自体激活雪旺细胞移植治疗急性脊髓损伤的实验研究

目的 探讨自体激活雪旺细胞（autologous activated Schwann cells,AASCs）移植治疗急性脊髓损伤的疗效.方法 通过结扎单侧隐神经从而激活自体雪旺细胞并进行体外分离、培养及纯化,测定其不同培养时期培养基中神经生长因子、脑源性神经营养因子含量的变化.Wistar大鼠90只,以纽约大学脊髓损伤打击器建立T10急性脊髓损伤模型.随机分为3组,每组30只：单纯DMEM移植对照组、自体未激活雪旺细胞（autologous Schwann cells,ASCs）移植组和AASCs移植组.对各组实验动物脊髓损伤后肢体功能的恢复情况进行行为学评分（BBB评分）、体感诱发电位与运动诱发电位（somatosensory evoked potential and motor evoked potentials,SEP ＆ MEP）、生物素标记的葡聚糖胺示踪皮质脊髓束（corticospinal tract,CST）观察,比较各组差异.结果 BBB评分4周以后组间比较差异有统计学意义（P＜0.05）,其中细胞移植组明显高于对照组（P＜0.05）;SEP、MEP潜伏期和波幅值8、12周后组间比较差异有统计学意义（P＜0.05）;CST标记距离损伤中心向头端方向0.6、1.2、1.8、2.4mm和距离损伤中心向尾端方向0.6、1.2、1.8mm组间比较差异有统计学意义（P＜0.05）.结论 AASCs可分泌大量神经营养因子,并能明显促进急性脊髓损伤后的轴突再生和肢体功能恢复.     

嗅神经髓鞘细胞移植促进大鼠横断脊髓轴突再生的研究

目的;观察嗅神经髓鞘细胞（OECs)对脊髓轴突再生的促进作用。方法：将分离,纯化与体外培养2周的SD大鼠OECs,移植于12只成年SD大鼠第10胸椎横断模型的脊髓两断端;8只对照动物注入DMEM／F12培养基,移植后2周和8周进行神经嗜银染色与髓磷脂碱性蛋白（MBP）组织化学和神经生长因子受体（NGFR）免疫组织化学研究。结果：OECs移植组2周后脊髓两端在形态学上呈现初步愈合现象,组织学观察显示,移植OECs与损伤脊髓组织整合良好,再生轴突长入断端组织,8周时再生轴安全检查明显增多,OECs与再生轴突均呈MBP和NGFR阳性表达。对照组两断端液化坏死,未见再生轴突。结论：OECs移植可保护损伤的脊髓并促进宿主脊髓轴突再生。     

许旺细胞与PLGA共同移植于大鼠全横断脊髓损伤的实验研究

目的观察许旺细胞与PLGA组织工程材料共同移植于大鼠全横断脊髓损伤处对脊髓修复的影响。方法许旺细胞与PLGA体外共同培养后行扫描电镜观察,将其共移植于大鼠横断性脊髓损伤处,不同时间行BrdU／MBP免疫组化、天青-美蓝染色、透射电镜、运动功能评分（BBB）、运动诱发电位（MEP）、股四头肌复合肌肉动作电位（CMAP）、体感诱发电位（SEP）检测。结果扫描电镜下许旺细胞在PLGA内生长良好。6个月后移植物内可见BrdU／MBP免疫组化双染阳性细胞,天青-美蓝染色和透射电镜观察可见新生的髓鞘及形成髓鞘的许旺细胞。BBB评分各组间无显著性差异,MEP和SEP检测移植后6个月所有大鼠波形仍未恢复,但T（11-12）椎间隙可记录到SEP波。结论许旺细胞与PLGA具有良好的组织相容性,共同移植于损伤脊髓可促进轴突生长及髓鞘再生,但没有与损伤远端联系。     

