【作者】 于海龙；

【导师】 卢世璧； 彭江；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2009， 博士

【摘要】 研究背景周围神经损伤是临床上的难题,目前依然以自体神经移植修复为主要手段,但由于自体神经来源有限且供区有较多并发症,故多年来医学界不断寻找能替代自体神经移植的生物材料。在众多的替代材料中,去细胞异体神经显示了良好的应用前景。化学法萃取去细胞异体神经(Chemically ExtractedAcellular Nerve,CEAN),在最大限度地去除免疫原性物质的同时,较好地保存神经内部结构和细胞外基质的活性成分,成为修复周围神经缺损理想材料之一。在我们前期的研究中,已经采用CEAN成功地修复了犬、猴的大段周围神经缺损,取得了良好效果,其有效修复长度范围可达8厘米。应用CEAN桥接周围神经损伤伤后,在再生神经纤维长入靶器官之前,患侧肌肉已发生严重萎缩,提高神经纤维再生速度,减轻肌肉萎缩对修复神经功能至关重要。此外,随着周围神经缺损长度的增加,神经修复效果降低,如何增加移植神经修复长度,已成为周围神经损伤研究重点之一。相关的研究发现,CEAN中存在有利于神经生长的成分,也有不利于神经再生的成分,将这些抑制神经再生的物质去除,并保留促神经再生的物质,将进一步提高CEAN修复周围神经缺损效果。研究目的1对大鼠CEAN蛋白成分进行电泳分析,了解CEAN中免疫原性蛋白残存情况;在CEAN切片上,接种背根神经节,观察CEAN结构对神经再生的影响。2为CEAN修复周围神经缺损补充缓释的外源性神经生长因子,观察该方法对神经再生生长速度、肌肉萎缩、神经组织再生情况的影响。3应用软骨素酶ABC去除CEAN中不利于神经再生的物质-硫酸软骨素蛋白多糖(Chondroitin Sulfate Proteoglycan,CSPG),修复大鼠坐骨神经长距离缺损,评价去除CSPG对修复缺损距离的影响。研究方法第一部分CEAN蛋白成分及结构特性按Sondell法制备大鼠的CEAN,将制备好的CEAN进行电泳分析,观察28-30 kDa区域的髓鞘蛋白的残留情况;将CEAN进行冰冻切片,接种鸡胚背根神经节,进行神经纤维荧光染色,观察神经纤维在CEAN切片上的生长方向。第二部分神经生长因子(NGF)复合缓释制剂对提高CEAN修复周围神经缺损的实验研究采用复乳溶媒蒸发—萃取法,以聚乳酸羟基/乙酸共聚物为材料制备NGF微球,与纤维蛋白胶混合制备成补充外源性NGF复合缓释制剂。体外进行NGF复合缓剂药物释放曲线,与鸡胚背根神经节共培养鉴定释放NGF的活性。体内实验,以桥接大鼠坐骨神经10mm缺损为模型,分为自体神经移植组(A组),CEAN+NGF复合缓释制剂组(B组);CEAN移植组(C组):CEAN移植+纤维蛋白胶组(D组),术后两周测量神经再生生长距离,术后4个月进行神经电生理、腓肠肌湿重、计算机图像分析等方面评价神经移植后的再生效果。第三部分去除CEAN中的CSPG对修复周围神经缺损的实验研究CEAN经软骨素酶ABC降解CSPG后,进行免疫组化染色,观察CSPG去除效果和对去细胞异体神经其它成分的影响。经软骨素酶ABC处理后的CSPG修复周围神经缺损为实验组(A组),桥接大鼠20mm周围神经缺损,对照组不作处理(B组),术后3个月,评价其修复周围神经效果。研究结果第一部分:CEAN电泳结果显示:28-30kDa区域髓鞘蛋白完全消失,化学萃取的去细胞神经可以完全去除髓鞘蛋白;背根神经节发出大量的神经纤维沿着CEAN基底膜管方向生长;而CEAN切片外侧仅有少量神经纤维生长。第二部分:体外实验证明,NGF复合缓释制剂可持续释放NGF达60天以上,释出的NGF可促进鸡胚背根神经节神经纤维突起生长,具有较强的生物学活性。术后2周A组神经再生距离比其他组长(P＜0.05),B组优于C和D组(P＜0.05);C、D组间无差异无统计学意义(P＜0.05)。术后16周,A、B、C及D组小腿三头肌收缩力恢复率分别为85.33%±5.59%、69.79%±5.31%、64.46%±8.49%、63.35%±6.40%;A组优于其他3组(P＜0.05),B、C、D组间差异无统计学意义(P＜0.05)。A、B、C及D组肌肉湿重恢复率分别为62.54%±8.25%、53.73%±4.56%、46.37%±5.68%、45.78%±7.14%;A组三头肌湿重恢复率好于其他3组(P＜0.05),B组好于C和D组(P＜0.05);C、D组间无差异无统计学意义(P＜0.05)。组织学图像分析结果为B组在有髓神经纤维数量,轴突直径,髓鞘厚度,均优于C、D组(P＜0.05),轴突直径小于与A组(P＜0.05)。第三部分:CSPG免疫组化染色结果显示,软骨素酶ABC可以明显去除CSPG,并保留促神经生长的Laminin有效成分。动物实验结果显示,术后2个月,两组都出现残足情况,A组仅一只大鼠出现残足,而B组6只大鼠出现残足。据Wall自残标准评分,两组进行等级资料秩和检验,P＜0.05,两者之间具有统计学差异。术后3个月,A组6只大鼠5只出现钳夹痛觉试验阳性,而B组仅1只大鼠出现阳性反应。进行四格表卡方检验,x~2=4.42,P＜0.05。两组之间具有显著性差异。在小腿三头肌肌张力恢复率和小腿三头肌肌湿重方面,两组比较,A组均优于B组。进行近端与远端吻合口HE染色A组神经纤维结构优于B组。结论1 CEAN中无残留引起免疫反应的髓鞘蛋白,保留的基底膜管结构对促神经纤维再生具有引导性作用。2 NGF复合缓释制剂可以保护NGF活性,可以提高CEAN修复周围神经缺损的神经再生速度。3软骨素酶ABC可以去除CEAN中CSPG,经软骨素酶ABC处理的CEAN可以增加修复周围神经缺损的长度。更多还原

【Abstract】 Background:The treatment of peripheral nerve injury is a tough problem clinically. The standard clinical approach for bridging nerve defects is an autologous nerve graft, or autograft.Disadvantages of the autograft include limitation of the source of autologous nerve and loss of function at the donor site.Therefore,many researchs are focused on developing proper biomaterials as an alternate to autograft.Chemically extracted acellular nerve allograft(CEAN) which removes immunogenicity such as Schwann cells and myelin while restoring the nerve structures such as basal lamina tubes and extracellular matrix,is a promising material alternative to autograft.In