星形胶质细胞对骨髓基质干细胞增殖及分化的作用研究

发布时间：2018-06-11 17:35

本文选题：星形胶质细胞 + 炎症　；参考：《山东大学》2009年博士论文

【摘要】： 近几十年来,中枢神经系统疾病(如创伤、神经退行性疾病、脑血管病等)的发病率逐年增加,其致残率和死亡率较高,而成体中枢神经系统自身的再生能力较弱,目前临床只能对症治疗,尚无有效的治疗方法。近年来,干细胞(stemcells)移植疗法逐渐成为治疗中枢神经系统疾病的一种有前途的治疗措施。干细胞是一类具有自我更新、自我复制能力和多向分化潜能的细胞,在合适的条件下,可以分化为不同类型的具有特定形态、特定分子标志和特殊功能的成熟细胞。骨髓基质干细胞(bone marrow stromal stem cells,BMSCs)是来源于骨髓的一种干细胞。BMSCs可从自体骨髓取材、来源充足、取材方便,是治疗性干细胞移植中比较理想的种子细胞。近年来研究发现,BMSCs不仅可分化为成骨细胞、软骨细胞、成肌细胞等间充质来源的细胞,在合适的培养条件下,BMSCs还可横向分化为神经细胞;并在不同的体内微环境或体外诱导条件下,分化为不同递质类型的神经细胞。因此,BMSCs有望成为干细胞移植治疗神经系统疾病的理想种子细胞,具有广阔的临床应用前景。星形胶质细胞是中枢神经系统内数量最多的一类细胞,也是中枢神经系统微环境的主要构成成分。研究发现,星形胶质细胞不仅对神经元提供营养、支持、保护、隔离作用,在神经元的发育、轴突再生、突触形成和信号传递以及干细胞的分化等方面也发挥着重要作用。实验证明,中枢神经系统损伤或发生神经退行性疾病后,炎症反应非常明显,星形胶质细胞的激活并分泌一系列炎性细胞因子是这种炎症反应的重要标志。这种炎症反应所形成的微环境对神经组织中固有的神经干细胞或移植的骨髓基质干细胞会产生什么影响,至今仍不得而知。本研究利用体外建立的星形胶质细胞炎症模型,研究了激活的星形胶质细胞对骨髓基质干细胞增殖及分化的影响,并对其作用机制进行了初步探讨。一、骨髓基质干细胞的分离、培养和体外扩增由于骨髓中细胞成分复杂,且BMSCs在骨髓中的含量很少,约10~5个骨髓单个核细胞中仅含2～5个BMSCs,因而BMSCs的分离、培养、纯化和体外扩增的难度较大。根据BMSCs密度比较低和容易贴壁生长的特点,首先用密度为1.077g/ml的淋巴细胞分离液将大部分的造血细胞去除,然后通过贴壁培养、定期换液去除悬浮生长的造血细胞,使用低糖DMEM培养液(含10%FBS)培养扩增。原代培养7天左右,BMSCs可形成明显的细胞集落;细胞生长至80%～90%融合时用0.25%胰蛋白酶-0.02%EDTA消化,传代培养。传至3代时,细胞基本纯化,形态趋于一致,呈长梭形。由于BMSCs缺乏专一的特异性抗原标志,本实验应用流式细胞仪检测了多种细胞表面抗原,以便确认BMSCs。检测结果显示:几乎所有的培养细胞,其表面抗原CD29和CD90呈阳性,说明培养的细胞为较纯的BMSCs。二、炎性星形胶质细胞条件培养基对BMSCS增殖及分化的影响在成功培养BMSCs的基础上,为模拟中枢神经系统体内炎性微环境下星形胶质细胞细胞对BMSCs增殖及分化的影响,我们首先分别收集加入脂多糖(LPS)培养12、36、72h后的星形胶质细胞上清液作为炎性星形胶质细胞条件培养基(astrocyte conditioned medium,ACM,分别称为12h ACM、36h ACM、72h ACM),同时收集无LPS的星形胶质细胞上清液作为正常星形胶质细胞条件培养基(normal ACM,N-ACM),然后将收集的条件培养基加入BMSCs的培养中,观察细胞的增殖及神经分化情况。细胞增殖实验结果显示,和N-ACM相比,炎性星形胶质细胞条件培养基(12hACM、36h ACM、72h ACM)可明显促进BMSCs的增殖,36h ACM的促增殖作用明显强于12h及72h ACM两组。神经分化结果表明,和N-ACM相比,炎性条件培养基可使BMSCs向神经元和神经胶质细胞分化率明显升高,36h ACM组的神经元阳性细胞比例明显高于12h ACM和72h ACM两组;而神经胶质细胞的分化率,12h ACM、36h ACM、72h ACM三组无明显差异。三、炎性星形胶质细胞条件培养基调节BMSCs增殖及分化的机制研究第二部分的实验结果表明,炎性星形胶质细胞可促进BMSCs的增殖及神经分化,炎性星形胶质细胞的这种促进作用可能是通过分泌的各种因子而实现的。本实验重点检测了白细胞介素-6(IL-6)。以往的实验证实,作为一种多功能的炎性细胞因子,IL-6在各种中枢神经系统创伤或疾病中的表达明显增加,并在神经发育及再生等过程中发挥重要作用。为明确IL-6在炎性星形胶质细胞诱导的BMSCs增殖及神经分化过程中的作用,我们首先检测了炎性星形胶质细胞合成和分泌IL-6的情况。ELISA检测结果显示,炎性星形胶质细胞培养基中IL-6的含量明显增多,且36h ACM中IL-6的含量明显高于12h和72h ACM,RT-PCR结果与此相一致。当在培养基中添加特异性IL-6抗体以阻断IL-6的作用时,BMSCs的增殖明显减少,BMSCs向星形胶质细胞方向分化的趋势也明显减弱,但BMSCs向神经元方向分化明显增多,说明IL-6可促进BMSCs的增殖及向星形胶质细胞方向分化,而抑制BMSCs向神经元方向分化。结论: 炎症激活的星形胶质细胞能明显促进骨髓基质干细胞的增殖及向神经方向分化,且其作用强度与作用时间密切相关,炎性星形胶质细胞分泌的炎性细胞因子IL-6参与了这一过程。
