【作者】 刘素芳；

【导师】 邢莹；

【作者基本信息】 郑州大学 ， 神经生物学， 2013， 博士

【摘要】 外周神经损伤是神经系统疾病中的常见形式,当神经损伤远端部位与神经元胞体失去联系后,即发生一系列神经脱髓鞘及神经降解的过程。目前许多研究致力于干细胞治疗外周神经损伤。虽然从形态学上发现神经结构已经恢复,但是恢复的神经纤维却并不能发挥相应的功能或者损伤部位神经纤维发生错配。并且大部分干细胞或诱导分化后的细胞在移植早期就趋向凋亡,因此提高移植细胞的存活、分化、迁移和促进神经网络形成以改善治疗效果迫在眉睫。研究表明,神经干细胞可以分化为雪旺细胞或者运动神经元,用来治疗外周神经损伤,并且成体神经干细胞主要存在于海马和室管膜下区,因此,为了观察干细胞治疗外周神经损伤的机制以及优化干细胞治疗外周神经损伤的效果,本研究首先观察了体外培养的海马神经干细胞诱导分化后的神经细胞在某些调控因素下的迁移、突起延伸及其机制,其次将海马神经干细胞作为种子细胞,采用坐骨神经损伤的SD大鼠动物模型,通过对细胞进行不同处理,优化神经干细胞治疗外周神经损伤的途径,从而观察神经干细胞对外周神经损伤的疗效。在神经系统发育和成熟过程中,神经细胞迁移与脑内氧浓度和相关细胞因子有关。本研究发现,体外培养的海马神经干细胞诱导后的神经细胞,缺氧(3%)预处理4h后,基质细胞衍生因子(stromal cell derived factor, SDF)1α表达上调。既然缺氧与神经细胞迁移有关,那么SDF1α是否与细胞迁移有关?缺氧和SDF1α是否影响神经细胞突起延伸以形成神经网络?缺氧和SDF1α是否有利于神经干细胞治疗外周神经损伤?以及这些作用的机制是什么?为了解决上述问题,本课题采用了两部分实验,即体内实验和体外实验进行观察。体外实验中采用了体外培养的海马神经干细胞诱导的神经细胞(简称神经细胞)进行观察,体内实验中采用了海马神经干细胞移植入体内(简称神经干细胞),观察治疗外周神经损伤的效果。体外实验观察了缺氧和趋化因子SDF1α对神经细胞的突起延伸、actin聚集等形态的影响,并且观察了信号分子ERK和有关通路在其中的作用。为了更进一步明确机制,通过沉默SDF1α的新型受体CXCR7,观察在缺氧条件下神经细胞对SDF1α的反应以及细胞的形态学变化。其次体内实验观察了坐骨神经损伤大鼠模型中,缺氧和趋化因子SDF1α预处理海马神经干细胞后移植入大鼠体内,观察动物的行为学变化和坐骨神经的恢复情况,以期评价和优化神经干细胞治疗外周神经损伤的途径和疗效。实验方法和结果：第一部分1.缺氧后体外培养的海马神经细胞SDF1α的表达：给予体外培养的神经细胞缺氧(3%O2)预处理4h,观察缺氧后0.5h、1h、12h、24h、36h后SDF1α的表达,结果显示随着缺氧时间的延长,SDF1α的表达逐渐增加,至缺氧后24h达到高峰。2.缺氧和SDF1对神经细胞轴突延伸、actin聚集和细胞迁移的影响：SDF1α作用24h后,神经细胞突起增多,轴突延伸,actin在突起的聚集增加,缺氧则导致神经细胞突起减少,轴突变短,actin在突起的聚集减少。但是在缺氧预处理后,给予SDF1α作用24h,则可以拯救这一状态,细胞突起增多,轴突延伸明显,actin在突起的聚集增加。缺氧预处理组和对照组神经细胞,在SDF1α分别作用0.5h,1h,12h,24h,36h后,随着SDFla作用时间延长,神经细胞迁移增加,但是缺氧预处理的神经细胞则显示出比对照组细胞对SDF1α较高的敏感性和反应性。3.缺氧预处理后CXCR7在海马神经细胞的表达：随着缺氧后时间的延长,从0.5h,1h,12h到24h,SDF1α的新型受体CXCR7表达逐渐增加,在缺氧后24h,CXCR7蛋白表达增加至对照未缺氧组的两倍之多,达到了所观察的时间段内的表达高峰。4.CXCR7对细胞形态,actin聚集和细胞迁移能力的影响：为了观察CXCR7对海马神经细胞突起和轴突延伸的影响,首先对神经细胞进行CXCR7shRNA预处理3天,阻断CXCR7的作用,然后加入5Ong/ml的SDFla作用24h和36h,观察细胞的突起生长和延伸,正常对照组细胞在SDF1α作用24h和36h下,轴突延伸明显,但阻断CXCR7的表达后,神经细胞突起减少,轴突延伸消失。阻断CXCR7后,突起内actin聚集减少,并且随之24h的SDF1α处理,也未能增加actin在突起的聚集。神经细胞迁移实验则显示,在SDF1α作用0.5h,1h,12h后,对照组和CXCR7shRNA处理组神经细胞迁移数量没有差异,但是经过慢性SDF1α处理(24h)后,CXCR7shRNA处理组神经细胞迁移数量较对照组下降。5.缺氧预处理后,CXCR7对细胞形态,actin聚集和细胞迁移能力的影响：将体外培养的海马神经细胞首先进行CXCR7shRNA转染,对照组神经细胞不进行CXCR7shRNA处理,3天后,所有细胞均行缺氧预处理4h,然后观察神经细胞形态的变化。结果显示,SDF1α处理24h后,单纯缺氧组细胞轴突延伸、突起延长、分支增多。但是CXCR7shRNA预处理后的缺氧组神经细胞,SDF1α则未能改变神经细胞的形态,表现为细胞突起较短,分支减少。神经细胞迁移实验结果显示,随着SDF1α作用时间延长,从0.5h,1h,12h,24h到36h,缺氧组神经细胞迁移明显,但是经过CXCR7shRNA预处理后的缺氧组神经细胞则迁移能力下降,并且表现出对SDF1α的反应性下降。6.CXCR7沉默和缺氧预处理对神经细胞内ERK信号的影响：缺氧和SDF1α均增加了神经细胞内ERK1/2磷酸化过程,但是CXCR7沉默后,神经细胞内ERK1/2磷酸化下降,且随之的SDF1α作用,也未能增加ERK1/2的磷酸化。由于在体外实验中观察到,SDF1α促进了海马神经干细胞诱导分化后的神经细胞的迁移和突起延伸,而缺氧增加了神经细胞SDF1α的反应,因此在体内实验中,采用了海马来源的神经干细胞作为种子细胞移植,治疗外周神经损。