【作者】 殷中罡；

【导师】 王沛；

【作者基本信息】 天津医科大学 ， 外科学， 2007， 博士

【摘要】 周围神经损伤后增殖的雪旺细胞提供了一个有利于轴突再生的环境。它可作为轴突生长的细胞底物，并可以分泌众多神经营养因子，表面表达细胞粘连分子和整合蛋白，产生层粘蛋白、纤连蛋白、胶原和蛋白聚糖等胞外基质分子。还分泌多种单核细胞趋化因子以刺激巨噬细胞向变性神经的募集，后者可清除轴突和髓鞘碎片，并清除抑制轴突生长的髓鞘相关糖蛋白(MAG)。由多种胞外基质成分构成的基底膜是周围神经再生所必需的生长底物。神经损伤后，损伤以远的基底膜的完整性能得以保留是神经再生的必要条件。在众多胞外基质分子中既有促进轴突生长的分子，如层粘蛋白、纤连蛋白和Ⅳ型胶原等，也有抑制轴突生长的分子，如硫酸软骨素蛋白聚糖(CSPGs)。后者同样存在于中枢神经系统中，并是抑制中枢神经损伤后再生的重要因素。周围神经损伤后胞外基质的重塑，即增加促轴突生长的分子，去除抑制分子，对于神经再生是十分重要的。软骨素酶ABC是针对葡糖胺聚糖(GAG)的特异性糖苷酶，可以分解CSPGs上的GAG侧链，从而降解CSPGs。利用软骨素酶ABC降解CSPGs来促进神经再生，改善功能恢复已经在众多中枢神经损伤的研究中得到证实，但有关其对周围神经再生的作用的研究还很少。此外软骨素酶ABC治疗对雪旺细胞的影响，以及对基底膜的其他成分和基底膜结构完整性的影响均不明了。本研究以大鼠为研究对象，建立自体坐骨神经移植模型，用软骨素酶ABC对移植的神经进行移植前预处理。通过电生理检查和组织学观察研究移植后神经再生的情况，并利用免疫组化技术观察软骨素酶ABC治疗对移植神经内雪旺细胞和基底膜层粘蛋白这两种主要的促再生因素的影响。方法：1．软骨素酶ABC治疗对移植神经再生的影响30只大鼠随机分为3组，每组10只。A组为空白对照组，B组为赋形剂组，C组为实验组(软骨素酶治疗组)。盐酸氯胺酮注射液腹腔注射麻醉。两侧臀部斜行切口。劈开臀肌，分离股二头肌和半膜半腱肌，暴露并游离各组双侧坐骨神经。在距离梨状肌下缘约8mm处向远端切除右侧坐骨神经5mm，两断端回缩后形成约10mm的间隙以接纳移植神经；切除左侧坐骨神经10mm作为移植神经。A组：切取左侧坐骨神经10mm直接移植于右侧缺损处。B组：切取左侧坐骨神经10mm，在100μl PBS(赋形剂)溶液中浸泡2h后移植于右侧缺损处。C组：切取左侧坐骨神经10mm，在100μl软骨素酶ABC溶液中(10U／ml)浸泡2h后移植于右侧缺损处。术后8周进行运动神经传导速度测定。电生理检查完毕后切取各组移植神经中段5mm的神经节段。每组各随机选取1个神经节段做透射电镜观察，其余神经节段制成横切片，做神经纤维Palmgren镀银染色，光学显微镜观察再生轴突的密度。2．软骨素酶ABC治疗对移植神经层粘蛋白／基底膜的影响16只大鼠随机分为2组，每组8只。A组为对照组，B组为实验组。手术方法同上。A组：切取左侧坐骨神经10mm直接移植于右侧缺损处。B组：切取左侧坐骨神经10mm，在100μl软骨素酶ABC溶液中浸泡2h后移植于右侧缺损处。术后5天取材。切取各组移植神经近段5mm的神经节段制成横切片，用多克隆抗层粘蛋白抗体标记层粘蛋白，观察层粘蛋白免疫反应阳性产物在移植神经内的分布概况。3．软骨素酶ABC治疗对移植神经雪旺细胞的影响16只大鼠随机分为2组，每组8只。A组为对照组，B组为实验组。手术方法同上。A组：切取左侧坐骨神经10mm直接移植于右侧缺损处。B组：切取左侧坐骨神经10mm，在100μl软骨素酶ASC溶液中浸泡2h后移植于右侧缺损处。术后14天取材。切取各组移植神经近段5mm的神经节段制成横切片，用多克隆抗S-100蛋白抗体标记雪旺细胞，观察移植神经内S-100免疫反应阳性细胞的密度。结果：1．软骨素酶ABC治疗对移植神经再生的影响术后8周时A、B、C三组移植神经的运动传导速度的差异没有统计学意义(P=0.381)，空白对照组与赋形剂组之间再生轴突数目的差异没有统计学意义(P=0.607)，实验组再生轴突的数目多于空白对照组和赋形剂组(P＜0.05)。2．软骨素酶ABC治疗对移植神经层粘蛋白／基底膜的影响术后5天时对照组与实验组的基底膜均呈现层粘蛋白阳性反应，且两组基底膜结构的完整性相似。3．软骨素酶ABC治疗对移植神经雪旺细胞的影响术后14天时两组大鼠移植神经均见S-100阳性染色细胞，分布较为均匀。对照组与实验组S-100阳性染色细胞的密度的差异没有统计学意义(P=0.945)。上述结果提示：1．软骨素酶ABC对移植神经做移植前预处理，可以促进近端神经向移植神经片段内长入。2．软骨素酶ABC对移植神经做移植前预处理没有影响移植后移植神经内雪旺细胞的数量，也没有破坏移植神经内层粘蛋白及基底膜的完整性，即没有破坏轴突再生的必要因素。因此利用软骨素酶ABC促进周围神经再生是一合理的治疗策略，对其进一步深入研究有助于改善周围神经损伤后的功能恢复。更多还原

【Abstract】 The proliferating Schwann cells following peripheral nerveinjury provide a favorable environment for axonal regeneration.They can act as the substratum for axonal growth, secrete a varietyof neurotrophic factors, express cell adhesion molecule andintegrin on their surface, and produce extracellular matrixmolecules including laminin, fibronectin, collagen, and variousproteoglycans. They can also secret a variety of monocytechemotactic factors to stimulate the recruitment of macrophagesinto the degenerating nerves. The recruiting macrophages can removethe debris of the degraded axons and myelin sheaths, and removemyelin-associated glycoprotein (MAG), which would otherwiseinhibit axonal growth. The endoneurial basal laminae that arecomposed of various extracellular matrix molecules areindispensable substratum for peripheral nerve regeneration. Afterperipheral nerve injury, the basal laminae distal to the injury canremain intact, which is prerequisite for nerve regeneration. Theextracellular matrix components are