NGF/MAG双基因共表达腺病毒修复大鼠周围神经损伤的实验研究

EFFECT OF REC OMBINANT C O-EXPRESSION ADENOVIRUS OF NERVE GROWTH FACTOR AND MYELIN ASSOCIATED GLYCOPROTEIN GENES ON RAT SCIATIC NERVE INJURY

目的构建携带NGF及髓磷脂相关糖蛋白（myelin associated glycoprotein,MAG）基因序列的双基因共表达腺病毒（adenovirus expressing NGF and MAG,Ad-NGF-MAG）,探讨其在大鼠坐骨神经损伤修复中的作用。方法将NGF和MAG共同克隆至5型腺病毒穿梭质粒p CA 13,并在HEK 293细胞中包装得到重组腺病毒Ad-NGF-MAG并测序鉴定。取雄性SD大鼠32只,体质量180~200 g;随机分成4组（n=8）：对照组（正常对照）、空病毒组（Ad组）、单独表达NGF组（Ad-NGF组）和共表达NGF、MAG组（Ad-NGF-MAG组）。Ad组、AdNGF组和Ad-NGF-MAG组大鼠制备右侧坐骨神经损伤模型后,分别于术侧腓肠肌注射空腺病毒、Ad-NGF及AdNGF-MAG（1×108 PFU）,隔天1次,共3次。对照组仅暴露右侧坐骨神经后关闭切口,术后对应时间点注射生理盐水10μL。术后31 d,分别行坐骨神经功能指数（sciatic nerve function index,SFI）、神经电生理检测;切取坐骨神经标本,行RT-PCR及Western blot检测NGF及MAG m RNA及蛋白表达水平,以及组织学观察。结果实验成功构建重组腺病毒Ad-NGF和Ad-NGF-MAG。32只大鼠术后均成活,切口Ⅰ期愈合。Ad-NGF-MAG组SFI、神经传导速度、诱发电位波幅、潜伏期均明显优于Ad-NGF组和Ad组,但未达对照组水平,比较差异均有统计学意义（P〈0.05）。Ad-NGF-MAG组内MAG m RNA和蛋白表达最高,NGF m RNA和蛋白表达高于对照组和Ad组,比较差异均有统计学意义（P〈0.05）。组织学观察显示,对照组神经连续性好,Ad组神经纤维层次混乱,Ad-NGF组神经纤维层次结构清晰,局部断端再生良好,但神经纤维结构紊乱;Ad-NGF-MAG组神经纤维生长有序,神经直径较Ad-NGF组粗,神经纤维结构良好。结论坐骨神经损伤后修复过程中,腺病毒介导NGF与MAG共表达,既可促进神经纤维生长,又可抑制神经异常分支形成,促进神经结构与功能的恢复。

Objective 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. Methods NGF and MAG gene sequences were cloned into shuttle plasmid p CA 13 of adenovirus type 5. After packed in HEK 293 cells, the recombinant Ad-NGF-MAG underwent sequence and identification. Thirty-two male Sprague Dawley rats were randomly divided into 4 groups（n= 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 × 108 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; m RNA 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. Results 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（P〈0. 05）. MAG m RNA and protein expressions of Ad-NGF-MAG group were the highest in all the groups（P〈0. 05）. The expressions of NGF m RNA and protein increased in Ad-NGF group and AdNGF-MAG group when compared with control group and Ad group（P〈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. Conclusion 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.