星形胶质细胞活化与脊髓损伤修复

脊髓损伤后星形胶质细胞活化并分泌多种细胞外基质共同组成的胶质瘢痕,是阻碍神经轴突再生的重要因素。星形胶质细胞活化与TGF-β、Rheb-mTOR等信号通路的激活密切相关,并受到细胞外基质中高分子量透明质酸抑制作用的影响。细胞周期调控是近年报道的抑制星形胶质细胞活化、促进轴突再生的重要手段,而降解星形胶质细胞活化后分泌的多种抑制性蛋白,尤其是硫酸软骨素蛋白多糖则早已受到关注。胶质瘢痕对于脊髓损伤的修复是一把双刃剑,因此干预星形胶质细胞活化的时机亦显得非常重要。     

神经元胞外基质网络在脊髓损伤修复作用中的研究进展

神经元胞外基质网络是由细胞外基质分子高度凝聚且环绕神经元形成的复杂的网络结构.在维持神经元性能、保护神经元免受有害物质的影响等方面起重要作用.然而,在脊髓损伤后,神经元胞外基质网络形成一道包裹在神经元外,限制神经可塑性的物理屏障,阻碍神经元轴突再生和髓鞘形成,同时,也会促进局部神经炎症吸收.本文主要阐述神经元胞外基质网...     

人脐血单个核细胞联合雪旺细胞修复脊髓损伤的实验研究

目的 探讨人脐血单个核细胞(haman cord blood mononuclear cells,HCMNCs)与大鼠雪旺细胞(Schwann cells,SCs)联合移植应用于大鼠脊髓损伤的疗效.方法 健康成年8周龄Wistar雌性大鼠40只,体重(200±30)g.利用Impactor Model Ⅱ型打击器制成T10脊髓损伤模型.40只大鼠随机分成4组,每组10只,即DMEM实验对照组、HCMNCs移植组、SCs移植组、HCMNCs+SCs联合移植组.用HE染色、顺行示踪染色及电镜观察脊髓损伤处轴突再生情况,对各组实验动物脊髓损伤后肢体功能的恢复情况进行行为学评分(BBB评分)及脚印分析实验,综合评估脊髓功能恢复程度.结果 HCMNCs+SCs联合移植治疗能够明显促进神经再生,改善功能,减少空洞形成.BBB评分和脚印分析实验显示各组疗效比较,差异有统计学意义.组织学和电镜观察神经轴突再生情况与功能学检查的结果 相吻合.结论 以分泌各种神经营养因子为特点的SCs为平台,联合移植后诱导HCMNCs向神经元及神经胶质细胞分化,可促进脊髓损伤的恢复.     

不同途径移植自体激活雪旺细胞修复大鼠脊髓损伤

目的 通过不同途径移植自体激活雪旺细胞(autologous activated Schwann cells,AASCs),评价各种移植途径在修复脊髓损伤中的作用.方法 结扎Wistar大鼠双侧隐神经,1周后取下结扎处远端神经,在体外分离、培养、传代、纯化和鉴定后获得供实验用AASCs.60只Wistar大鼠制成T_(10)脊髓损伤模型后随机分为三组,1周后将预先用Hoechst33342标记的AASCs移植到三组大鼠体内:Ⅰ组,尾静脉移植;Ⅱ组,鞘内移植(经蛛网膜下腔);Ⅲ组,局部损伤处移植.术后采用BBB评分评价大鼠功能恢复.3个月后行BDA皮质脊髓束顺行示踪标记.标记2周后处死动物,取出损伤处脊髓行快速冰冻切片,行Cy3荧光探针染色、神经丝蛋白200(NF200)和HE染色.结果 AASCs在体外可稳定传4代以上,并表达S-100抗原.从术后第4周开始,BBB评分在各组间差异有统计学意义.至实验结束时,HE染色显示Ⅲ组中损伤空洞明显小于其余两组,NF200免疫组化染色阳性面积占总面积百分比各组间差异有统计学意义.BDA神经示踪显示,Ⅲ组中有较多的再生轴突通过脊髓损伤区,横断面上再生轴突的免疫组化阳性面积各组间差异有统计学意义.结论 局部损伤处移植AASCs可以有效保证移植细胞的数量,AASCs通过分泌多种营养因子和桥接损伤轴突再生的作用促进大鼠脊髓损伤后的功能恢复.     

