雪旺细胞与周围神经组织工程

应用组织工程学原理和技术方法研制组织工程化人工神经、修复临床周围神经缺损,是一种创新的研究。人工神经要求以具有良好生物相容性的可降解高分子聚合物为载体与有活性的细胞、细胞因子结合而成,具有特定三维结构和生物活性,可替代自体神经移植体修复神经缺损。其中雪旺细胞(Schwann cell,SC)是周围神经组织工程研究的重要种子细胞,本文就有关SC的研究作一综述。 SC的作用 SC是周围神经系统特有的胶质细胞,亦称神经膜细胞(neurilemmal cell,neurolemmocyte)或鞘细胞(sheath cell),由Theodor Schwann于1839年首先描述。SC在周围神经的发生、发育、形态、功能维持方面起着重要作用,支持和保护轴突,维持轴突的良好微环境;形成髓鞘,对有髓纤维起着绝缘作用,加速神经轴突的传导;对神经轴突有营养代谢作用。在周围神经损伤、再生与修复中,SC也起着关键作用。概括说来,主要包括以下几方面: 1.周围神经损伤后,远段神经瓦勒变性,SC分裂增殖、形成Bungner氏带,并和巨噬细胞共同吞噬变性的轴突与髓鞘碎屑,SC及SC基底膜(basal lamina,BL)管一起共同为再生轴突提供一个生长通道;     

周围神经中许旺细胞特异性标记物

周围神经损伤后,其近、远端神经纤维将发生瓦勒氏变性(Wallerian degeneration)。许旺细胞(Schwann Cell)起源于神经嵴细胞,是周围神经的种子细胞。目前周围神经损伤的修复,主要围绕神经内部结构重建,促进神经纤维再生进行。通过特异性标记物标记、识别移植的许旺细胞,对神经移植效果的评价至关重要。本文就许旺细胞比较常见的特异性标记物进行综述,以期为许旺细胞在周围神经损伤及组织工程方面的应用提供帮助。     

雪旺氏细胞和脊髓腹侧神经元联合培养重组“组织块”的发现

瓦勒氏变性神经来源的雪旺氏细胞，作为细胞滋养层和胚胎脊髓腹侧运动神经元联合培养，可维持神经元存活，长出神经突起，两周后电镜发现雪旺氏细胞可以包绕神经突起，形成髓鞘，有神经营养作用；并意外发现雪旺氏细胞和神经元分离细胞联合培养，至第９日联合细胞层出现“脱壁一卷曲”，进而形成“组织块”样物，后者又可重新长出细胞突起。可望为雪旺氏细胞和脊髓神经元联合移植修复脊髓损伤，提供一种有形的移植物。     

Laminin等因子失活后巨噬细胞的行为及对周围神经再生的影响

用Ｗｉｓｔａｒ大鼠观察Ｌａｍｉｎｉｎ．Ｆｉｂｒｏｎｅｃｔｉｎ或ＣｏｌｌａｇｅｎⅣ失活时神经段内巨噬细胞的行为及对周围神经再生的影响。结果表明：巨噬细胞的快速清除，有利于再生轴突的良好生长；但巨噬细胞促轴突生长的能力，只表现在轴突再生早期，受基膜成份失活影响而长期滞留在组织内的巨噬细胞对轴突生长并无促进作用。认为位于基底膜管外的单个再生轴突，可能因没有雪旺氏细胞或基膜的保护和营养作用，而遭巨噬细胞侵     

不同时间差速粘附分离`纯化瓦勒变性神经的雪旺细胞

不同时间差速粘附分离、纯化瓦勒变性神经的雪旺细胞顾立强，朱家恺雪旺细胞（ＳｃｈｗａｎｎＣｅｌｌ，ＳＣ）培养是周围神经再生研究深入细胞、分子水平的要求之一，但有关瓦勒变性神经ＳＣ培养报道甚少。本文报道用不同时间差速粘附法分离、纯化成年大鼠瓦勒变性神经的...     

