背根节神经嵴干细胞的分离培养和分化的研究

发布时间：2018-03-24 10:13

  本文选题：躯干神经嵴干细胞　切入点：背根神经节　出处：《苏州大学》2011年博士论文

【摘要】：组织工程技术为周围神经损伤的治疗带来了新的希望,种子细胞的优化精选是组织工程技术的关键。施旺氏细胞是周围神经系统的主要结构和功能细胞,是公认的种子细胞,然而其来源有限。躯干神经嵴干细胞,可分化为组成周围神经系统的绝大部分神经元和胶质细胞,是周围神经系统的原基,因此将躯干神经嵴干细胞应用于神经组织工程有着良好的应用前景。 干细胞的分化受到体内外多种因素的调节,具有多向性和不确定性,应用神经干细胞移植来治疗神经系统损伤或疾病需要有目的地引导神经干细胞向我们所需要的特定细胞类型分化。因此,诱导神经干细胞的定向分化是一项具有重要意义的研究。bFGF具有多功能的特性,即它既能促进神经元的分化,也能促进神经胶质细胞的分化并且它的这种功能变化是和体内发育时相的变化一致的。即在神经发育的早期促进神经元分化,而在发育晚期则促进神经胶质细胞分化。周围神经系统中,bFGF是周围神经系统干细胞分化为施旺氏细胞祖细胞的正向决定因素,因此利用bFGF定向诱导背根节神经干细胞沿施旺氏细胞方向分化将是一个很好的尝试。本文首先体外分离培养背根节神经干细胞,然后体外诱导其沿施旺氏细胞方向分化及对其功能鉴定;在此基础上对bFGF体外诱导神经干细胞沿施旺氏细胞分化的机制进行研究。 第一部分背根节神经干细胞的培养,鉴定及定向诱导其沿施旺氏细胞方向分化 目的: 建立成体大鼠背根节神经干细胞的培养方法及定向诱导其沿施旺氏细胞方向分化的方法,为周围神经系统再生提供真正的周围神经系统来源的种子细胞。 方法: 1.取成年SD大鼠的背根神经节,体外悬浮培养获得原代神经干细胞球后,利用无限稀释法获得单细胞克隆,传代2次后对其干细胞特性进行鉴定,主要包括特异性标志物,增殖率及分化潜能; 2.将克隆化的神经干细胞球分5组诱导分化,即NGF组;bFGF组;NRG组;FBS组;及对照组。诱导7 d后分别行S100免疫细胞化学和Westernblot。免疫细胞化学鉴定时随机选取10个视野统计各组的S100阳性细胞比例;同时提取蛋白通过Western blot检测各组S100的表达水平。 3. bFGF诱导背根神经节干细胞分化的短时程和长时程效应检测,即一组加入分化培养液24 h后洗去bFGF继续培养;另一组则始终用含有bFGF的分化培养液。7 d后行S100免疫细胞化学和Westernblot。 4. BrdU标记检测bFGF作用24小时内细胞的增殖:在加入bFGF诱导细胞分化的同时加入BrdU标记液,24 h后免疫细胞化学检测。 5.利用免疫细胞化学,传代培养,体外成髓鞘实验等方法对bFGF诱导所获得的细胞进行细胞学和功能学的鉴定;通过双向电泳技术比较原代培养的施旺氏细胞和由背根节干细胞诱导分化的细胞蛋白表达谱差异。 结果: 1.通过体外分离培养的方法以及单克隆实验获得了稳定传代的背根节神经干细胞。它表达神经嵴干细胞特异性Marker,而且具有较高的增殖率和分化潜能。 2.诱导细胞分化的5组中的bFGF处理组, S100β阳性细胞的比例高于其他各组相比,Western Blot结果与此一致。 3.诱导背根神经节干细胞分化的短时程和长时程效应检测结果显示在bFGF在作用24 h时间内影响细胞的分化,而对细胞的增殖没有影响。 4.分化的细胞具有施旺氏细胞的形态学和功能学特征。 结论: 我们成功建立了培养背根节神经干细胞的方法及定向诱导其向施旺氏细胞分化的方法,为周围神经再生提供了真正周围神经系统来源的种子细胞。 第二部分bFGF诱导神经干细胞沿施旺氏细胞方向分化的机制研究 碱性成纤维细胞生长因子(bFGF)属于有丝分裂原,通过与成纤维细胞生长因子受体(FGFRs)结合,激活MAPK,PI3K,PLCγ等信号通路,在影响细胞增殖、分化及生存方面起关键作用。成纤维生长因子受体有4种,配体与不同的受体结合会产生不同的效应。神经干细胞发育分化过程中的基因调控是一个复杂的过程,主要由快速反应基因和转录因子的激活来完成。 目的: 探讨bFGF对背根节神经干细胞沿施旺氏细胞方向分化的过程中结合的受体及可能的信号转导机制以及所涉及的快速反应基因和转录因子调控作用,以揭示其诱导分化的机制。 方法: 1. RT-PCR和Western Blotting检测成纤维生长因子受体在神经球中的表达,化学交联和免疫共沉淀检测与bFGF结合的受体。 2. Western Blotting检测bFGF下游的主要的信号通路被激活情况及分化过程中参与的信号转导通路。 3.检测bFGF激活的信号转导通路所起的生物学效应。在加入bFGF的同时加入MEK抑制剂U0126, PI3K抑制剂LY294002及BrdU标记液,收取细胞蛋白或固定细胞,Western Blotting或组化检测细胞的增殖与分化情况。 4.利用TransAMTM Assay kits (Active Motif)报告实验检测bFGF作用的细胞的快速反应基因c-jun, c-fos和Egr2(Krox20)的DNA的结合活性的变化;半定量RT-PCR检测与雪旺氏细胞分化相关的转录因子Mash-1及Sox10的表达变化。 结果: 1. RT-PCR和Western Blot结果显示背根节神经干细胞高表达FGFR1-3,而低表达FGFR4。体外化学交联和免疫共沉淀实验结果以及细胞内免疫共沉淀实验结果证明bFGF与FGFR1结合激活下游信号通路。 2.与对照组相比,bFGF刺激可持续激活Erk1/2及Akt的磷酸化,表明Erk1/2和PI3K/AKT两条信号通路在bFGF的生物功能中起作用。 3.背根节神经干细胞体外分化24小时,bFGF在不影响分化细胞的增殖和存活的情况下通过ERK1/2信号通路促进细胞向雪旺氏细胞方向分化。bFGF的刺激可在背根节神经干细胞内引起一系列的反应,包括ERK1/2和PI3K/AKT通路的激活,以及快速反应基因c-Jun的活化和转录因子Mash-1的下调。如果在bFGF处理的同时用ERK1/2上游蛋白MEK1/2的抑制剂U0126阻断ERK1/2的磷酸化,则bFGF介导的c-Jun的活化和转录因子Mash-1的下调及细胞沿施旺氏细胞分化被抑制。与之相对的,如果在bFGF处理的同时用PI3K的抑制剂LY294002阻断AKT的磷酸化,则对则bFGF介导的c-Jun的活化和转录因子Mash-1的下调及细胞沿施旺氏细胞分化并无影响。 结论: bFGF绑定FGFR1受体通过Erk1/2信号通路引起快速反应基因Jun的激活,下调转录因子Mash-1的基因表达水平,完成其在干细胞分化过程中的信号转导和生物功能。
[Abstract]:Tissue engineering technology brings new hope for the treatment of peripheral nerve injury, the optimization selection of seed cells is the key technology of tissue engineering. Schwann cells are the main structure and function of peripheral nervous system cells, seed cells is recognized, but its source is limited. The trunk neural crest stem cells, can differentiate into around the nervous system of most neurons and glial cells, peripheral nerve system primordia, will therefore trunk neural crest stem cells in neural tissue engineering has a good application prospect.
