神经生长因子促进再生坐骨神经中血管生成的量效关系

摘要

背景:对于神经生长因子的促血管生成作用的研究,目前还处于探索阶段.目的:探讨神经生长因子对再生坐骨神经中血管形成的剂量效应及其机制.设计:随机对照动物实验.单位:解放军第三二四医院神经内科,解放军第三军医大学大坪医院野战外科研究所三室.材料:健康成年Wistar大鼠32只,雌雄不拘,体质量200～250 g.硅胶管(上海新亚医用橡胶厂出产);神经生长因子(美国Sigma公司).方法:实验于2003-08／2005-02在解放军第三军医大学大坪医院野战外科研究所三室完成.实验分组:采用随机数字法将大鼠分成4组:生理盐水组、50,100,500 ng神经生长因子组,每组8只.实验干预:切除大鼠后肢坐骨神经,造成10 mm缺损,用单通道的硅胶管桥接大鼠坐骨神经缺损,在桥接管内给药.生理盐水组注入5μL生理盐水.50,100,500 ng神经生长因子组注入20 mg／L的神经生长因子2.5,5,25μL.实验评估:在第30天时,用免疫组织化学的方法检测大鼠再生坐骨神经新生血管内皮细胞中CD34、vWf、血管内皮生长因子、trkA的表达,并作形态计量分析.主要观察指标:各组大鼠神经再生普通病理及组织化学染色观察.结果:32只大鼠全部进入结果分析.①各组大鼠神经再生病理切片结果:术后30 d的坐骨神经近远端完全连接,硅胶管内坐骨神经粗壮程度为:500 ng神经生长因子组＞50 ng、100 ng组＞生理盐水组.各神经生长因子组再生的坐骨神经的纤维数目和排列规则程度要好于生理盐水组,术后30 d可见明显的血管生成.②免疫组织化学染色观察:3种不同剂量的神经生长因子组与生理盐水组比较,4种抗原的表达皆有显著性差异(P＜0.05);500 ng神经生长因子组4种抗原的表达与100 ng和50 ng神经生长因子组比较增加(CD34:94.2±6.4,74.2±10.9,77.0±11.0;vWf:116.2±20.0,72.0±13.1,68.0±9.7;TrkA:105.4±10.6,57.8±11.5,58.8±6.5;血管内皮生长因子:89.0±3.0,48.6±7.4,35.2±2.9;P＜0.05或P＜0.01).结论:神经生长因子能促进再生周围神经的血管生成,且具有剂量效应;其机制可能与神经生长因子促进trkA、血管内皮细胞生长因子的表达密切相关.%BACKGROUND: Effect of nerve growth factor(NGF)on angiogenesis of regenerative perpheral nerve is still researched up to now.OBJECTIVE:To evaluate the dose-response relationship and mechanism of NGF on angiogenesis of regenerative sciatic nerve in rats.DESIGN:Randomized controlled animal study.SEFFING:Department of Neurology,the 324 Hospital of Chinese PLA;the Third Laboratory,Institute of Battle Surgery,Daping Hospital of the Third Military Medical University of Chinese PLA.MATERIALS:A total of 32 healthy adult Wistar rats weighing 200-250 g and of both genders were selected in this study.Silica gel conduit was provided by Shanghai Xinya Medical Rubber-goods Factory and NGF was provided by Sigma Company,USA.METHODS:The experiment was carried out in the Third Laboratory,Institute of Battle Surgery,Daping Hospital Affiliated to the Third Military Medical University of Chinese PLA from August 2003 to February 2005.Experimental grouping:All rats were randomly divided into 4 groups,including saline group,50,100 and 500 ng NGF groups with 8 rats in each group.Experimental intervention:Sciatic nerves were cut to establish defect of 10 mm in length,and then the defect was bridged with silica gel conduit in the left latter limb.After the bridging,rats in the saline group were perfused with 5 μL saline,and rats in the NGF groups were perfused with 2.5,5 and 25μL NGF(20 mg/L),respectively.Experimental evaluation:On the 30th day,immunohistochemical technique was used to detect the expressions of CD34,vWf,vascular endothelial growth factor(VEGF)and trkA in newborn vascular endothelial cells of regenerative sciatic nerve;meanwhile,their forms were performed with quantitative analysis.MAIN OUTCOME MEASURES:Pathological and immunohistochemical changes of nerve regeneration of rats in each group.RESULTS:All 32 rats were involved in the final analysis,①Results of pathological sections of nerve regeneration in rats:Distal end of sciatic nerve was completely bridged at 30 days after operation and thickness of sciatic nerve in silica gel conduit lined in 500 ng NGF group＞50 and 100 ng NGF group＞saline group.Numbers of fiber and regularity of arrangement of sciatic nerve were superior in NGF groups to those in saline group,In addition,angiogenesis was obviously observed at 30 days after operation.②Results of immunohistochemical staining:There were significant differences among three NGF groups as compared with saline group in the expressions of four antigens (P＜0.05).Expressions of four antigens were increased in 500 ng NGF group as compared with those in 100 and 50 ng NGF groups(CD34:94.2±6.4,74.2±10.9,77.0±11,0;vWf:116.2±20.0,72.0±13.1,68.0±9.7;TrkA:105.4±10.6,57.8±11.5,58.8±6.5;VEGF:89.0±3.0,48 6±7.4,35.2±2.9;P＜0.05 or P＜0.01).CONCLUSION: NGF can promote angiogenesis of the regenerative peripheral nerves and show a dose-response relationship,while its mechanism may be related to promoting expressions of TrkA and VEGF.