脊髓损伤的细胞移植治疗

目前，神经科学的发展已经改变了以往认为脊髓损伤（SCI）后神经轴突不可能再生的观点，并将脊髓损伤的修复分为四个方面：（1）细胞存活；（2）神经轴突的再生；（3）再生轴突的正确走向；（4）正确的、有功能的神经突触联系的建立。近年来，应用活细胞和组织移植修复脊髓损伤并提供治疗因子是研究热点。现将细胞移植治疗脊髓损伤的概况综述如下。     

带蒂神经植入脊髓后许旺细胞的存活与迁移

目的：探讨神经组织移植后许旺细胞存活、增殖及诱导轴突再生的能力。方法：成鼠胸髓损伤后,分别移植带血管蒂正中神经（VN组）和游离正中神经（PN组）,术后8周行组织学检查和形态计量分析。结果：带血管蒂正中神经组移植神经与脊髓连接紧密,有较多新生轴突,许旺细胞大量存活和增殖,NF,S－100阳性反应均明显高于游离正中神经组。结论：带血管蒂周围神经移植较接近生理状态,许旺细胞存活并向脊髓实质内迁移和大量增殖,其诱导损伤轴突再生的能力也优于游离周围神经移植。     

嗅鞘细胞移植修复脊髓损伤的研究进展

脊髓损伤(spine cord injury,SCI)是骨科领域常见的疾病,损伤后果严重,给受害者个人和社会都带来了巨大负担。其治疗一直是科学家和临床医生面临的颇具挑战性的问题。以往一直认为脊髓损伤后,将引起损伤段以下永久性感觉、运动和括约肌功能丧失。然而近年来研究发现,嗅神经鞘细胞(olfactory ensheathing cells,OECs)不仅能促进轴突再生,且能引导再生的轴突重新进入中枢神经系统,从而与靶器官形成新的功能连接[1]。因此,OECs被认为是细胞移植修复SCI中很有希望的种子细胞之一,并且具有潜在的临床应用价值。1OECs促进SCI修复的功能OE…     

Schwann cell transplantation for repair of the adult spinal cord

The Schwann cell is one of the most widely studied cell types for repair of the spinal cord. These cells play a crucial role in endogenous repair of peripheral nerves due to their ability to dedifferentiate, migrate, proliferate, express growth promoting factors, and myelinate regenerating axons. Following trauma to the spinal cord, Schwann cells migrate from the periphery into the injury site, where they apparently participate in endogenous repair processes. For transplantation into the spinal cord, large numbers of Schwann cells are necessary to fill injury-induced cystic cavities. Several culture systems have been developed that provide large, highly purified populations of Schwann cells. Importantly, the development of in vitro systems to harvest human Schwann cells presents a unique opportunity for autologous transplantation in the clinic. In animal models of spinal cord injury (SCI), grafting Schwann cells or peripheral nerve into the lesion site has been shown to promote axonal regeneration and myelination. However, axons do not regenerate beyond the transplant due to the inhibitory nature of the glial scar surrounding the injury. To overcome the glial scar inhibition, additional approaches such as increasing the intrinsic capacity of axons to regenerate and/or removal of the inhibitory molecules associated with reactive astrocytes and/or oligodendrocyte myelin should be incorporated. Clearly, Schwann cells have great potential for repair of the injured spinal cord, but they need to be combined with other interventions to maximize axonal regeneration and functional recovery.     

