【作者】 佟雷；

【导师】 佟晓杰；

【作者基本信息】 中国医科大学 ， 人体解剖与组织胚胎学， 2009， 博士

【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(P<0.01)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(P>0.05)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。更多还原

【Abstract】 PrefaceSchwann cells (SCs) are the main neuroglial cells of the PNS. They play essential roles in the regeneration of peripheral nerves after injury. Regeneration mechanism of peripheral nerve injury is very complicated and affected by many factors. Treatment by direct end-end surgical repair is used to minor defects and longer gaps depend on autologous nerve grafts. The therapy of long gap peripheral nerve injuries is clinically limited because of grafts availability and donor-site morbility. Tissue engineering techniques which promote the beneficial endogenous responses to nerve injury could provide an alternative repair strategy. Schwann cells can release neurotrophic factors and play a critical role in peripheral nerve regeneration. Moreover, it has been known for many years that they are capable of sustaining remyelination of CNS axons, and unlike oligodendrocytes, their myelin does not present a target for autoimmune demyelinating diseases. However, the requirement for nerve donor material evokes additional morbility and the production of sufficient numbers of SCs is a prerequisite for applying these cells for grafting.Neural stem cells are pluripotent cells capable of self-renewal and generation of multiple, distinct cell types, including neurons, astrocytes, oligodendrocytes of central nervous system and hemocytes, myocytes of other systems. Neural stem cells are experimentally used for the source of transplantation to the disease models and proper donor-cells for regeneration of central nervous system because these cells have high proliferative activation, long-term cultivation potential, the potential in an undifferentiated state before differentiation into other cells and low immunogenicity. A number of studies have now demonstrated that neurospheres from rat adipose-derived stem cells could differentiate into Schwann cells. However, it is not known up to now whether neural stem cells from central nervous system could be induced into Schwann cells.Mitogenic activated protein kinase pathway (MAPK) plays a crucial role in growth and differentiation. There are three major forms of MAPK pathway in mammals, including extracellular signal regulated kinase (ERKs), c-jun N-terminal kinase (JNKs) and P38 MAPK. ERKs are basic signal pathway in mediation of cell proliferation, differentiation and apoptosis. JNKs and P38 pathway are activated by several conditions, such as environmental stress, cytokine, growth factors. The effects of MAPK in different cell lineages are controversial and it is not known up to now whether MAPK had any effects in the process that neural stem cells differentiated into Schwann cells.In this study, we investigated whether neural stem cells from hippocampus of neonatal rats could differentiate into Schwann cells in the media containing HRG, bFGF, PDGF-AA. We cultured neural stem cells in the media containing inhibitors of ERKs, JNKs and P38 MAPK in order to know the effects of the three MAPK pathways in the differentiated process. It would supply theory and experimental foundation of harvesting large number of Schwann cells and clinical therapy for nerve injury by cell transplantation.MethodsThis study cultured neural stem cells of hippocampus from neonatal rats in the serum-free media. Monoclone was used to purify neural stem cells identified by immunofluorescence staining. The level of S-100 and P75 in the differentiated cells were detected by immunofluorescence staining and western blot. The level of P0, Krox-20 and Oct-6 mRNA in the differentiated cells were evaluated by RT-PCR. The function of differentiated cells was evaluated by lengths of neurites co-cultured with Schwann-like cells. The effects of inhibitors of ERK、JNK、P38 pathways in the differentiation of neural stem cells were detected by western blot.Results1. Culture and identification of neural stem cells of hippocampus of neonatal ratsNeural stem cells dissociated from hippocampus of neonatal rats were suspended growing, neural spheres-like and have intensive light refraction. Passaged cells grew faster than primary culture. The cultured monoclonal spheres expressed Nestin, and expressed NSE, GFAP and Galc one week after induced differentiation.2. Differentiation of neural stem cellsThe shapes of neural stem cells changed in the media containing HRG, RA, FSK, and PDGF-AA. Differentiated cells expressed S-100 and P75 detected by immunofluorescence staining and western blot. The expressions of P0、Krox-20 and Oct-6 mRNA in the Schwann cells and differentiated cells were consistent with the results reported by Marie.3. The effects of proliferation and apoptosis of neural stem cells induced by inhibitors of ERK1/2、P38、JNK pathways in different concentrationThere were no significant differences among the groups that the concentration of inhibitors was 5μM. The cells in P38 group increased gradually and have significant difference with control group. There was necrosis in the cells of central spheres and connected in the flake in P38 group 3 weeks after culture. However, cells in ERK group and JNK group were died 2 weeks after culture. Apoptosis of cells was detected by TUNEL staining. The proportion of apoptosis cells in P38 groups was lower than control group, and that in ERK group and JNK group were higher when the concentration of inhibitors was 10μM. However, there were no significant differences among the four groups that the concentration of inhibitors was 5μM.4. The expression of phosphorylated ERK, phosphorylated P38, phosphorylated JNK and total ERK, total P38, total JNKThe level of phosphorylated ERK of neural stem cells increased gradually and reached the peak 1 hour after adding inducer. Then its level decreased and recovered to normal. The levels of phosphorylated ERK, phosphorylated P38 and phosphorylated JNK were decreased evidently after adding inhibitors and maintained in a normal level for a long time.5. The results of differentiation Schwann-like cells from neural stem cells after adding inhibitorsThe percentage of Schwann-like cells in the ERK group was lower than control group and there was no significant difference between control group and P38 group, JNK group.更多还原