携带NGF基因腺病毒治疗大鼠坐骨神经损伤的实验研究  

EFFECT OF ADENOVIRUS EXPRESSING NGF ON SCIATIC NERVE INJURY IN RATS

作　　者：陈渝[1,2] 王大勇[2] 王祖贵[2] 翁政[2] 邓忠良[1] 

机构地区：[1]重庆医科大学附属第二医院骨科,重庆400010 [2]重庆市第九人民医院骨科

出　　处：《中国修复重建外科杂志》2009年第8期947-953,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：重庆市北碚区科委资助项目(2008-10)~~

摘　　要：目的构建携带NGF基因序列的5型腺病毒(adenovirus expressing NGF,Ad-NGF),探讨其在促进大鼠坐骨神经神修复与再生中的作用。方法将NGF基因序列克隆至5型腺病毒穿梭质粒pCA13,并在HEK-293细胞中包装得到重组腺病毒Ad-NGF并测序鉴定。雄性SD大鼠32只,体重180～200g,随机分为4组(n=8)。切断大鼠右侧坐骨神经并缝合,建立坐骨神经损伤模型。模型制备后,A组及C组分别于术侧腓肠肌注射Ad-NGF及不携带NGF基因序列的空腺病毒载体,1×108PFU/次,隔天1次,共3次;B组及D组分别于术侧腓肠肌注射NGF(200U/d)及生理盐水(100μL/d),连续3周。术后第31天检测坐骨神经功能指数、神经电生理检测,取吻合处及其远端1cm坐骨神经,采用RT-PCR及Westernblot技术定量检测大鼠损伤坐骨神经中NGFmRNA及蛋白的表达水平,并行组织学及免疫组织化学、透射电镜观察。结果实验成功构建了携带NGF基因序列的5型腺病毒Ad-NGF。A组坐骨神经功能指数、神经传导速度、诱发电位波幅及潜伏期与B、C、D组比较,差异均有统计学意义(P<0.05);B、C、D组间比较差异无统计学意义(P>0.05)。RT-PCR及Westernblot检测显示,A组NGFmRNA及蛋白表达量与B、C、D组比较,差异均有统计学意义(P<0.05);B、C、D组间比较差异无统计学意义(P>0.05)。组织学及免疫组织化学观察示A组坐骨神经再生情况明显优于其他组。透射电镜观察示A组坐骨神经横切面轴突直径、髓鞘厚度、神经纤维个数与B、C、D组比较,差异有统计学意义(P<0.05);B、C、D组间比较差异无统计学意义(P>0.05)。结论Ad-NGF提供了一种在周围神经损伤局部获得大量NGF的新方法,且可有效促进大鼠坐骨神经损伤后的修复。Objective To construct adenovirus expressing NGF （Ad-NGF） and to investigate its promotive effect on the reparation and regeneration of sciatic nerve injury in rats. Methods NGF gene sequence was cloned into shuttle plasmid pCA13 of adenovirus type 5. After packed in HEK-293 cells, the recombinant adenoviruses-Ad-NGF underwent sequence identification. Thirty-two male SD rats weighing 180-200 g were randomly divided into 4 groups （n=8 rats per group）. Sciatic nerve injury model was established by disconnecting and direct suturing the right sciatic nerve in the rat. The right gastrocnemius muscle of group A and C received Ad-NGF injection and adenovirus vector without NGF gene sequence injection, respectively, and 1 × 10^8 PFU/per time was given every other day for three times. Group B and D received NGF injection （200 U/d） and normal saline （100 μL/d）, respectively, for 3 weeks. The effect of various treatments on injured sciatic nerve was evaluated by performing sciatic nerve function index and nerve electrophysiology detections 31 days after operation. Meanwhile, the sciatic nerve in the anastomosis and at the site 1 cm distal to the anastomosis were obtained, and underwent RTPCR and Western blot analysis for detecting NGF mRNA and protein expression level in the injured sciatic nerve in the rats. Histology, immunohistochemistry, and transmission electron microscope observations were conducted. Results Ad-NGF carrying NGF gene sequence was constructed successfully and confirmed by sequence analysis. The sciatic nerve function index, nerve conduction velocity, evoked potential amplitude, and latent period of group A was better than those of other groups （P 〈 0.05）, and there were no significant differences among group B, C, and D （P 〉 0.05）. RT-PCR and Western blot detection： the expression levels of NGF mRNA and protein in group A were greater than those of group B, C, and D （P 〈 0.05）, and no significant differences were noted among group B, C, and D （P 〉 0.05）. Histo

关 键 词：坐骨神经损伤 NGF 腺病毒 神经修复 大鼠 

分 类 号：R651.3[医药卫生—外科学]