Chen Yu, Deng Zhongliang, Weng Zheng, Chen Shimou, Tong Wei
The Fisrt Department of Orthopedics, Chongqing Ninth People's Hospital, Chongqing, 400070, P. R. China.
Department of Orthopedics, the Second Affiliated Hospital of Chongqing Medical University.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2016 Aug 8;30(8):1026-1033. doi: 10.7507/1002-1892.20160206.
To construct recombinant adenovirus expressing nerve growth factor (NGF) and myelin associated glycoprotein (MAG) (Ad-NGF-MAG) and to investigate its effect on repair and regeneration of sciatic nerve injury in rats.
NGF and MAG gene sequences were cloned into shuttle plasmid pCA13 of adenovirus type 5. After packed in HEK293 cells, the recombinant Ad-NGF-MAG underwent sequence and identification. Thirty-two male Sprague Dawley rats were randomly divided into 4 groups (=8): control group (normal control), adenovirus vector group (Ad group), Ad-NGF group, and Ad-NGF-MAG group. The sciatic nerve injury model was established by transection of the right sciatic nerve; then, the empty adenovirus vector, Ad-NGF, and Ad-NGF-MAG were injected into the gastrocnemius muscle of the affected limb at a dose of 1×10 PFU every other day for 3 times in Ad group, AdNGF group, and Ad-NGF-MAG group, respectively. The right sciatic nerve was exposed only, and then the incision was closed in the control group. The sciatic nerve function index (SFI) was measured, and neuro-electrophysiology was observed; mRNA and protein expressions of NGF and MAG were detected by RT-PCR and Western blot; and histological examination was performed at 31 days after operation.
Recombinant adenovirus vectors of Ad-NGF and Ad-NGF-MAG were constructed successfully. All rats survived and incision healed by first intension. The SFI, nerve conduction velocity, evoked potential amplitude, and latent period of Ad-NGF-MAG group were significantly better than those of Ad group and Ad-NGF group (<0.05). MAG mRNA and protein expressions of Ad-NGF-MAG group were the highest in all the groups (<0.05). The expressions of NGF mRNA and protein increased in Ad-NGF group and AdNGF-MAG group when compared with control group and Ad group (<0.05). Histological examination showed that the nerve had good continuity in control group; nerve fibers disarranged in Ad group; neurons connections formed in some nerve fibers of Ad-NGF group, but nerve fibers arrange disorderly; and the growth of the nerve were ordered and wellstructured in Ad-NGF-MAG group.
Ad-NGF-MAG can effectively promote the growth of the nerve and inhibit the form of abnormal branches, facilitating the repair of sciatic nerve injury in rats.
构建表达神经生长因子（NGF）和髓鞘相关糖蛋白（MAG）的重组腺病毒（Ad-NGF-MAG），并研究其对大鼠坐骨神经损伤修复和再生的影响。
将NGF和MAG基因序列克隆到5型腺病毒穿梭质粒pCA13中。在HEK293细胞中包装后，对重组Ad-NGF-MAG进行测序和鉴定。32只雄性Sprague Dawley大鼠随机分为4组（每组8只）：对照组（正常对照）、腺病毒载体组（Ad组）、Ad-NGF组和Ad-NGF-MAG组。通过切断右侧坐骨神经建立坐骨神经损伤模型；然后，Ad组、Ad-NGF组和Ad-NGF-MAG组分别每隔一天以1×10⁹ PFU的剂量向患侧肢体的腓肠肌注射空腺病毒载体、Ad-NGF和Ad-NGF-MAG，共注射3次。对照组仅暴露右侧坐骨神经，然后缝合切口。测量坐骨神经功能指数（SFI），观察神经电生理；通过RT-PCR和Western blot检测NGF和MAG的mRNA和蛋白表达；术后31天进行组织学检查。
成功构建了Ad-NGF和Ad-NGF-MAG重组腺病毒载体。所有大鼠均存活，切口一期愈合。Ad-NGF-MAG组的SFI、神经传导速度、诱发电位幅度和潜伏期均明显优于Ad组和Ad-NGF组（P<0.05）。Ad-NGF-MAG组的MAG mRNA和蛋白表达在所有组中最高（P<0.05）。与对照组和Ad组相比，Ad-NGF组和Ad-NGF-MAG组的NGF mRNA和蛋白表达增加（P<0.05）。组织学检查显示，对照组神经连续性良好；Ad组神经纤维排列紊乱；Ad-NGF组部分神经纤维形成神经元连接，但神经纤维排列无序；Ad-NGF-MAG组神经生长有序且结构良好。
Ad-NGF-MAG能有效促进神经生长，抑制异常分支形成，促进大鼠坐骨神经损伤的修复。