第二部分：1.动物分组：将成年雄性SD大鼠随机分为6组,分别为假手术组、手术组、神经干细胞治疗组、缺氧预处理神经干细胞治疗组、SDF1α预处理神经干细胞治疗组、SDF1α和缺氧两因素预处理神经干细胞治疗组。假手术组只暴露坐骨神经,但不行坐骨神经钳夹损伤；手术组按前述方法行坐骨神经钳夹；神经干细胞治疗组,采用神经干细胞局部移植治疗坐骨神经损伤；缺氧预处理干细胞治疗组,采用缺氧预处理神经干细胞4h,后移植入坐骨神经损伤阶段；SDF1α预处理神经干细胞治疗组,采用SDF1α预处理神经干细胞24h后,局部移植入损伤部位,观察坐骨神经损伤恢复情况；SDF1α和缺氧两因素预处理神经干细胞治疗组,采用缺氧预处理神经干细胞4h后,随之SDF1α处理24h,然后将神经干细胞移植入坐骨神经损伤处。神经干细胞移植在神经损伤的同日进行。然后选取神经干细胞移植后1w,2w,3w和4w进行行为学评价和免疫组化分析。2.步态行为学测试：步态分析采用Catwalk XT分析软件进行。电脑会自动记录动物的完整运动并且自动获得最大接触平均压力(max contact mean intensity, MCMI)、爪印面积(print area, PA),步长(stride length, SL)等有关动物运动和感觉功能恢复的指标。MCMI反映了动物在最大接触下对玻璃板的平均压力,PA反映了动物在玻璃板上行走时的接触面积,SL反映了动物行走时步与步之间的距离。实验分别于手术/干细胞移植后1w、2w、3w、4w对动物进行步态分析,观察坐骨神经损伤后运动和感觉功能恢复情况。由于假手术组动物在第1w即完全愈合,而损伤组动物无论是否接受神经干细胞治疗,动物在术后1w内均无法行走,因此本实验从术后1w开始观察动物行为学变化。MCMI、PA口SL分析结果显示,缺氧和SDF1α两因素预处理的神经干细胞组显示出在6组中最好的治疗效果,在第2w就达到了假手术组的水平,而神经干细胞治疗组,无论是否经过缺氧或SDF1α预处理,均在第3周或以后才达到假手术组的水平,而手术组,在第4w仍然没有恢复到假手术组的水平。3.免疫组化分：3.1坐骨神经损伤节段的雪旺细胞标识物S100的表达：免疫组织化学结果显示,在术后2w,缺氧和SDF1α两因素预处理的神经干细胞治疗组显示出类似于假手术组的结果,雪旺细胞标志物S100强着色；在术后4w,除了手术组,其他神经干细胞治疗组无论是否经缺氧或SDFlα预处理,均显示出雪旺细胞标志物S100的强着色。提示损伤的坐骨神经已经恢复。3.2坐骨神经逆向标记：本实验中采用Fluoro-Ruby染料(橘红色)逆向标记坐骨神经,观察神经修复情况。该物质是荧光素的一种,经神经分支的末梢吸收,循轴突逆行输送至胞体。在荧光显微镜下可看到胞体内呈现荧光标记物。术后2w脊髓内Fluoro-Ruby P日性细胞数结果显示,缺氧和SDF1α两因素预处理神经干细胞组脊髓内Fluoro-Ruby阳性细胞数达到了假手术组的水平,而神经干细胞组、缺氧预处理神经干细胞组和SDF1α预处理神经干细胞组脊髓内Fluoro-Ruby阳性细胞数虽然均较手术组有显著性差异,但是均未能达到假手术组的水平。术后2w DRG内Fluoro-Ruby阳性细胞数结果显示,缺氧和SDF1α两因素预处理神经干细胞组DRG内Fluoro-Ruby阳性细胞数达到了假手术组的水平,而神经干细胞组、缺氧预处理神经干细胞组和SDF1α预处理干细胞组DRG内Fluoro-Ruby日性细胞数虽然均较手术组有显著性差异,但是均未能达到假手术组的水平。术后4w脊髓内Fluoro-Ruby阳性细胞数结果显示,神经干细胞治疗组,无论是否经过缺氧或SDF1α预处理,脊髓内Fluoro-Ruby阳性细胞数均达到了假手术组的水平,而手术组的脊髓内Fluoro-Ruby日性细胞数则未能达到假手术组的水平。术后4w DRG内Fluoro-Ruby阳性细胞数结果显示,神经干细胞治疗组,无论是否经过缺氧或SDF1α预处理,DRG内Fluoro-Ruby阳性细胞数均达到了假手术组的水平,而手术组的DRG内Fluoro-Ruby阳性细胞数则未能达到假手术组的水平。结论：1缺氧和SDF1α可诱导海马神经细胞突起增多,轴突延伸,actin聚集和ERK磷酸化增加,并且沉默CXCR7可减弱神经细胞缺氧后对SDF1α的反应。2缺氧和SDF1α预处理增加了神经干细胞治疗外周神经损伤的效果。更多还原

【Abstract】 Previous studies have demonstrated that nerve damage results in rapid disruption of nerve function, which is a serious issue in clinics. Neural stem cells (NSCs) have been shown to promote peripheral nerve regeneration. However, some results still show low efficiency of transplanted cells in vivo and some miscommunication after nerve recovery. So it is necessary to optimize stem cell therapy strategy and improve microenvironment so as to increase therapy efficiency. Previous studies have shown that chemotactic factor stromal-cell derived factor1α (SDF1α) promotes cell