【作者】 李瓦里；

【导师】 阚世廉； 宫可同； 张建兵；

【作者基本信息】 天津医科大学 ， 外科学， 2009， 博士

【摘要】 目的：临床中的周围神经损伤比较常见。一般分为神经传导障碍、轴索中断及神经中断。临床处理都需要后期的固定。那么为什么固定的时间为3-4周?在这固定期间神经的微观变化都有哪些?神经修复和时间的相关性如何?探讨周围神经挤压损伤后轴突对损伤后的潜在反应，对于帮助神经再生和修复的治疗非常关键。明胶酶参与了细胞外基质的降解、细胞的移行及增殖等有关的神经受损之后修复的所有病理及生理过程，在神经损伤修复后的过程中发挥了重要的作用。本实验以大鼠为研究对象，研究的目的是为了探讨坐骨神经损伤后，神经修复的时间过程中微观的病生理变化，探讨明胶酶A(MMP-2)和明胶酶B(MMP-9)是否有表达以及其可能发挥的作用。方法：我们用实验的方法将大鼠的双侧的坐骨神经进行暴露，一侧钳夹持续一分钟，另一侧作为正常对照组(暴露的时间与对侧相同)，从而建立了大鼠坐骨神经挤压损伤的动物模型。方法一、30只大鼠分为1天、3天、1周、2周和4周共五组。称重后盐酸氯胺酮注射液腹腔注射麻醉，150mg／kg。俯卧位固定四肢于手术台上。双侧腰、臀和股部剃毛，碘伏消毒，铺无菌纱布。两侧臀部各做一斜行切口，并向股后侧延伸。劈开臀肌，分离股二头肌和半膜半腱肌，暴露各组的双侧坐骨神经。在距离梨状肌下缘约8mm处，用反向血管钳钳夹一分钟(左侧为钳夹组，右侧为正常对照组，在手术区域和显露区域丝线标志准确位置)，所有组的正常对照组仅仅暴露，时间与钳夹组相同。0／1丝线缝合臀肌和皮肤。苏醒后送回饲养室给予动物普通饮食，同等条件下饲养。在大鼠坐骨神经挤压损伤后1天、3天、1周、2周和4周时随机取6只大鼠，进行深度麻醉下心脏灌注0．01M的PBS后取材，石蜡包埋切片，光镜下观察损伤后各个时间段内受损神经的病理变化；利用明胶酶谱的测定方法，测定损伤后大鼠坐骨神经组织内的明胶酶A(基质金属蛋白酶2，MMP-2)和明胶酶B(基质金属蛋白酶9，MMP-9)的活性变化；方法二、15只大鼠随机分为1天、7天和4周共三组。手术方法同一。在大鼠坐骨神经挤压损伤后1天、7天和4周时，深度麻醉下心脏灌注0．01M的PBS后，分离大鼠双侧标志处的坐骨神经，切取神经长度约5-8mm，OCT包埋，立刻放入盛有4℃异戊烷的容器内，浸入液氮2分钟后，放入-25℃冰冻切片机内，切成10微米厚的纵行坐骨神经冰冻切片，粘贴于载玻片上，室温下风干，需做原位酶谱的直接在玻片上滴加反应缓冲液。免疫组织化学染色-20℃冰箱保存待用。用免疫组织化学和原位酶谱的方法观察MMPs在损伤神经轴突中的表达位置及其功能。用于神经胶质酸性蛋白(GFAP)和硫酸软骨素蛋白多糖(CSPG)免疫组织化学的大鼠，在损伤后1周，4周，经4％多聚甲醛心脏灌注后，取大鼠双侧标志处的坐骨神经长约各5-8mm，常规石蜡包埋，纵行切片，了解硫酸软骨素蛋白多糖与瘢痕组织及明胶酶之间可能存在的关系。结果：1．大鼠坐骨神经挤压伤之后有不同种类的明胶酶的表达，即明胶酶A和明胶酶B。在挤压伤后的各个阶段光镜下观察，有不同的细胞参与此过程，这与以往的报道一致。明胶酶谱检测的结果显示正常的大鼠坐骨神经组织内有明胶酶A的表达，没有发现明胶酶B的表达。在坐骨神经挤压伤之后的一天内有明胶酶B的迅速表达，而且表达较高。此时光镜下可见到受损区域内有很少的细胞核及血细胞，周围可看到局部中性粒细胞的出现。在伤后的三天可看到起到吞噬作用的炎细胞，至一周时可见减少，与明胶酶B呈现正相关。明胶酶A在受损伤后的表达水平有轻微的增加，到一周时候表达的最高。此时光镜下见到炎细胞较前减少，两周时细胞恢复较多，四周时已经接近正常。Western blot分析进一步确认所消化的条带是明胶酶A和明胶酶B。2．原位酶谱和免疫组织化学的结果显示基质金属蛋白酶中的明胶酶在损伤区域的边缘有较高的表达，不仅仅如此而且和受损区域内的硫酸软骨素蛋白多糖(CSPG)呈现负相关的趋势。结论：1．大鼠坐骨神经损伤后的再生与修复不仅仅有巨噬细胞、雪旺氏细胞等细胞因子存在，同时还有基质金属蛋白酶类的明胶酶参与。在神经受损的第一天，神经周围本身的中性粒细胞有一定程度的反应，此时明胶酶B迅速的上调，保持高水平至三天左右，而此时渗入性的炎细胞大量出现，一周后明胶酶B下降，而明胶酶A表达升高，至四周时明胶酶A、B表达接近正常。提示明胶酶B参与了神经受损后反应的前期阶段，即第一周；而明胶酶A参与了神经修复再生的后期阶段。2．神经的修复过程宏观上看是髓鞘的退变与再生过程，微观上是众多的细胞因子综合调控的结果。同时有大量降解细胞外基质成分的酶类参与。整个的神经修复过程是4周左右的时间，与临床神经处理术后固定时间吻合。3．损伤的坐骨神经中心部分有沉积的硫酸软骨素蛋白多糖的存在，明胶酶可能是降解了此成分，从而促进髓鞘的再生过程。更多还原