脊髓损伤修复研究进展

脊髓损伤治疗至今仍是世界性医学难题。随着医学的发展和临床治疗的需求,学者们越来越关注脊髓损伤后修复的实验研究,以更好地为临床服务。该文就脊髓损伤修复研究中信号转导通路、神经营养因子、细胞移植、剔除神经生长抑制因子及基因治疗等方面作一综述。     

干细胞移植治疗脊髓损伤的研究进展

创伤引起的脊髓损伤通常导致严重的肢体瘫痪,目前,药物疗法和康复疗法治疗脊髓损伤的效果并不明显.近年来,干细胞移植因其理论上的可行性及实验中可观的效果引起国内外学者广泛关注,用于移植的干细胞包括胚胎干细胞、神经干细胞、间充质干细胞及诱导性多能干细胞等.笔者查阅了国内外相关文献,在本文中将对不同干细胞移植治疗脊髓损伤的现状及前景作一讨论和分析.     

Characteristic improvement of the function following Schwann cell transplantation for demyelinated spinal cord

Transplantation of Schwann cells (SCs) induced remyelination of demyelinated rat dorsal column (DC) axons and improved conduction. To investigate the difference between oligodendrocyte (OL) and SC myelination in conductive functions of axons, we compared normal DCs, demyelinated DCs, demyelinated DCs remyelinated by SC transplantation, and normal dorsal roots. All of the axons was originated from dorsal root ganglion neurons. Dorsal roots of adult rats were demyelinated at T11 by X-ray irradiation and ethidium bromide, and transplanted with SCs (3 x 10(4)) of adult rats. Three weeks later, the spinal cord was removed and pinned in a recording chamber and compound action potentials (CAPs) were recorded, to investigate conduction properties (conduction velocity and response after high frequency stimulation). Normal DCs or dorsal roots were recorded in same manner. Following transplantation of SCs, histological examination revealed SC-like patterns of remyelination in demyelinated DCs. SC transplantation improved significantly conduction properties compared to demyelinated axons, but less than normal DC. Moreover, remyelinated axons by SC transplantation showed as low amplitude of CAP as dorsal roots, but lower conduction velocity than dorsal roots. Though anatomical difference and/or time after transplantation influenced the conduction, these result suggested that SC myelination resulted in lower amplitude of CAP than OL, and SC remyelination might be insufficient for conduction velocity.     

Functional considerations of stem cell transplantation therapy for spinal cord repair

Stem cells hold great promise for therapeutic repair after spinal cord injury (SCI). This review compares the current experimental approaches taken towards a stem cell-based therapy for SCI. It critically evaluates stem cell sources, injury paradigms, and functional measurements applied to detect behavioral changes after transplantation into the spinal cord. Many of the documented improvements do not exclusively depend on lineage-specific cellular differentiation. In most of the studies, the functional tests used cannot unequivocally demonstrate how differentiation of the transplanted cells contributes to the observed effects. Standardized cell isolation and transplantation protocols could facilitate the assessment of the true contribution of various experimental parameters on recovery. We conclude that at present embryonic stem (ES)-derived cells hold the most promise for therapeutic utility, but that non-neural cells may ultimately be optimal if the mechanism of possible transdifferentiation can be elucidated.     

乙交酯-丙交酯共聚物支架-细胞复合体移植对大鼠损伤脊髓的修复作用

目的 观察神经干细胞、雪旺细胞和组织工程材料乙交酯-丙交酯共聚物共移植后对大鼠损伤脊髓形态和功能的修复作用.方法 36只Wistar大鼠,随机数字法分为乙交酯-丙交酯共聚物移植组、神经干细胞/乙交酯-丙交酯共聚物绀和神经干细胞+雪旺细胞/乙交酯-丙交酯共聚物组.体外培养、鉴定胚胎脊髓源神经干细胞和雪旺细胞,制备和构建乙交酯-丙交酯共聚物支架细胞复合体并移植到大鼠脊髓T9半横断损伤部位,应用BBB行为评分和电生理技术在术后4、12周评价大鼠脊髓功能的恢复情况;应用透射电镜、HE染色和免疫组织化学染色方法在形态结构上观察轴突和髓鞘再生情况,以及神经干细胞在脊髓内的存活、迁移和分化情况.结果术后4、12周,细胞移植组的BBB评分较对照组明显提高(P＜0.05);细胞移植组的体感诱发电位和运动诱发电位波幅较对照组都有所好转.术后12周移植材料正中横断面透射电镜可见新生的无髓及有髓神经纤维;脊髓标本免疫组织化学染色显示移植的神经十细胞呵以在宿主脊髓内存活、迁移并分化成神经元和少枝胶质细胞,未分化成星形胶质细胞.结论 神经干细胞、雪旺细胞和组织工程材料乙交酯-丙交酯共聚物共移植可以促进半横断损伤的大鼠脊髓轴突再生,改善肢体的运动功能.     