recovery from hypoxic injury. However, the role of SDF1α in neural stem cell therapy for peripheral nerve injury remains largely unknown. In this study, experiments were divided into two parts, in vivo and in vitro.In the first part, neurons induced from hippocampal progenitor cells were pre-conditioned in hypoxia for4h and subsequently monitored to investigate the effect of SDFla on cell repair after hypoxia. We assessed neuronal morphology, actin filament polymerization and migration capability. SDFla protein levels increased significantly1h after hypoxia compared to control group (P<0,01), and it reached to a peak level at24h after hypoxia. Moreover, SDFla incubation promoted neurite outgrowth and actin filament polymerization both in normoxic and hypoxic cells.. Cell migration showed a time-dependent increase with SDF1α stimulation in both groups, and hypoxic cells illustrated a significantly augmented migration at0.5h,1h and12h after SDFla application compared to normoxic cells (P<0.01). CXCR7expression also increased time-dependently after hypoxia and demonstrated a two-fold upregulation compared to that in the control group at24h after hypoxia. With CXCR7silencing, axon elongation and actin filament polymerization induced by SDFla were inhibited markedly both in normoxic and hypoxic cells. CXCR7silencing also led to reduced hypoxic cell migration at0.5h,1h,12h,24h and36h after SDFla application (P<0.01), but it failed to reduce normoxic cell migration induced by SDF1α at0.5h,1h and12h (P>0.05). SDF1α stimulation for24h led to higher ERK1/2phosphorylation compared to the control group, and ERK1/2phosphorylation increased more in hypoxic cells than that in normoxic cells. In conclusion, this study suggests that CXCR7plays an important role in cell repair processing induced by SDFla, and that CXCR7silencing attenuates cell adaptive response to acute SDFla stimulation (≤12h) after hypoxia.In the second part, rat sciatic nerve crush model was made by giving the sciatic nerve crush (1cm width at500g for lmin) to observe the neural stem cell therapy. Neural stem cells derived from hippocampus were injected into the crush-injured segment of the sciatic nerve immediately after crush to observe the effects of NSC transplantation on neural regeneration. Animals were divided randomly into6groups:1) sham group,2) surgery group,3) NSCs group,4) hypoxia pretreated-NSCs group,5) SDFla pretreated-NSCs group,6) hypoxia plus SDFla pretreated-NSCs group. Gait