复苏的骨髓间充质干细胞的体外培养和多向分化潜能的研究

发布时间：2018-06-10 07:37

  本文选题：骨髓间充质干细胞 + 培养　； 参考：《浙江大学》2005年硕士论文

【摘要】：骨髓中除含有能分化发育成各种血细胞的造血干细胞之外,还含有可产生非造血 组织的间充质干细胞(Mesenchymal stem cells,MSCs)。MSCs一方面支持骨髓中造 血干细胞的生存、生长和分化。 另一方面, 经许多体外实验证明,它们是一种具有 多向分化潜能的细胞,具有极强的自我复制能力,在特定的条件下可以分化为中胚层 起源的多种组织和细胞(如:成骨细胞、软骨细胞、肌细胞等),甚至可以分化为跨胚 层的细胞(如神经细胞)。MSCs具有取材方便,不存在免疫排斥反应,而且易于进行 细胞扩增,易于临床应用的特点,是一种重要的组织工程种子细胞。由于MSCs具有独 特的细胞增殖分裂模式,使外源基因易于导入和表达,从而使其有望成为一种在人类 医学上潜在的细胞治疗和基因治疗的靶细胞,在多种造血以外组织的缺陷性疾病、退 行性疾病和遗传性疾病的细胞治疗和基因治疗中具有重要的应用前景。 但是MSCs在体外长期培养后也会遇到一些不利因素,诸如基因漂流,细胞衰老, 分化,表型不稳定性,污染或者是培养箱故障等,而这些均会影响对MSCs的应用。因 此,适时地冻存MSCs是将来储备细胞种子的一种重要手段。然而,冻存后MSCs的扩 增和分化能力如何,值得进一步研究。 本实验对成人的骨髓间充质干细胞(human mesenchyma].stem cells,hMSCs)进 行了体外分离、培养和初步的细胞诱导分化,并对其生长方式进行观察,旨在建立一 种分离、培养hMSCs并诱导其向软骨细胞、脂肪细胞和神经细胞转化的有效方法,为 hMSCs作为组织工程的细胞来源提供实验依据。同时为检测冻存的hMSCs在复苏并传 至15代后是否仍具有向软骨、脂肪和神经元多向分化的潜能,我们在通过黏附方法从 骨髓中获得hMSCs并进行冻存的基础上,复苏细胞并传至第15代,然后在相应的条件 培养液作用下依次诱导成软骨细胞,脂肪细胞和神经元细胞,观察其形态学变化以及 在对应的诱导液的作用下胶原Ⅱ、三油酸甘油酯、巢蛋白(Nestin)和神经元特异性烯 醇化酶(neuron-specific enolase,NSE)的表达,从而研究冻存细胞的扩增和分化潜 能。研究结果表明,冻存的hMSCs在复苏后仍是具有单一特征的、均质的细胞群。其
[Abstract]:The bone marrow contains not only hematopoietic stem cells which can differentiate into various blood cells, but also mesenchymal stem cells. MSCs can support the survival, growth and differentiation of hematopoietic stem cells. On the other hand, many experiments in vitro have proved that they are a kind of cells with multidirectional differentiation potential, and have a strong ability to replicate themselves. A variety of tissues and cells (such as osteoblasts, chondrocytes, myocytes, etc.) that can differentiate into mesoderm origin under certain conditions, and even cells that can be differentiated into transembryonic layer cells (e.g., neural cells. MSCs) have the advantages of being able to obtain materials, such as osteoblasts, chondrocytes, myocytes, and so on. It is an important seed cell of tissue engineering that there is no immune rejection, and it is easy to carry out cell expansion and is easy to be applied in clinic. Because MSCs have a unique cell proliferation and division mode, exogenous genes can be easily introduced and expressed, which makes MSCs a potential target cell for cell therapy and gene therapy in human medicine. MSCs have important application prospects in cell therapy and gene therapy for various diseases other than hematopoiesis, degenerative diseases and genetic diseases. However, MSCs may encounter some unfavorable factors after long-term culture in vitro. Gene drift, cell senescence, differentiation, phenotypic instability, contamination, or incubator failure all affect the use of MSCs. Therefore, cryopreservation of MSCs is an important method to store cell seeds in the future. However, the ability of expansion and differentiation of MSCs after cryopreservation is worthy of further study. In this experiment, MSCs from adult bone marrow mesenchymal stem cells (MSCs) were isolated in vitro, cultured and preliminarily differentiated. The purpose of this study was to establish an effective method to isolate, culture and induce the transformation of hMSCs into chondrocytes, adipocytes and nerve cells, and to provide experimental evidence for hMSCs as a cell source of tissue engineering. At the same time, in order to detect whether cryopreserved hMSCs still have the potential to differentiate into cartilage, fat and neurons after resuscitation and transmission to 15 generations, we obtained hMSCs from bone marrow by adhesion method and frozen them. The resuscitation cells were transferred to the 15th passage, then induced chondrocytes, adipocytes and neuronal cells in turn under the corresponding conditioned medium. The morphological changes of the cells and collagen 鈪，

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