大鼠骨髓间充质干细胞的分离培养及对T淋巴细胞免疫功能的影响

发布时间：2018-03-25 09:40

本文选题：骨髓间充质干细胞　切入点：分离培养　出处：《山东农业大学》2008年硕士论文

【摘要】： 近年来的研究发现,骨髓间充质干细胞具有可塑性,在特定的条件下可分化为各胚层来源的组织细胞,称为横向分化(transdifferentiation),这一发现不仅对于细胞生物学理论的发展有重大意义,对于各种因素导致的组织和器官病变以及遗传缺陷性疾病的细胞替代治疗也具有深远的影响。由于骨髓MSCs易于分离培养和体外扩增,可自体移植无免疫排斥反应等特点,因此利用骨髓MSCs横向分化潜能,可以根据需要将其诱导分化为特定的组织细胞,用以修复受损的组织或器官,这也将为免疫功能紊乱的治疗提供了另一条途径。在骨髓MSCs应用过程中,它与血管内皮细胞或组织细胞及细胞外基质(extracellular matrix, ECM)的粘附是发挥修复治疗作用的第一步。研究证明,粘附分子在细胞与细胞、细胞与细胞外基质间粘附过程中起着重要的作用。因此,探讨骨髓MSCs的黏附分子的生物学及分子学基础就显得尤为重要。近年来愈来愈多的研究还显示了MSCs具有免疫调节特性,使其在免疫抑制方面具有可预期的理论意义和使用价值。就此,本课题第一部分系统研究了骨髓MSCs的分离培养、体外扩增及黏附分子测定,第二部分重点研究了骨髓间充质干细胞对T淋巴细胞免疫功能的影响。试验一:大鼠骨髓间充质干细胞的分离培养及黏附分子测定目的:探讨体外分离培养和扩增大鼠间充质干细胞(MSCs)的最佳条件,并对其进行表型鉴定及黏附分子测定。方法:采用全骨髓直接贴壁法获得原代大鼠骨髓MSCs,差速贴壁结合消化控制法纯化细胞,Ⅰ型胶原作为细胞外基质扩增MSCs;镜下观察细胞生物学形态及生长状态,流式细胞仪检测第4代MSCs的细胞周期,免疫细胞化学方法对其进行表型鉴定与细胞黏附分子检测。结果:原代培养的MSCs呈圆形、梭形、多角形等,24 h内即可见少量细胞体壁伸展,7～8 d左右可达90%融合,纯化扩增后的MSCs呈均匀一致的长梭形,24 h内细胞已基本贴壁,传代周期为5 d左右。流式细胞仪检测显示,第4代MSCs约有80%处于G0/G1期。免疫细胞化学显示,MSCs CD34、CD45表达阴性,CD29、CD105、CD166、VLA-4、P-selectin表达阳性。结论:本实验方法能很容易地获得大量高纯度的MSCs,并表达一定的黏附分子,为MSCs进一步临床应用打下了良好的基础。试验二:骨髓间充质干细胞对T淋巴细胞免疫功能的影响目的:探讨大鼠骨髓间充质干细胞对T淋巴细胞免疫功能的影响方法:采用试验一的方法分离获得MSCs,尼龙棉柱法从外周血分选出T淋巴细胞; MTT法测定MSCs对T淋巴细胞增殖的影响。结果:MSCs形态均匀一致,生长曲线显示增殖能力强;尼龙棉柱法分离的单个核细胞其CD3阳性细胞高表达。MSCs各组及其上清与T淋巴细胞共培养,其增殖百分率呈明显下降趋势。结论:实验能获得性能可靠的MSCs及T淋巴细胞;MSCs及其上清对T淋巴细胞增殖表现为抑制效应并呈剂量及浓度依赖性。
[Abstract]:Recent studies have found that bone marrow mesenchymal stem cells have plasticity, differentiation of the endoderm derived tissue cells in specific condition, called transdifferentiation (transdifferentiation), this discovery not only has great significance for the development of the theory of cell biology, cell replacement therapy for a variety of factors that lead to tissue and organ disease and genetic defect disease also has far-reaching implications. Because the bone marrow MSCs easy to be isolated and amplified in vitro, autologous transplantation without immune rejection and other characteristics, so the use of bone marrow MSCs horizontal differentiation potential can be induced to differentiate into specific tissue cells to repair damaged tissues or organs, the the treatment of immune dysfunction provides another way.
In the bone marrow in the process of MSCs application, its cells and vascular endothelial cells or tissue and extracellular matrix (extracellular matrix, ECM) adhesion is the first step to play a role in the treatment of repair. Studies have shown that adhesion molecules in cells and cells, cells and extracellular matrix adhesion plays an important role in the process. Therefore, it is very important to investigate the biological and molecular basis of adhesion molecule MSCs in bone marrow.
In recent years, more and more studies have shown that MSCs has the characteristics of immunomodulatory, which has the expected theoretical significance and the use value in the area of immunosuppression.
In this regard, the first part of the project studied the isolation and culture of bone marrow MSCs, expansion and adhesion molecules in vitro. The second part focused on the effect of bone marrow mesenchymal stem cells on T lymphocyte immune function.
Experiment 1: isolation and culture of rat bone marrow mesenchymal stem cells and determination of adhesion molecules
Objective: To explore the best conditions for the isolation, culture and amplification of rat mesenchymal stem cells (MSCs) in vitro, and to identify the phenotypic and adhesion molecules of the rat mesenchymal stem cells.
Methods: using the whole bone marrow adherent primary MSCs rat bone marrow, differential adhesion purified by digestion control cells, collagen as extracellular matrix MSCs amplification; observation of cell biology, morphology and growth status under microscope, detection of the fourth generation of MSCs cell cycle by flow cytometry, immunohistochemistry methods detect the phenotype and cell adhesion molecules on it.
Results: the cultured MSCs were round, fusiform, polygon, 24 h within the small amount of cell wall extension, about 7~8 d up to 90% MSCs after amplification and purification of fusion were uniform long spindle, 24 h cells have basic adherent passage cycle was about 5 d. The results of flow cytometry detection, the fourth generation of MSCs is about 80% in the G0/G1 phase. The expression of MSCs, CD34, CD29, CD45 negative expression of CD105, CD166, VLA-4, P-selectin positive expression.
Conclusion: this method can easily get a lot of high purity MSCs and express some adhesion molecules, which lays a good foundation for further clinical application of MSCs.
Test two: the effect of bone marrow mesenchymal stem cells on the immune function of T lymphocyte
Objective: To investigate the effect of bone marrow mesenchymal stem cells (MSCs) on the immune function of T lymphocytes in rats
Methods: MSCs was obtained by the method of experiment one. T lymphocytes were separated from peripheral blood by nylon cotton column method, and the effect of MSCs on the proliferation of T lymphocytes was measured by MTT.
Results: the morphology of MSCs was uniform and the growth curve showed a strong proliferation ability. The CD3 positive cells isolated from nylon monocytic cells were highly expressed.MSCs, and the proliferation percentage of each group and its supernatants and T lymphocytes co cultured showed a significant downward trend.
Conclusion: the reliable performance of MSCs and T lymphocytes can be obtained, and MSCs and its supernatant can inhibit the proliferation of T lymphocytes in a dose and concentration dependent manner.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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