神经干细胞诱导分化为施万样细胞及其信号转导机制探讨

发布时间：2018-05-28 01:09

本文选题：神经干细胞 + 施万细胞　；参考：《中国医科大学》2009年博士论文

【摘要】： 前言施万细胞(Schwann cells, SCs)是周围神经系统结构和功能的主要细胞,在周围神经的发育和再生中起着重要的作用。周围神经损伤后,施万细胞进行增殖、迁移,并能产生多种神经营养因子,促进和引导远侧神经断端的生长,向不同的周围神经支架移植施万细胞能加快神经再生,因而施万细胞已经应用于临床神经系统再生和脱髓鞘疾病模型的治疗。然而,体外培养施万细胞的来源有限,培养周期长,并且容易受增殖更快的成纤维细胞污染和排斥,所以施万细胞的分离、纯化并达到治疗的数量十分困难,临床上的应用受到限制。因此,寻找一种容易获取、增殖周期短且免疫原性小的施万细胞来源,对周围神经损伤的修复治疗具有重要的意义。神经干细胞(neural stem cells, NSCs)存在于中枢神经系统的多个部位,有自我更新和多向分化能力,能分化为神经元、星形胶质细胞、少突胶质细胞等中枢神经系统细胞以及血细胞、肌细胞等其他系统细胞。神经干细胞因具有很高的增殖活性并能进行长期培养、在分化为其他细胞之前一直保持其表型不变、免疫原性低等优点被用于中枢神经系统疾病的细胞治疗,由于其具有优秀的重塑神经组织的潜能,被认为是神经系统再生合适的供体细胞。有报道,脂肪干细胞源性神经球能分化为施万样细胞,但是体外培养的中枢神经系统来源的神经干细胞是否能分化为施万细胞尚未见报道。促分裂原活化蛋白激酶(mitogenic activated protein kinase pathway, MAPK)通路对生长和分化极为重要,哺乳动物体内存在的主要的三条MAPK通路：细胞外信号调节激酶(extracellular signal regulated kinase, ERK)通路、c-jun氨基末端激酶(c-jun N-terminal kinase, JNK)通路、P38通路。其中,ERK是调节细胞生长增殖、分化、和凋亡的最基本信号途径,JNK、P38通路可被应激刺激、细胞因子、生长因子等激活。在不同的细胞系中,MAPK通路起到何种作用一直存在争议,此通路是否在神经干细胞诱导为施万样细胞过程中起到作用目前尚未见报道。本研究采用无血清培养技术培养新生大鼠海马神经干细胞,通过单细胞克隆培养、Nestin免疫荧光染色及诱导分化能力进行鉴定。通过向培养液中添加heregulin-β1、碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)、血小板源性生长因子(platelet-derived growth factor-AA, PDGF-AA)等诱导剂,新生大鼠海马神经干细胞可以分化为施万样细胞,进一步实验应用细胞外信号调节激酶(extracellular signal regulated kinase, ERK)通路、c-jun氨基末端激酶(c-jun N-terminal kinase, JNK)通路、P38通路通路抑制剂进行诱导分化干预,探讨这3条通路在神经干细胞向施万样细胞诱导中的作用,对获得大量的施万样细胞及临床上进行神经缺损的细胞治疗提供理论和实验基础。实验方-法本实验通过无血清培养技术体外培养新生大鼠海马神经干细胞,应用单细胞克隆技术对培养的神经干细胞进行纯化,应用免疫荧光染色对所培养细胞进行神经干细胞鉴定,应用免疫荧光染色和Western Blot技术测定分化后神经干细胞S-100和P75蛋白的表达情况,RT-PCR技术检测P0、Krox-20、Oct-6 mRNA表达；通过与神经元共培养的方法检测施万样细胞的功能,应用Western Blot、免疫荧光染色技术检测ERK、JNK、P38通路抑制剂对神经干细胞诱导分化的影响。实验结果 1、新生大鼠海马神经干细胞的分离培养和鉴定新生大鼠海马分离的神经干细胞呈神经球样,悬浮生长,折光性强,传代后细胞较原代培养细胞增殖加快,单细胞克隆培养形成的克隆球表达Nestin,诱导分化1w后细胞表达NSE、GFAP及Galc。 2、神经干细胞诱导分化结果向培养液中添加HRG、RA、FSK、PDGF-AA等诱导剂后,新生大鼠海马神经干细胞的形态发生改变,免疫荧光染色及Western Blot检测显示分化后细胞表达胶质细胞特异性标志：S-100和P75。应用RT-PCR检测P0、Krox-20和Oct-6mRNA在SCs、dNSCs及NSCs中的表达情况。结果显示,dNSCs和SCs中均有PO、Krox-20和Oct-6 mRNA的表达,SCs中的mRNA表达与相关报道相符。 3、不同浓度ERK1/2、P38、JNK抑制剂对神经干细胞增殖和凋亡影响当抑制剂浓度为5μM时,各组间未见明显差异。而当抑制剂浓度为10、15μM时,P38组可见干细胞球逐渐增大,与对照组相比有明显差异。3w时球中心细胞坏死,多个克隆球连接成片状。ERK组、JNK组2w时全部死亡。TUNEL法检测细胞凋亡,当抑制剂浓度为10μM时,与对照组相比, P38组细胞凋亡比例明显减低,而ERK、JNK组细胞凋亡比例明显升高。当抑制剂浓度为5μM时,与对照组相比,此3组细胞凋亡比例无明显变化。 4、应用诱导剂后磷酸化及总ERK、P38和JNK的表达情况 Western blot显示,神经干细胞在加入诱导剂后1h即可见磷酸化ERK1/2水平升高,并持续增加,8h左右达到高峰,此后逐渐降低,恢复正常。加入抑制剂后,与加入前相比,相应各组的ERK、P38和JNK的磷酸化水平显著降低,并长时间维持在较低水平。 5、加入抑制剂后,神经干细胞向施万细胞诱导分化结果 3w后,与其它组相比,ERK组施万样细胞所占百分比明显减少(P0.01)而P38组和JNK组与对照组相比,施万样细胞百分比未见明显变化(P0.05) 结论 1、FSK、RA、HRG、PDGF-AA、bFGF诱导剂能诱导新生大鼠海马神经干细胞分化为施万样细胞。 2、神经干细胞源性施万样细胞表达胶质细胞标志性蛋白：S-100和P75,表达P0、Krox-20、Oct-6等施万细胞标志性mRNA;分泌促进神经元轴突生长的可溶性营养因子。 3、神经干细胞分化为施万样细胞过程中磷酸化ERK表达增强,ERK信号转导通路被激活。 4、在神经干细胞分化为施万样细胞过程中,加入ERK信号转导通路抑制剂U0126,能够抑制神经干细胞分化为施万样细胞。P38、JNK信号转导通路抑制剂SB203580和SP600125对神经干细胞分化为施万样细胞无明显作用。
