腺病毒介导的NT-3基因在大鼠坐骨神经的表达

Expression of adenoviral mediated NT- 3 genes in Schwann cells of sciatic ne rve in the rats

目的观察腺病毒介导的神经营养素-3(neurotrophin-3,NT-3)基因在大鼠坐骨神经雪旺细胞(Schwanncells,SCs)的表达。方法NT-3重组腺病毒在293细胞中培养繁殖并测定滴度后,直接注入大鼠损伤修复的坐骨神经内,不同时间点取材,采用免疫组织化学染色检测NT-3蛋白的表达,并用LEICAM550型图像分析仪对坐骨神经切片NT-3免疫组化染色强弱进行定量评价。结果坐骨神经损伤修复后直接注射Ad-NT-3,2d后出现NT-3免疫组化染色阳性产物,主要位于吻合口附近,阳性产物呈平行条纹状排列,7d时显著增加(与2d组相比,P<0.01),14d和28d时有所下降(与7d组相比,P<0.01),两者差异无显著性意义(P>0.05),但与2d组相比,仍维持在较高的水平(P<0.01)。而正常坐骨神经、损伤修复后注射Ad-LacZ或生理盐水的坐骨神经NT-3免疫组化染色结果为阴性。结论NT-3基因能通过腺病毒介导转入损伤修复后周围神经的SCs并表达NT-3蛋白,为腺病毒介导神经营养因子基因治疗促进周围神经损伤再生提供了初步的理论和实验依据。

Objective To investigate the expression of neurotrophin- 3 (NT- 3) gene in Schwann cells (SCs) of rat sciatic nerve introduced by adenovir al vector in vivo. Methods Recombinant adenoviral vector for NT- 3 was propagat ed in 293 packaging cells and was titered by tissue culture infections dose 50 ( TCID50) method. Ad- NT- 3 was injected intraneurally into the rat sciatic nerv e following axotomy of the sciatic nerve which was repaired after the immediate injury. Immunohistochemical staining was employed to determine the expression of NT- 3 in SCs in rat sciatic nerve, and the expressive intensity difference was measured with LEICA M550 imagine analysis system on the tissue slides. Results Two days after injection of NT- 3 into the rat nerve, positive stain in the SCs were apparent in the vicinity of anastomosis. There was significant increase of the amount of NT- 3 expression 7 days following injection of Ad- NT- 3 (vs. 2 days group P< 0.01). The amount of NT- 3 expression decreased 14 to 28 days following injection of Ad- NT- 3 (vs. 7 days group P< 0.01). There was no sign ificant difference between the 14 and 28 days group (P >0.05). Compared with the 2 days group, the 14 and 28 days groups still maintained a relatively high leve l of NT- 3 (P< 0.01). The intact nerves and repaired nerves, which were injecte d with Ad- LacZ or physiological saline served as control, showed no NT- 3- p ositive SCs. Conclusion It was shown that an adenoviral vector could be used to efficiently direct the expression of NT- 3 gene in SCs of rat peripheral nerves following nerve injury and repair. The results suggested that neurotrophic fact ors could be introduced into SCs by adenoviral vector to promote peripheral nerv e regeneration. [