nerve grafting,muscles may show irreversible atrophy and lose function before axonal regeneration into the distal targets.For CEAN,the speed of recovery of nerves regeneration must be increased and muscle atrophy decreased.In addition,the results of repiring the peripheral nerve defect with CEAN became worse while the length of peripheral nerve defect increasing.It becomes an important research subject that how to increase the length of repairing peripheral nerve defect.Objective:1.Electrophoretic analysis of rat CEAN protein is to be performed to know about the protein of CEAN.Dorsal root ganglia of chicken are cultured onto the surface of CEAN slice in order to observe the effect of CEAN structure on the fiber regeneration. 2.Sustained of nerve growth factor is to be deliveried for CEAN,used to repair injured nerves,to evaluate effect of the method on the speed of nerve regeneration, muscular atrophy and nerve tissue regeneration.3.The protein of Chondroitin Sulfate Proteoglycan(CSPG) in CEAN is to be degraded by Chondroitin enzyme ABC,and the peripheral nerve large defects is to be repaired with these CEAN in order to evaluate the effect of degrading Chondroitin sulfate proteoglycan of CEAN on the length of repairing nerve defects. Methods:ⅠThe protein and structure characteristics of CEAN The CEAN were perpared according to Sondell method and electrophoresed to observed whether the electrophoresis strip of 28-30kDa(Myelin Protein) exited or not.Dorsal root ganglia of chicken was cultured onto the surface of CEAN slice and dyed with nerve fiber fluorescence to observe the orientation of nerve fiber growth on the surface of CEAN slice.ⅡExperiment on the effect of controlled nerve growth factor on peripheral nerve defect repaired by CEANThe microspheres of NGF were prepared drug microsphere technology and fixed with the fibrin gels to make the complicated controlled release NGF.The Wister rats were randomly divided into four groups:Autograft group(A group),acellular nerve allograft+the double controlled release NGF(B group),acellular nerve allograft(C group) and acellular nerve allograft+fibrin glue(D group).The nerve regeneration length was measured 2 weeks after operation.The effects of peripheral nerve regeneration were evaluated by neural electrophysiology,the recovery rate of triceps surae muscular tension and weight and histological assessment 4 monthes after operation.ⅢEffect of CSPG degraded by Chondroitin enzyme ABC in CEAN on repairing peripheral nerve defectCEAN of which chondroitin sulfate proteoglycan was degraded by chondroitin enzyme ABC was immunohistochemistrily dyed with anti-CSPG in order to observe the remain of chondroitin sulfate proteoglycan and the other elements in CEAN.The Wister rats which left sciatic nerve was cut for 20 mm defect were randomly divided into two groups:CEAN degraded by chondroitin enzyme ABC(A group),CEAN(B group).The result of nerve regeneration was evaluated 3 monthes after operation.Results:ⅠElectrophoretic analysis of rat CEAN protein showed that the electrophoresis strip of 28-30kDa was totally disappeared.The large number of nerve fibers growed from DRG along the basement membrane of CEAN,while as the little nerve fibers grew outsite CEAN.