【Abstract】 Objective: To investigate the potential response that the axon of the peripheralnerve after crushing injury and it is very important to comprehend the regenerationand reparation of the injuried nerves.The gelatinases participated all the pathologyand physiology processes of the reparations of the injuried nerves includingdegradation of the extracellular, migration and proliferation of the cells,they aresignificant during the recovery of injuried nerves.This experiment is to probe thatthere are whether or not the expression of the gelatinase A and B after the nervecrushing injuried meanwhile the role of them.Method:we exposed the hibateral sciatic nerves of the rats by means ofexperimental modes,one side was being retained for sixty seconds,the other was justbeing exposed for same time to be the nomal contrl,so we established the rats’ischiadic nerve crushing injuried models.Method 1.All the rats were allocated into five groups,each group was sixrats.They were weighed and intraperitoneally injected the keanaesthesiad to gotanaesthesia,the dosage was 150mg/kg.The for legs were fixed on the operating-table,shaving the waist,buttocks and thighs,sterilized with povidone iodine,spread thesterilitas gauze.Incised an oblique cut on the bilateralis buttocks and extended to thepost of the femoris.Splited open the gluteus,biceps femoris,semi-membrane andsemitendinosus,exposed the bilateral sciatic nerves.The nerves were ringed clamp forsixty seconds just about 8mm far from the inferior border of musculus piriformis,theleft side was the experimental group and the other side was the normal group andmarked the operative and exposed regions with suture silks.All the rats of normalgroups was cut open to just expose for the same time as that the experimentalgroups’.The gluteus and skins were sutured with 0/1 silks.When they were awake,sentback to the breeding room and gave the animal full diets to breed under thecoordinate conditions.The six rats were sacrificed randomly after the nerve crushinginjury on the first day,three day,one week,two weeks and four weeks,infused 0.01MPBS to draw the materials from the bilateral sciatic nerves,utilized the assay methodof gelatinase enzymogram to mensurate the change of activity of the matrix metalloproteinase 2/9;Method 2.All 15 rats were allocated to three groups,one day,seven day and 4weeks,each group was five rats.The operation method was same to the anteriorexperiment. The five rats were sacrificed randomly after the nerve crushing injury onthe first day,one week and four weeks,infused 0.01 M PBS to draw the materials fromthe bilateral sciatic nerves,the incised nerve length was 5-8mm,OTC imbeded and putinto the container filled 4℃isopentane liquid immediately,immerged the liquidnitrogen about two minutes,then put into 25℃freezing mictotome and incided thenerve into 10mm thick frozen section portaitly,pasted them on glass slide,air dryingunder the room temperature.The specimen which needed in situ zymograms wasdropwised straightly reaction buffer.The specimen that needed immunohistochemicalstains was conserved in -20℃refrigerator.We observed the expressive sites andfunctions of matrix metalloproteinase in the injured nerve axon.Those specimens thatneeded GFAP and CSPG immunohistochemical imbedded in paraffin routinely andcut sheet to comprehend the possible relations among CSPG,scar tissue and thegelatinases.Result:1.Variety classes gelatinases were expressed after the rats’ ischiadic nervecrushing injury,that was gelatinase A and B.The result of the enzymogram showed thenormal rat ischiadic nerve disply the expression of gelatinase A but not B.Theexpression of gelatinase B after injured 24 houres extremely higher, contradictory thegelatinase A slightly increased post traum and highest on seven days afterinjured. Western blotting inspected the strap were gelatinase A and B.2.The results of the in situ zymograms and immunohistochemical showednot only gelatinase can be expressed in borderline of the injured area but also betaken on negative correlation to the CSPG in the injured area.Conclusion:1.Not only the macrophagocyte, Schwann cells et al cytokines existed aroundinjured nerve axon,but also the gelatinases participated in the regernaeration andreparation of the rat’s sciatic nerve injured.2. Macroscopicly the reparation of the nerve was the process of regeneration anddegeneration of the myelin sheath,in micro it was the result that masses of cell factors regulated them syntheticly.3.There were chondroitic acid proteoglycan in the center of the injured nerve,thegelatinase maybe degradated them and promoted the regenerative process.更多还原