composed of axongrowth-promoting molecules, such as laminin, fibronectin andⅣtype collagen, as well as growth-inhibiting molecules, such aschondroitin sulfate proteoglycans (CSPGs). The latter also ispresent in central nervous system, and is one of the major inhibitingfactors for axonal regeneration after central nervous injury. Theremodeling of the extracellular matrix, e. g. enhancing the axongrowth-promoting molecules and removing the inhibiting molecules,is very important for nerve regeneration. It has been known thattreatment with chondroitinase ABC which can degrade the GAG sidechains of CSPGs specifically can enhance nerve regeneration and functional recovery after central nervous system injury. Thestudies about the role of chondroitinase ABC on peripheral nerveregeneration are quite rare. Whether the treatment withchondroitinase ABC has effects on Schwann cells, other componentsof basal laminae, and the structural integrity of the basal laminaeremain unresolved.In this study, we established the autograft model of rat sciaticnerve, and treated the nerve grafts with chondroitinase ABC beforethey were interposed into the nerve gaps to increase the degradationof CSPGs. Axonal regeneration into the grafts was assessed byelectrophysiological test and morphologic study. The effects oftreatment with chondroitinase ABC on the Schwann cells and basallamina laminin of the nerve grafts, which are the major axongrowth-promoting factors, were assessed by immuno-histochemicaltechnique.Methods:1. Effect of treatment with chondroitinase ABC on axonalregeneration in nerve graftsThirty rats were randomised into three groups containing 10 ratseach. Group A, controls group (no treat); Group B, vehicle treatgroup; Group C, chondroitinase ABC treat group.Under anesthesia with intraperitoneal ketamine hydrochloride,the sciatic nerves on both sides were approached through a glutealmuscle-splitting incision and isolated free of surrounding fascia.A 5mm segment of the right sciatic nerve was excised distally fromthe point that was 8 mm distal to the inferior border of thepiriformis. This made a 10 mm gap defect after retraction of thenerve ends. A 10 mm segment of the left sciatic nerve was excisedto act as the graft. In group A, the 10 mm segment of the left sciaticnerve was interposed into the gap defect of the right sciatic nerveimmediately. In group B, the 10 mm segment of the left sciatic nervewas incubated in 100μ1 of PBS (vehicle) for 2 h before grafting into the defect of the right sciatic nerve. In group C, the 10 mmsegment of the left sciatic nerve was incubated in 100μl ofchondroitinase ABC (10U/ml)for 2h before grafting.At postoperative 8 weeks, the MCV was measured for each nervegraft. After electrophysiological test,, a 5 mm nerve segment wasexcised from the midway trunk of the graft. One nerve segment wasselected randomly from each group and was performed transmissionelectron microscopic investigation. The other segments of eachgroup were made into transverse sections and stained with Palmgrenmethod for light microscopic investigations. The numbers of theregenerating axons in the