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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 a recombinant adenovirus expressing adenovirus expressing adenovirus expressing adenovirus expressing human myelin associated glycoprotein (MAG) and adenovirus expressing adenovirus vector (Ad-NGF-MAG) for the repair of sciatic nerve in rats effect. Methods NGF and MAG were co-cloned into shuttle plasmid p CA 13 of adenovirus type 5, and recombinant adenovirus Ad-NGF-MAG was packaged in HEK 293 cells and sequenced. 32 male SD rats weighing 180-200 g were randomly divided into 4 groups (n = 8): control group (normal control), empty virus group (Ad group), NGF group (Ad-NGF group) And co-expressed NGF, MAG group (Ad-NGF-MAG group). The right sciatic nerve injury model was prepared in Ad group, AdNGF group and Ad-NGF-MAG group. Ad-NGF and AdNGF-MAG (1 × 108 PFU) , A total of 3 times. In the control group, only the right sciatic nerve was exposed and the incision was closed. After the operation, 10 μL of normal saline was injected. At 31 days after operation, the sciatic nerve function index (SFI) and electrophysiological examination were performed respectively. The sciatic nerve specimens were obtained and the expression of NGF and MAG m RNA and protein were detected by RT-PCR and Western blot. . Results The recombinant adenovirus Ad-NGF and Ad-NGF-MAG were successfully constructed. Thirty-two rats survived after surgery, and incisions healed in the first period. Ad-NGF-MAG group SFI, nerve conduction velocity, evoked potential amplitude and latency were significantly better than the Ad-NGF group and Ad group, but did not reach the level of the control group, the difference was statistically significant (P <0.05). The mRNA and protein expressions of MAG m RNA and protein in Ad-NGF-MAG group were higher than those in control group and Ad group (P <0.05). Histological observation showed that the neurological continuity of the control group was good, the nerve fibers of Ad-NGF-MAG group were disorderly, the structure of nerve fibers in Ad-NGF group was clear, Orderly, nerve diameter than Ad-NGF group thick, good nerve fiber structure. Conclusion Adenovirus-mediated co-expression of NGF and MAG can promote the growth of nerve fibers, inhibit the formation of abnormal nerve branches and promote the recovery of nerve structure and function during the repair process after sciatic nerve injury.