雪旺氏细胞分泌的神经营养活性物质的提取及体外生物活性研究

雪旺氏细胞（ＳＣ）分泌多种神经营养因子促进周围神经再生，但ＳＣ分泌的各种蛋白质对周围神经再生的影响尚未完全阐明。为此，从成年大鼠瓦勒氏变性坐骨神经中分离出ＳＣ，经培养获得ＳＣ无血清条件培养液（ＳＣ－ＳＦＣＭ）。通过ＰＭ１０超滤浓缩、Ｄｉｓｃ－ＰＡＧＥ和Ｂｉｏｔｒａｐ电洗脱，从ＳＣ－ＳＦＣＭ中分离出Ｄ蛋白带，分子量在４３～６７Ｋｄ之间。经ＭＴＴ检测，Ｄ带蛋白在２５～５０ｎｇ／ｍｌ浓度时对脊髓前角神经元表现出明显的体外成活作用。Ｄ蛋白带可能是一种有别于已知的ＳＣ源神经营养物质，其活性浓度达到了神经营养因子分子检测水平，值得深入研究     

雪旺氏细胞植入硬脊膜管修复周围神经缺损的实验研究

本文报道两组10mm大鼠坐骨神经缺损,分别用含雪旺氏细胞和不含雪旺氏细胞的硬脊膜管桥接修复,术后2、4个月行光镜、电镜、神经电生理和计算机图像分析检测,证实植入雪旺氏细胞者,再生神经生长及成熟较早,肢体功能恢复较快,雪旺氏细胞和硬脊膜管结合,对周围神经再生有更大的促进作用。     

多功能生长因子Pleiotrophin与周围神经损伤的研究进展

原癌基因Pleiotrophin于1989年被发现,在肿瘤发生发展和中枢神经系统内起着重要作用.最近的研究表明:Pleiotrophin分子信号很可能参与周围神经损伤后脊髓神经元的保护、促进轴突再生、轴突再生导向、骨骼肌的神经再支配4个环节,并可能在轴突再生导向和骨骼肌的神经再支配中发挥关键作用.随着研究的深入,Pleiotrophin基因有望成为提高周围神经修复效果和重建神经肌连接的基因调控靶点.     

雪旺氏细胞分泌的神经营养活性物质的提取及体外生物活…

雪旺氏细胞（ＳＣ）分泌多种神经营养因子促进周围神经再生，但ＳＣ分泌的各种蛋白质对周围神经再生的影响尚未完全阐明。为此，从成年大鼠瓦勒氏变性坐骨神经中分离出ＳＣ，经培养获ＳＣ无血清条件培养液（ＳＣ－ＳＦＣＭ）。通过ＰＭ１０超滤浓缩、Ｄｉｓｃ－ＰＡＧＥ和Ｂｉｏｔｒａｐ电洗脱，从ＳＣ－ＳＦＣＭ中分离出Ｄ蛋白带，分子量在４７－６７Ｋｄ之间，经ＭＴＴ检测，Ｄ带蛋白在２５－５０ｎｇ／ｍｌ浓度时对脊髓产角神经元     

嗅鞘细胞移植治疗脊髓损伤的研究进展

脊髓损伤的治疗一直是医学界的难题。脊髓损伤后神经元轴突再生困难,同时神经胶质瘢痕阻碍再生轴突的通过。嗅鞘细胞是一种特殊的神经胶质细胞,具有中枢神经系统（CNS）星形胶质细胞和外周雪旺氏细胞的特性,许多基础与临床研究发现嗅鞘细胞是神经系统中具有促进神经轴突再生,引导轴突正确生长的独特细胞,取得了一定的成果,为脊髓损伤的修复带来了希望。     

Cyclosporin A affects axons and macrophages during Wallerian degeneration

A traumatic injury of a peripheral nerve leads to Wallerian degeneration. It includes the recruitment of macrophages and the phagocytosis of myelin and the remnants of axons. We have previously studied the recruitment of macrophages and now wished to determine if the immunosuppressant cyclosporin A (CsA) affects the number of macrophages at the site of nerve injury. The primary target of CsA is T-cells, but it may also have an effect on mononuclear phagocytes which exert a key role during Wallerian degeneration. Rats were divided into two groups: CsA-treated animals and control animals. Following transection of the sciatic nerve in the treatment group, the animals received 5 mg/kg CsA subcutaneously. The groups were further subdivided into a freely regenerating nerve group and a sutured nerve group. The number of macrophages and MHC class II positive cells were counted 3 days, 7 days, 2 weeks, 4 weeks, and 8 weeks posttransection; also CD4, CD8, IL-2 receptor positive cells, B cells, and the axonal sprouting were studied. In the CsA-treated group, there were more macrophages in the distal areas under 8 weeks than in the controls (p < 0.05); thus, the clearance of macrophages is delayed in the CsA-treated rats compared to the control rats. In the proximal area, the difference in macrophage number did not gain statistical significance. Additionally, CsA retarded axonal degeneration. CsA affects number of macrophages during Wallerian degeneration, while retarding axonal degeneration and subsequent reinnervation. Its mechanism of action appears to involve either direct or indirect via T-cells-mediated responses.     