ⅡThe NGF was detected from the complicated controlled release drug of NGF at least 60 days and the bioactivity of released NGF stimulated the neuritis growth of chick dorsal root ganglion.At second week after nerve repair,the datum showed that the length of nerve regeneration in autografting was larger than the other groups (P＜0.05),NGF-treated acellular grafting better than acellular grafting alone and acellular grafting with fibrin glue(P＜0.05),and there were no difference between acellular grafting alone and acellular grafting with fibrin glue.Four months after nerve repair,nerve regeneration was assessed functionally and histomorphometrically. The percentage of the triceps surae muscular tension in autografting were better than the other groups(P＜0.05).There were no significant differences between the other three groups(P＜0.05).The percentage of the triceps surae weight in autografting were better than the other groups(P＜0.05),and that in NGF-treated acellular grafting better than the other two control group(P＜0.05).There were no significant differences between acellular grafting alone and acellular grafting with fibrin glue (P＜0.05).Based on the histological observation,the image analysis indicated that axonal diameter,axon number and myelin thickness in NGF-treated acellular grafting were better than those in acellular grafting alone and acellular grafting with fibrin glue (P＜0.05).There were no significant differences between NGF-treated acellular grafting and autografting.ⅢThe slice of CEAN degraded by chondroitin enzamy ABC was immunohistochemistrily dyed:the CSPG could be degraded by chondroitin enzamy ABC and the laminin could be perserved.Animal experiment evaluated by functional and histological accessment 12 weeks after operation showed that there was autotomy in both groups.There was only one of 6 rats that had autotomy foot in A group but 6 of 8 rats in B group.Assessment of autotomy was carried out using the scale described by Wall,there were significant difference between both groups(P＜0.05). Five of six rats had positive pain reaction test in A group while as 1 of 8 rats did in B group.There were significant difference between them according to Chi-square test of four-fold table was performed(P＜0.05).The percentage of the triceps surae muscular tension and triceps surae weight in A group were better than the B group(P＜0.05). Nerve fibers structure dyed by HE staining were better in A group than in B group.Conclusions:1 There was no myelin protein remained in the CEAN,while the basement membrane structure of CEAN can induce the nerve fiber growing.2 NGF bioactivity can be protected by NGF complicated controlled release drug. The speed of peripheral nerve regeneration can be improved repaired by CEAN with NGF complicated controlled release drug.3 CSPG can be removed from the CEAN degraded by chondroitin enzyme ABC. Chondroitinase treatment increases the effective length of acellular nerve grafts.更多还原