvironment can inhibit MSCs migration from the stem cell niche in the role, but which is a specific part of bone marrow cells, extracellular matrix and cytokines play a role remains to be further studied.

【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782

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