Chen Yu, Wang Dayong, Wang Zugui, Weng Zheng, Deng Zhongliang
Department of Orthopedics, Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Aug;23(8):947-53.
To construct adenovirus expressing NGF (Ad-NGF) and to investigate its promotive effect on the reparation and regeneration of sciatic nerve injury in rats.
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 x 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 microL/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 RT-PCR 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.
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). Histology and immunohistochemistry observation showed that the regeneration of the sciatic nerve in group A was obvious superior to that of other groups. Transmission electron microscopy observation suggested there was significant difference between group A and groups B, C, and D in terms of axonal diameter of sciatic nerve cross-section, myelin sheath thickness and nerve fiber number (P < 0.05), and there were no significant differences among group B, C, and D (P > 0.05).
Ad-NGF can effectively promote the repair of sciatic nerve injury in rats, and is a new method for obtaining large amounts of NGF in the area of injured peripheral nerve.
构建表达神经生长因子（NGF）的腺病毒（Ad-NGF），并研究其对大鼠坐骨神经损伤修复和再生的促进作用。
将NGF基因序列克隆到5型腺病毒穿梭质粒pCA13中。在HEK-293细胞中包装后，对重组腺病毒-Ad-NGF进行序列鉴定。将32只体重180-200 g的雄性SD大鼠随机分为4组（每组n = 8只大鼠）。通过切断并直接缝合大鼠右侧坐骨神经建立坐骨神经损伤模型。A组和C组的右侧腓肠肌分别接受Ad-NGF注射和无NGF基因序列的腺病毒载体注射，每隔一天给予1×10(8) PFU/次，共3次。B组和D组分别接受NGF注射（200 U/d）和生理盐水（100 μL/d），持续3周。术后31天通过进行坐骨神经功能指数和神经电生理检测评估各种治疗对损伤坐骨神经的影响。同时，获取吻合处及吻合处远端1 cm处的坐骨神经，进行RT-PCR和蛋白质印迹分析，以检测大鼠损伤坐骨神经中NGF mRNA和蛋白表达水平。进行组织学、免疫组织化学和透射电子显微镜观察。
成功构建了携带NGF基因序列的Ad-NGF，并经序列分析证实。A组的坐骨神经功能指数、神经传导速度、诱发电位幅度和潜伏期均优于其他组（P < 0.05），B、C、D组之间无显著差异（P > 0.05）。RT-PCR和蛋白质印迹检测：A组中NGF mRNA和蛋白的表达水平高于B、C、D组（P < 0.05），B、C、D组之间无显著差异（P > 0.05）。组织学和免疫组织化学观察显示，A组坐骨神经的再生明显优于其他组。透射电子显微镜观察表明，A组与B、C、D组在坐骨神经横截面积、髓鞘厚度和神经纤维数量方面存在显著差异（P < 0.05），B、C、D组之间无显著差异（P > 0.05）。
Ad-NGF可有效促进大鼠坐骨神经损伤的修复，是在损伤周围神经区域获得大量NGF的新方法。