[Abstract]:Preface
Schwann cells (SCs) is the main cell of the structure and function of the peripheral nervous system. It plays an important role in the development and regeneration of the peripheral nerve. After the peripheral nerve injury, Schwann cells proliferate, migrate, and can produce a variety of neurotrophic factors to promote and guide the growth of the distal nerve broken ends and to the different surrounding gods. Schwann cells have been applied to the treatment of the regenerative and demyelinating disease models of the clinical nervous system. However, the source of Schwann cells in vitro is limited, the culture cycle is long, and the proliferation and rejection of fibroblasts are easy to grow, so the isolation and purification of Schwann cells It is very difficult to achieve the number of treatment, and the clinical application is limited. Therefore, it is of great significance to find a source of Schwann cells, which is easy to obtain, the proliferation cycle is short and the immunogenicity is small, and it is of great significance for the repair and treatment of peripheral nerve injury.
Neural stem cells (NSCs) exists in many parts of the central nervous system and has the ability of self renewal and multidirectional differentiation. It can differentiate into neurons, astrocytes, oligodendrocytes and other central nervous system cells, as well as blood cells, and muscle cells. Neural stem cells have high proliferation activity. It can be cultured for a long time and keep its phenotype unchanged before differentiation into other cells. The advantages of low immunogenicity are used in the cell therapy of central nervous system disease. Because of its excellent potential of remolding the nerve tissue, it is considered to be a suitable donor cell for the regeneration of the nervous system. It can differentiate into Schwann like cells, but it is not reported whether neural stem cells derived from central nervous system can differentiate into Schwann cells in vitro.
The mitogenic activated protein kinase pathway (MAPK) pathway is very important for growth and differentiation. There are three main MAPK pathways in mammals: extracellular signal regulated kinase (kinase, ERK) pathway, and amino terminal kinase. JNK) pathway, P38 pathway. In which, ERK is the most basic signaling pathway to regulate cell proliferation, differentiation, and apoptosis. JNK, P38 pathway can be stimulated by stress, cytokines, growth factors, etc. in different cell lines, the role of MAPK pathway has been controversial, whether this pathway is induced by neural stem cells for Schwann like cells. The role of the course has not yet been reported.
