【作者】 孙晓红；

【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， 2005， 博士

【摘要】 前言 近年来,周围神经损伤组织工程修复的研究取得了较大的进展。动物实验与临床研究主要聚焦于寻找理想的人工神经移植替代物、神经导管和种子细胞。施万细胞是周围神经系统的主要胶质细胞,是由神经嵴前体细胞演化而来,形成有髓神经纤维的髓鞘和无髓神经纤维的内膜,参与维持轴突周围微环境的稳定,保证神经纤维正常功能的行使,在哺乳动物周围神经损伤后,远端发生华勒氏变性(Wallerian degeneration),轴索和髓鞘完全崩解,唯有施万细胞分裂增殖,形成Bungner带,引导再生轴突生长,在缺乏施万细胞的情况下,神经再生的能力非常有限,这就限制了无施万细胞的神经移植材料如肌肉,静脉或硅胶管修复神经缺损的效果。组织工程神经移植修复神经缺损要有实际效果,需要在短期内获得大量增殖的施万细胞,目前在各种动物施万细胞体外培养研究中,多采用新生动物的外周神经或背根神经节,培养方法采用植块或分散培养,本实验通过用乳鼠的神经组织为材料,体外分离、培养、纯化施万细胞以及荧光标记等进行了研究,为进一步应用组织工程化神经修复周围神经缺损提供实验基础。 神经导管有非降解或可降解两种,神经移植物有自体神经、同种异体神经及异种神经移植物,作为桥接神经缺损的支架有各自的优点与缺点。周围神经组织工程研究的重点之一是构建适合施万细胞长期存活、发挥生理功能的细胞外基质。同时,培养、种植一定数量与高纯化度的具有分泌多种神经营养因子活性的施万细胞也是提高修复神经损伤效果的关键。自体神经移植后血-神经屏障的建立及远端的趋化作用形成理想的神经生长环境,具有非神经材料所不具备的优越性,但是自体神经来源受限。而同种异体神经移植修复神经缺损,具有来源充足、对患者不产生副损伤、各种类型的神经段都可以得到的优点,其存在的主要障碍是免疫排斥反应。我们先前的研究应用低渗-除垢剂脱细胞方法处理大鼠坐骨神经,脱掉其施万细更多还原

【Abstract】 Great progress was made on the study of injury and rehabilitating of peripheral nerve by tissue engineering in the recent years. The use of an artificial nerve conduit containing viable Schwann cells (SCs) is one of the most promising approaches to repair nerve injuries. SCs is the key ganglia cells which derived from pre - neurologic cell. Predegenerated nerves, so - called Wallerian degeneration, which occurs after nerve injuries, contain infiltrating macrophages and fibroblasts. Many investigators demonstrated that the presence of Schwann cells in the regenerating milieu benefits nerve regeneration. The Schwann cell -coated rolled grafts provided a modestly enhanced regenerative milieu, as evidenced by improved functional recovery. This is attributable to the known neuro-trophic properties of Schwann cells, both soluble and surface proteins augment the axonal migration process. In this study, we have developed a novel technique to isolate and purify SCs from neonatal rat peripheral nerves for an artificial nerve conduit, which is shown to have an effect on SC proliferation and survival. The combination of this technique and the altered medium promoted the migration and proliferation of SCs selectively by utilizing the supporting cells of SCs instead of discarding them by changing the culture dishes and media. The objective was to place adherent Schwann cells in artificial nerve grafts and to assess regeneration through the Schwann cell — laden grafts compared with that through acellular grafts and autografts. Schwann cells were isolated from neonatal SD rats. Using an artificial nerve conduit containing viable Schwann cells(SCs) is a more promising method for repairing peripheral nerve injuries than the use of a conduit alone tissue engineering offers some advantages over al-lograft nerve transplantation, it uses biodegradable materials and autologous cells that do not cause antigenic reactions, and it has already been applied to clinical fields. Thus, obtaining a large number of viable SCs in a short period ( which is necessary for the application of tissue engineering) is necessary for a clinical use. The nerve grafts are divided into allogeneic nerve grafts and heterogenous nerve grafts those have their benefits and disbenefits in bridging nerve gaps respectively . In addition, amount of cultured and highly purified Schwann cells play a key role as a seed cell in rehabilitating of peripheral nerve injury because of their secreting numerous neurotrophic factors. Presently, the autologous nerve grafting is an important method in reconstructive surgery of peripheral nerve lesions. However, there are still unresolved problems. The availability of auto-grafts is limited and the harvesting of autografts result in donor site morbidity such as sensory defects, scarring and neuroma formation. So the study of ideal graft is an important subject of peripheral nerve surgery. These results highlight the role of Schwann cells in nerve regeneration. Regenerative results approaching autograft levels in the Schwann cell laden group suggest that this methodology may ultimately be useful in clinical nerve repair.MATERIALS AND METHODSSchwann cells were isolated from SD rats and enzymatically digested with collagenase and dispase in vitro. Expression was monitored by immunohisto-chemical staining of the S -100 protein for Schwann cells and marked by Ho-echst33342. We made natural biodegradable nerve graft which could be cocul-tured with SCs. Morphological change can be observed by transmission electron microscope (TEM) , scanning electron microscope ( SEM ) and fluorescent microscope. Rats were randomly divided into three groups; autografts, acellular grafts, or Schwann cell - laden grafts. After injury and implantation, the animals were tested at intervals for return of function in 13 - weeks or 18 - weeks. Axon measurements were obtained in blinded fashion for fiber number, density, andaverage diameter as well as percentage of neural tissue in the total cross section by TEM and SEM. All groups were designed to investigate the motor functional recovery and NCV\ AMP N AREA of regenerated nerve after resconstruction by histochemical methods and electrophysiologic test. The data were expresses as mean ± SD and analyzed by SPSS10. 0 statistical package after taking a photograph under the microscope. Statistical comparisons were made using t - test or F - test required for statistical significance.RESULTS1. SCs performed mononuclear with two polar after culturing 24hr, then a-mount of regular SCs displayed activity and migration with a fairly stable orientation after 48 hr. The volumn of SCs becomes bigger according to the time changed. We can observe the morphological proformance with standard fluorescent microscope, owing to SCs nuclear staining labeled by Hoechst 33342. The expression of S - 100 of SCs was examines exceeding 90% by ABC immunohis-tochemical method.2. The axons and Schwann cell sheath disappear in the cross section of AR-SN. In the longitudinal section, typical long basal membrane consuit can be observed. The morphological change is also detective in toluidine blue stained specimen, while the group of transplanting SCs can observe the amount of mono-nuclear with two polar. The feature of cultures SCs shows shoulder by shoulder , head to head.3. After ARSN with cultured SCs bridged the gap of rat sciatic nerve gap, no anastomotic disruptions and no fractured conduits were observed. On gross inspection, all specimens contained neural regenerates. Based on the histomor-phometric data presented in Table. There was no significant difference in number of the regenerated nerve fibers, the thickness and the acre of regenerated my-elinated nerve between the experimental group and autograft group( P > 0.05). Whereas, there was significant difference between ARSN with SCs and ARSN(P< 0. 05 ) There was no significant difference between experimental group and control group in terms of the examined parameters of regeneration by electro-更多还原