grafts were counted and compared betweenthree groups.2. Effect of treatment with chondroitinase ABC on laminin/basallamina of nerve graftsSixteen rats were randomized into two groups containing 8 ratseach. Group A, controls group (no treat); Group B, chondroitinasetreat group.Surgical technique was the same as above-mentioned. In group A,the 10 mm segment of the left sciatic nerve was interposed into thegap defect of the right sciatic nerve immediately. In group B, the10 mm segment of the left sciatic nerve was incubated in 100μl ofchondroitinase ABC (10U/ml)for 2h before it was grafted.At postoperative 5 days, a5 mm nerve segment was excised fromthe proximal trunk of the graft and made into transverse sections.Polyclonal anti-laminin antibody was used to label basal laminae.3. Effect of treatment with chondroitinase ABC on Schwann cellsin nerve graftsSixteen rats were randomized into two groups containing 8 ratseach. Group A, controls group (no treat); Group B, chondroitinasetreat group.Surgical technique was the same as above-mentioned. In group A,the 10mm segment of the left sciatic nerve was interposed into the gap defect of the right sciatic nerve immediately. In group B, the10 mm segment of the left sciatic nerve was incubated in 100μl ofchondroitinase ABC (10U/ml) for 2h before it was grafted.At postoperative 14 days, a5mm nerve segment was excised fromthe proximal trunk of the graft and made into transverse sections.Polyclonal anti-S-100 antibody was used to label Schwann cells. Thenumbers of Schwann cells were counted and compared between twogroups.Results:1. Effect of treatment with chondroitinase ABC on axonalregeneration in nerve graftsThere was no significant difference in MCV between the threegroups at postoperative 8 weeks (P=0.381). The difference in numberof regenerative axons between group A and B was insignificant (P=0.607). Group C had more regenerating axons than group A and B,and the difference was significant (P＜0.05).2. Effect of treatment with chondroitinase ABC on laminin/basallamina of nerve graftsAt 5 days, both group A and B revealed intense laminin labelingin grafts, and the two groups were similar in structural integrityof basal laminae of grafts.3. Effect of treatment with chondroitinase ABC on Schwann cellsin nerve graftsAt 14 days, both group A and B revealed positive S-100 labelingin grafts. No significant difference was found between group A andB in number of Schwann cells in grafts (P=0.945).The above-mentioned results suggest:1. Treating nerve autografts before they are grafted withchondroitinase ABC can enhance axonal regeneration into the grafts.2. Treating nerve autografts before they are grafted withchondroitinase ABC does not influence the number of Schwann cellsin the grafts, and does not impair the laminin and the structural integrity of basal laminae of the grafts, i.e. does not impair themajor axon growth-promoting factors.In conclusion, treatment with chondroitinase ABC is a logicalstrategy to enhance peripheral nerve regeneration. The furtherresearch in this field may improve functional recoveryI afterperipheral nerve injury.更多还原