Effects of the immunomodulator linomide on macrophage migration and myelin phagocytic activity in peripheral nerve trauma: an experimental study

Wallerian degeneration after peripheral nerve transection leads to the phagocytosis of degenerated myelin and axon components by macrophages. These phagocytes are recruited from the systemic circulation and Wallerian degeneration may therefore be used as a model for myelin removal by hematogenous macrophages, a feature that is also a hallmark of demyelinating diseases of the central and peripheral nervous system. The immunomodulator linomide has been shown to be effective in the treatment of experimental demyelinating diseases although the exact mode of its action is not yet defined. The present study investigated the effect of linomide on monocyte invasion and myelin phagocytosis after sciatic nerve transection. Linomide had a dual effect in Wallerian degeneration. Monocyte migration from the circulation to the damaged nervous system was significantly reduced. Additionally, the myelin phagocytic capacity of macrophages was impaired, finally resulting in a significant delay in the removal of myelin. The present experiments may provide an explanation for the effects of linomide during the course of demyelinating diseases of the nervous system.     

Acceleration of recovery after injury to the peripheral nervous system using ultrasound and other therapeutic modalities

Taken together, these studies show the promise of various therapeutic modalities for the noninvasive treatment of peripheral nerve injury. Further progress on these promising methods requires determining the biologic mechanisms responsible for the ability of these modalities to enhance peripheral nerve recovery. Necessary investigations include validation or refutation of the hypothesis that these therapies act on various aspects of the natural healing process. Examples include cellular and molecular processes involved in promoting Wallerian degeneration and the rate and specificity of axonal regeneration and remyelination and muscle reinnervation, processes that are distributed between the regenerating nerve itself, the pathway of the regenerating axon, and the target of the regenerating nerve. An increased understanding of the biologic mechanisms underlying the enhancement of peripheral nerve recovery after injury would lend greater insight into the cellular and molecular mechanisms involved in successful nerve regeneration and muscle reinnervation. This increased understanding may also result in clinically beneficial treatments for peripheral nerve disorders.     

Role of Schwann cells in the regeneration of penile and peripheral nerves

Schwann cells (SCs) are the principal glia of the peripheral nervous system. The end point of SC development is the formation of myelinating and nonmyelinating cells which ensheath large and small diameter axons, respectively. They play an important role in axon regeneration after injury, including cavernous nerve injury that leads to erectile dysfunction (ED). Despite improvement in radical prostatectomy surgical techniques, many patients still suffer from ED postoperatively as surgical trauma causes traction injuries and local inflammatory changes in the neuronal microenvironment of the autonomic fibers innervating the penis resulting in pathophysiological alterations in the end organ. The aim of this review is to summarize contemporary evidence regarding: (1) the origin and development of SCs in the peripheral and penile nerve system; (2) Wallerian degeneration and SC plastic change following peripheral and penile nerve injury; (3) how SCs promote peripheral and penile nerve regeneration by secreting neurotrophic factors; (4) and strategies targeting SCs to accelerate peripheral nerve regeneration. We searched PubMed for articles related to these topics in both animal models and human research and found numerous studies suggesting that SCs could be a novel target for treatment of nerve injury-induced ED.     

Cellular activity of resident macrophages during Wallerian degeneration

The resident macrophages have been accepted as an important component of the peripheral nervous system as Schwann cells. To elucidate their role during Wallerian degeneration without interference from extrinsic hematogenous macrophages, we designed a culture system to investigate the behavior of resident macrophages in vitro. A total of 75 adult male Lewis rats were used; 2. 5-cm-length sciatic nerve explants were harvested. There were three groups. In the culture groups, the nerve explants were incubated in Dulbecco's modified Eagle's medium (DMEM) only or in DMEM supplemented with 2 microm forskolin and 10 microg/ml pituitary extract (mitogenic medium for Schwann cells). In vivo predegenerated nerves and normal nerves were used as the positive and negative controls, respectively. The observation periods extended to 3 weeks. Hematoxylin and eosin (H&E) stain was employed to estimate overall cell number in nerve explants. Macrophages were labeled with ED1; S-100 immunostaining was used to evaluate the presence of Schwann cells during Wallerian degeneration. Trichrome stain and toluidine blue stain were used to visualize the fate of myelin. In the culture groups, the number of resident macrophages increased continuously, although there were significantly fewer resident macrophages than hematogenous macrophages after 3 days of Wallerian degeneration (P < 0.01). Morphologically, resident macrophages contained densely small ED1-positive granules within their cytoplasm, even at later stages of observation, whereas hematogenous macrophages contained typical large ED1-positive foam vacuoles characteristic of their mature phagocytic ability. The cellular activity of Schwann cells was well preserved in the mitogenic medium; however, myelin removal was not significantly enhanced as compared with the DMEM groups (P > 0.05). The clearance of myelin debris was shown to be incomplete in culture groups as compared with the complete removal of myelin debris in the in vivo groups. Resident macrophages were actively involved in Wallerian degeneration, but their phagocytic and proliferation ability was limited. Schwann cells played an adjunctive role during the removal of myelin debris.     