[Abstract]:In recent decades, the incidence of central nervous system diseases (such as trauma, neurodegenerative disease, cerebrovascular disease, etc.) has increased year by year, its rate of disability and mortality is high, but the regeneration ability of the adult central nervous system is weak, and there is no effective treatment in clinical treatment. In recent years, stem cell (stemcells) transplantation therapy This method has gradually become a promising treatment for central nervous system diseases.
Stem cells, a class of cells with self renewal, self replicating and pluripotent differentiation potential, can differentiate into different types of mature cells with specific morphologic, specific molecular markers and special functions under suitable conditions. Bone marrow stromal cells (bone marrow stromal stem cells, BMSCs) are a dry fine derived from bone marrow. Cell.BMSCs can be extracted from autologous bone marrow, with sufficient sources and convenient materials. It is an ideal seed cell in therapeutic stem cell transplantation.
In recent years, it has been found that BMSCs can not only differentiate into osteoblasts, chondrocytes, myoblasts and other mesenchymal stem cells. Under appropriate culture conditions, BMSCs can also differentiate into neural cells transversely, and differentiate into different neurotransmitter types in different microenvironment or in vitro induced conditions. Therefore, BMSCs is expected to become a potential source. The ideal seed cells for stem cell transplantation in the treatment of nervous system diseases have broad clinical application prospects.
Astrocytes are the largest number of cells in the central nervous system, and are also the main components of the central nervous system microenvironment. It is found that astrocytes not only provide nutrition, support, protection, isolation, neuron development, axonal regeneration, synapse formation and signal transmission, and stem cell differentiation in neurons. And it also plays an important role.
The experimental results show that the inflammatory reaction is very obvious after the central nervous system is damaged or the neurodegenerative disease is occurring. The activation and secretion of a series of inflammatory cytokines from astrocytes is an important sign of this inflammatory reaction. The microenvironment formed by this inflammatory reaction is an inherent neural stem cell in the nerve fabric or the bone marrow base of the transplant. The effect of mesenchymal stem cells is still unknown.