Adult olfactory ensheathing cell transplantation for acute spinal cord injury

Cellular transplantation strategies have been explored for the treatment of spinal cord injury. In particular, olfactory nerve ensheathing cell (OEC) transplantation has been reported to improve functional outcome following injury. We investigated the effect of OEC transplantation using cells derived from adult animals on the restoration of function following a contusion injury to the spinal cord. The NYU impactor was used to create a moderate to severe spinal cord injury in 17 rats. Hoescht stained cultured OECs derived from adult rats (n = 7) or culture medium alone (n = 10) were injected into the injury site immediately following injury. Histological and functional outcomes were measured using immunohistochemistry and the Basso, Beattie, and Bresnahan (BBB) scale. All animals transplanted with OECs were found to have surviving Hoescht positive cells within the spinal cord when sacrificed 6 weeks following injury. Immunohistochemical staining of the explanted cords revealed that the surviving cells stained positively for nerve growth factor receptor. Functional outcomes were not different between the transplanted and control groups. OECs transplanted immediately following a contusion injury to the spinal cord survive during the first 6 weeks following injury. These cells do not appear to influence functional outcome during the first 6 weeks following injury. Additional studies are required in order to definitively determine the utility of this type of cellular transplantation for spinal cord injury.     

经蛛网膜下腔细胞移植治疗脊髓损伤的研究进展

促进脊髓损伤（SCI）后神经再生及脊髓神经功能修复的研究已有大量文献报道,而这些研究多是以细胞移植为背景,其中多种细胞在促进轴突再生、长距离延伸、再髓鞘化方面的优势被学者关注[1～3]。随着大量研究的开展,细胞种类选择、细胞移植途径、移植时间窗等问题亦越来越突出。开放手术经脊髓实质内细胞移植损伤大,颅内感染、出血等并发症发生率较高;而蛛网膜下腔细胞移植损伤小、可重复移植、临床操作简单且部分已经用于临床,故研究[4～6]较多。经蛛网膜下腔细胞移植治疗SCI主要有两种方法：通过脑室移植[7～13]和腰椎穿刺移植[14～16],综述如下。     

脊髓干细胞移植对脊髓损伤后神经功能的影响

目的探讨胚胎脊髓神经干细胞移植对大鼠脊髓损伤后神经功能恢复的意义。方法160只SD大鼠随机分为空白组,假手术组,脊髓损伤组,细胞移植组,分别在细胞移植后1、2、4周应用斜板实验和Tarlov评分对脊髓损伤后功能恢复进行评价,应用nestin标记观察移植后干细胞的存活情况。结果移植后1周、2周、4周,移植组和对照组斜板试验结果分别为(38．30±0．84)°、(18．50±0．76)°；jm(39．40±0．78)°、(19．70±0．66)°；(45．00±0．81)°、(22．30±0．69)°；Tarlov评分分别为3．37±0．45、2．32±0．34；3．45±0．38、2．41±0．43；3．63±0．47、2．45±0．48；有统计学意义(P＜0．01),免疫组织化学观察可见在损伤的脊髓组织中有神经干细胞的存活。结论胚胎脊髓干细胞移植对脊髓损伤后神经功能恢复有促进作用。