Effect of liposome-mediated macrophage depletion on Schwann cell proliferation during Wallerian degeneration

The axolemma is considered a well-established mitogen, responsible for Schwann cell proliferation during Wallerian degeneration in the peripheral nerve. However, very little is known about the role of macrophages in Schwann cell proliferation. To test the possible influence of macrophages on Schwann cell proliferation during Wallerian degeneration, macrophages were depleted by dichloromethylene diphosphonate (CI2MDP)-containing liposomes in two-month old C57BL/6J mice. CI2MDP-containing liposomes were injected into the mice intravenously prior to inducing Wallerian degeneration. The injection was repeated every other day to maintain macrophage depletion. Physiologic saline was injected into the control mice. To assess macrophage depletion in vitro, cells were isolated from sciatic nerves at 1, 2, 3, 5, and 7 days post-transection (DPT) and Mac-1 positive cells attached to coverslips were counted. In an in vivo study, Mac-1 positive cells were counted on sciatic nerve sections at the same time points. Throughout the course, the number of Mac-1 positive cells in macrophage-depleted mice was less than that in the control mice both in vivo and in vitro. Schwann cell proliferation was assessed by an in vitro system that reflects in vivo status at the time of cell isolation. Schwann cells were isolated from sciatic nerves at the same time points and proliferation rate was measured by thymidine autoradiography. The proliferation rate was mildly suppressed in macrophage-depleted mice, especially for the initial 3 DPT; however, the pattern of proliferation was not significantly different from controls. These results suggest that macrophages contribute to Schwann cell proliferation during Wallerian degeneration however, their contribution may be relatively limited.     

Techniques for primary nerve repair

This article reviews the anatomy of the peripheral nerve, the pathophysiology of nerve injury, and Wallerian degeneration. It reviews the factors for deciding on immediate or delayed primary nerve repair and discusses the concept of longitudinal excursion of peripheral nerves about joints and the techniques for achieving an appropriate tension-free repair. The techniques of primary nerve repair, epineurial repair, and group fascicular repair are reviewed along with techniques for matching fascicles intraoperatively.     

Cellular mechanisms of rejection and regeneration in peripheral nerve allografts

A model of rejection and regeneration of peripheral nerve allografts in rats is presented. A 2.5-cm segment of 28 right sciatic nerves was transplanted orthotopically from LEW.1W to DA and from DA to LEW.1W. With a microsurgical technique, proximal and distal coaptations were performed. In an autologous control group the same surgical procedure was applied. Evaluation included clinical estimation of motor recovery and macroscopic appearance of the graft, electrophysiological examination, conventional histology, and immunohistology. The latter concentrated on demonstration of monomorphic and polymorphic determinants of MHC class I and II antigens and of macrophages. By functional, electrophysiological, and histological parameters it was demonstrated that after rejection a certain degree of regeneration took place in the allografts. Both rejection and subsequent regeneration were studied in detail by immunohistology. During the course of Wallerian degeneration MHC class I expression on myelin sheaths could be demonstrated. When the rejection response occurred, additional MHC class II expression on myelin sheaths and on vascular endothelial was observed. Recipient specific class I-positive macrophages were infiltrating the graft from the epineurium and the coaptation sites, and were later present at the sites of myelin degradation. At 6 weeks postoperatively donor-specific MHC products were no longer detectable, but recipient-specific Schwann cells were present in the allograft tissue. We conclude that a rejection response renders a peripheral nerve allograft acellular but does not destroy the nerve architecture, still enabling it to function as an axon conduit. The regeneration in the rejected allograft however lacks the positive neurotropic and -trophic influence physiologically provided by viable Schwann cells.     

The microstructure of peripheral nerves

An understanding of the normal peripheral nerve and its associated structures illuminates the events following a peripheral nerve injury. There are two overlapping stages: Wallerian degeneration followed by regeneration (Figure 10). Ideally, treatment interventions should be initiated early and take into account both of these phenomena. Although the physician cannot control the “biological battlefield” raging inside a damaged nerve, realistic goals according to Hall include: (1) therapy to keep more neurons alive; (2) encourage axons to cross longer interstump gaps; (3) maximize the accuracy of target reinnervation; and (4) manage the neuropathic pain.⁷