In this study, the effect of activated astrocytes on the proliferation and differentiation of bone marrow stromal cells was studied by using a model of astrocyte inflammation established in vitro, and the mechanism of its action was preliminarily discussed.
Isolation, culture and in vitro expansion of bone marrow stromal cells
Because the cell composition of bone marrow is complex and the content of BMSCs in bone marrow is few, about 2~5 BMSCs in 10~5 bone marrow mononuclear cells is only contained, so the separation, culture, purification and in vitro amplification of BMSCs are difficult.
According to the characteristics of low density and easy adherent growth of BMSCs, the most of the hematopoietic cells were removed by the lymphocyte separation solution with density of 1.077g/ml, and then the hemopoietic cells were removed by adhesion culture, and the suspension growing hematopoietic cells were removed regularly, and the low sugar DMEM culture solution (including 10% FBS) was used to cultivate and expand. The BMSCs could be formed for about 7 days in the primary culture. The cell colonies were obvious; when the cells grew to 80% ~ 90% fusion, the cells were digested with 0.25% trypsin -0.02%EDTA and were cultured. When the cells were passed to the 3 generation, the cells were basically purified, and the morphology tended to be a long shuttle. Because of the lack of specific antigen markers of BMSCs, a variety of cell surface antigens were detected by flow cytometry in order to confirm BMSCs. The results showed that almost all the cultured cells showed positive surface antigen CD29 and CD90, indicating that the cultured cells were more pure BMSCs..
Two, the effect of conditioned medium of inflammatory astrocytes on proliferation and differentiation of BMSCS
On the basis of the successful culture of BMSCs, in order to simulate the effect of astrocyte cells on the proliferation and differentiation of BMSCs in the inflammatory microenvironment of the central nervous system, we first collected astrocyte supernatant after adding lipopolysaccharide (LPS) to culture 12,36,72h as an inflammatory astrocyte conditioned medium (astrocyte conditione). D medium, ACM, called 12h ACM, 36h ACM, 72h ACM), and collects the astrocyte supernatant without LPS as a normal astrocyte conditioned medium (normal ACM,), and then the conditioned medium is added to the cultured culture to observe the cell proliferation and neural differentiation.
The results of cell proliferation assay showed that the inflammatory astrocyte conditioned medium (12hACM, 36h ACM, 72h ACM) could significantly promote the proliferation of BMSCs, and the proliferation promoting effect of 36h ACM was significantly stronger than that of 12h and 72h ACM two. The cell differentiation rate was significantly higher. The percentage of neurons positive cells in 36h ACM group was significantly higher than that of 12h ACM and 72h ACM two groups, while the differentiation rate of neuroglia cells, 12h ACM, 36h ACM, 72h ACM three groups were not significantly different.
Three, conditioned medium of inflammatory astrocytes regulate BMSCs proliferation and differentiation.
The experimental results in the second part suggest that inflammatory astrocytes can promote the proliferation and nerve differentiation of BMSCs. This promotion of inflammatory astrocytes may be achieved through various factors secreted. This experiment focuses on interleukin -6 (IL-6). Previous experiments have proved that it is a multifunctional inflammatory cell. Factors, the expression of IL-6 in various central nervous system trauma or diseases is significantly increased and plays an important role in the process of nerve development and regeneration. In order to clarify the role of IL-6 in the process of BMSCs proliferation and nerve differentiation induced by inflammatory astrocytes, we first detected the synthesis and secretion of IL-6 in inflammatory astrocytes. The results of.ELISA detection showed that the content of IL-6 in the inflammatory astrocyte culture medium increased significantly, and the content of IL-6 in 36h ACM was significantly higher than that of 12h and 72h ACM, and RT-PCR results were in accordance with this phase. The trend of differentiation was also markedly weakened, but the differentiation of BMSCs to neurons was significantly increased, indicating that IL-6 could promote the proliferation of BMSCs and differentiate into astrocytes, and inhibit the differentiation of BMSCs into neurons.
Conclusion:
Inflammation activated astrocytes can obviously promote the proliferation and differentiation of bone marrow stromal cells to the nerve, and their action intensity is closely related to the time of action. Inflammatory cytokine IL-6 secreted by inflammatory astrocytes is involved in this process.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

【参考文献】