In this study, the rat hippocampal neural stem cells were cultured with serum-free culture. Nestin immunofluorescence staining and differentiation ability were identified by single cell clone culture. Heregulin- beta 1, basic fibroblast growth factor (basic fibroblast growth factor, bFGF), and platelet derived growth factors were added to the culture medium. Platelet-derived growth factor-AA (PDGF-AA) and other inducers, neonatal rat hippocampal neural stem cells can differentiate into Schwann like cells, and further experimental application of extracellular signal regulated kinase (extracellular signal regulated kinase, ERK) pathway, c-jun amino terminal kinase (c-Jun N-terminal) pathway, pathway pathway The inhibitors were induced and differentiated to explore the role of these 3 pathways in the induction of neural stem cells to Schwann cells, and to provide theoretical and experimental basis for obtaining a large number of Schwann cells and the clinical treatment of neural defects.
Experimental recipe - Method
In this experiment, the rat hippocampal neural stem cells were cultured in vitro by serum-free culture, and the cultured neural stem cells were purified by single cell cloning technology. Immunofluorescence staining was used to identify the neural stem cells in the cultured cells. Immunofluorescence staining and Western Blot technique were used to determine the S-100 of neural stem cells after differentiation. And the expression of P75 protein, RT-PCR technique was used to detect the expression of P0, Krox-20, Oct-6 mRNA, and the function of the Schwann cells was detected by co culture with neurons. The effects of ERK, JNK, P38 pathway inhibitor on the differentiation of neural stem cells were detected by Western Blot and immunofluorescence staining.
experimental result
1, isolation, culture and identification of neural stem cells from hippocampus of neonatal rats
The neural stem cells isolated from the hippocampus of the newborn rats were neuroglobule, suspended and refracted, and the proliferation of the cells was faster than that of the primary cultured cells. The cloned cells formed by single cell clone culture expressed Nestin, and the cells expressed NSE, GFAP and Galc. after the induction of 1W differentiation.
2, neural stem cells induce differentiation
After adding the inducers of HRG, RA, FSK, and PDGF-AA to the culture medium, the morphological changes of the neural stem cells in the hippocampus of the newborn rats were changed. The immunofluorescence staining and the Western Blot detection showed that the cells expressed the specific markers of glial cells after the differentiation: S-100 and P75. used RT-PCR detection P0, Krox-20 and Oct-6mRNA were expressed and expressed. The results showed that PO, Krox-20 and Oct-6 mRNA were expressed in dNSCs and SCs, and mRNA expression in SCs was consistent with relevant reports.
3, the effects of different concentrations of ERK1/2, P38 and JNK inhibitors on proliferation and apoptosis of neural stem cells
When the inhibitor concentration was 5 M, there was no significant difference between each group. When the inhibitor concentration was 10,15 M, the P38 group showed that the stem cell balls gradually increased. Compared with the control group, there was a significant difference between the cells of the central cell necrosis at.3w and the multiple cloned spheres connected to a group of.ERK groups. The total death.TUNEL method was used to detect the apoptosis when the JNK group 2W, when the concentration of the inhibitor concentration was determined. When compared with the control group, the percentage of apoptotic cells in the P38 group was significantly lower than that of the control group, while the proportion of apoptosis in the group of ERK and JNK was significantly higher than that of the control group. When the inhibitor concentration was 5 mu M, there was no significant change in the percentage of apoptotic cells in the 3 groups compared with the control group.
4, phosphorylation and the expression of total ERK, P38 and JNK after induction.
Western blot showed that after adding inducer to the neural stem cells, the level of phosphorylated ERK1/2 increased and continued to increase, and the 8h reached the peak at about 8h, and then gradually decreased and returned to normal.
After adding inhibitors, the phosphorylation levels of ERK, P38 and JNK in the corresponding groups decreased significantly, and maintained at a low level for a long time.
5, after the inhibitor was added, the neural stem cells differentiated into Schwann cells.
After 3W, compared with other groups, the percentage of Schwann cells in group ERK decreased significantly (P0.01), but there was no significant change in the percentage of Schwann cells in the P38 and JNK groups (P0.05).
conclusion
1, FSK, RA, HRG, PDGF-AA and bFGF inducers can induce neural stem cells to differentiate into Schwann cells in neonatal rats.
2, neural stem cells derived Schwann cells express glial cell marker proteins, S-100 and P75, to express the marker mRNA of P0, Krox-20, Oct-6 and other Schwann cells, and secrete soluble nutrient factors that promote the growth of neuron axons.
3, the expression of phosphorylated ERK increased during the differentiation of neural stem cells into Schwann like cells, and the ERK signal transduction pathway was activated.
4, during the differentiation of neural stem cells into Schwann like cells, the addition of ERK signal transduction pathway inhibitor U0126 can inhibit the differentiation of neural stem cells into Schwann like cells.P38, and the JNK signal transduction pathway inhibitor SB203580 and SP600125 have no obvious effect on the differentiation of neural stem cells into Schwann like cells.
【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329.28

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