The differentiation of stem cells in vivo is regulated by many factors, with diversity and uncertainty, neural stem cell transplantation for treatment of nervous system injury or disease is necessity to induce neural stem cells to specific cell type differentiation we need. Therefore, induction of differentiation of neural stem cells is an important characteristic the study of the meaning of.BFGF with multi functions, which can promote the differentiation of neurons, but also can promote the differentiation of glial cells and the function of its changes and development in vivo was consistent. In the early stage of neural development promotes neuronal differentiation, whereas in the late development promote the differentiation of glial cells. In the peripheral nervous system, bFGF is the peripheral nervous system stem cells differentiate into Schwann cells was positive determinants of progenitor cells, so the use of bFGF induced dorsal root ganglion Stem cells Schwann cell differentiation is a very good attempt. This paper firstly isolated and cultured in vitro DRG neural stem cells in vitro, then induce Schwann cell differentiation and its functional identification; on the basis of bFGF in vitro induction of neural stem cells and Schwann's cell differentiation mechanism along to conduct the research.
The first part of the culture of dorsal root ganglion neural stem cells, identification and directional induction of its direction differentiation along Schwann cells
Objective:
Methods to establish adult rat dorsal root ganglion neural stem cells and to orientate their differentiation along Schwann cells, so as to provide real peripheral nerve system seed cells for regeneration of peripheral nervous system.
Method:
1. the dorsal root ganglia of adult SD rats were cultured in vitro, and then the primary neural stem cell balls were obtained after suspension culture. The single cell clones were obtained by infinite dilution method, and their stem cell characteristics were identified after 2 passages, including specific markers, proliferation rate and differentiation potential.
2. cloned NSCs were divided into 5 groups: differentiation, NGF group; bFGF group; NRG group; FBS group; and the control group. S100 positive cells were induced after 7 d S100 immunocytochemistry and Westernblot. immunocytochemistry were performed at 10 randomly selected groups of proteins through statistical perspective; the expression level of Western blot was detected by S100 extraction at the same time.
3. bFGF induced short time and long term effects of DRG differentiation, namely, a group of 24 h after adding differentiated culture fluid, washed bFGF and continued to culture; the other group always used bFGF containing differentiation medium,.7 D, S100 immunocytochemistry and Westernblot..
4. BrdU markers were used to detect the proliferation of bFGF cells in 24 hours, while adding bFGF to induce cell differentiation and adding BrdU marker, and immunocytochemical detection after 24 h.
Passage 5. by immunocytochemistry, culture in vitro myelination experiment method was used to identify the cytological and functional studies on bFGF induced cells obtained by two-dimensional electrophoresis; expression comparison of primary cultured Schwann cells and DRG by differentiation of stem cells protein spectrum difference.
Result:
1., a stable passage of dorsal root ganglion neural stem cells was obtained through in vitro isolation and culture and monoclonal experiments. It expressed specific Marker of neural crest stem cells, and had high proliferation and differentiation potential.
2. the proportion of S100 beta positive cells in the bFGF treated group of 5 groups induced by cell differentiation was higher than that in the other groups, and the results of Western Blot were consistent with this.
3., the short-term and long-term effects of induction of DRG differentiation showed that bFGF could affect cell differentiation within 24 h time, but had no effect on cell proliferation.
4. the differentiated cells have morphological and functional characteristics of Schwann cells.
Conclusion:
We successfully established a method for culturing dorsal root ganglion neural stem cells and directed the induction of differentiation into Schwann cells, providing a real peripheral nerve system seed cell for peripheral nerve regeneration.
Study on the mechanism of bFGF induced neural stem cells differentiation along Schwann cells in the second part of the neural stem cells
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