analysis by Catwalk testing and histopathology assessment were performed to systematically evaluate the nerve regeneration among different groups. Our results showed that injection of NSCs produced a significant improvement in max contact mean intensity (MCMI), print area (PA) and stride length (SL) compared to the untreated group at2weeks after crush (P<0.01).In addition, hypoxia pretreated-NSCs group and SDF1α pretreated-NSCs group showed improved therapy results in MCMI, PA and SL compared to the NSCs alone group (P<0.01). Moreover, hypoxia plus SDF1α pretreated-NSCs group showed the best results among these groups, and the functional recovery in this group reached to a similar level as shown in the sham group. At4weeks after crush, the animals in NSCs group, hypoxia pretreated-NSCs group, SDF1α pretreated-NSCs group, and hypoxia plus SDF1α pretreated-NSCs group showed normal behaviors in gait analysis. At2weeks after crush, histopathology assessment showed that the highest expression of Schwann cells marker S100was in the cross sections of sciatic nerves in hypoxia plus SDF1α pretreated-NSCs group. In addition, at4weeks after crush, all NSC treatment groups showed higher expression of S100compared to that in the surgery group. In our retrograde labeling experiments, Fluro-Ruby was directly injected at4-5mm distal from the crush cite in the tibial nerve. The L3-S2segments of the spinal cord and L4dorsal root ganglia (DRG) were harvested and sections were made by cryostat. At2weeks after crush, the number of positive cells in the spinal cord and L4DRG in the hypoxia plus SDFla pretreated-NSCs group reached to a similar level as shown in the sham group and no significant difference was observed between the two groups. Moreover, at4weeks after crush, the number of positive cells in the spinal cord and L4DRG in all NSC treatment groups showed no significant difference with that in the sham group. In conclusion, SDF1α and hypoxia may increase the efficiency of neural stem cell transplantation by promoting nerve regeneration and functional recovery after sciatic nerve crush injury.Conclusions:1. Hypoxia and SDFla may promote neurite outgrowth, actin filament polymerization, and ERK phosphorylation. And CXCR7silencing may reduce the cell response to SDF1α after hypoxia.2. Hypoxia and SDF1α may increase the efficiency of neural stem cell transplantation by promoting nerve regeneration and functional recovery after sciatic nerve crush injury.更多还原