摘要目的 观察胶原/丝素导管介导雪旺细胞联合神经干细胞所构建的组织工程化神经修复大鼠10 mm坐骨神经缺损的效果.方法 体外分离培养乳鼠坐骨神经雪旺细胞(SCs),并做S-100蛋白免疫荧光鉴定;从孕14 ～ 15 d的SD大鼠体内取出子鼠,分离纯化获得原代神经干细胞(NSCs)进行体外培养.实验分为4组,每组10只:自体神经移植组(A组)、胶原/丝素导管介导雪旺细胞联合神经干细胞移植组(B组)、胶原/丝素导管移植组(C组)、单纯损伤组(D组).SCs-NSCs与胶原/丝素导管联合培养14 d后,行扫描电镜观察.分别将3种不同移植物桥接于大鼠坐骨神经10 cm缺损处,并在12周后进行大体观察、电生理学检测、形态学观察及计量学分析.结果 术后12周,桥接组都不同程度地实现了坐骨神经缺损再通,且实验动物未出现明显排斥及炎性反应.神经电生理学检测坐骨神经复合肌动作电位(CMAPs)波幅分别为:未手术正常侧(21.00±1.83) mV,A组(15.00±1.12) mV,B组(13.00±1.06) mV,C组(6.00±0.58) mV,透射电子显微镜再生神经纤维髓鞘厚度分别为:A组(0.80±0.15) μm,B组(0.70±0.11) μm,C组(0.30±0.07) μm,D组(0.25±0.06) μm.统计结果表明:A组与B组之间差异无统计学意义(P＞0.05),但两者相对C组及D组差异有统计学意义(P＜0.05).结论 SCs和NSCs能够在胶原/丝素导管上共同生长分化,生物相容性良好.胶原/丝素导管介导雪旺细胞联合神经干细胞所构建的组织工程化神经对坐骨神经缺损的修复具有良好的桥接和促神经生长作用.

abstractsObjective To observe the repair effect of collagen/silk fibroin conduit transplanted with co-cultured Schwann cells (SCs) and neural stem cells (NSCs) for bridging 10 mm sciatic nerve defect by tissue engineering technique in rats.Methods SCs were isolated and purified from the sciatic nerve of neonatal SD rats,and were identified by immunocytochemical labelling for S100 protein.NSCs were derived from SD rats of gestation for 14-15 days.The 40 SD rats was randomly divided into 4 groups:autograft group (A),SF/collagen with cells graft group (B),SF/collagen graft group (C) and non-grafted group (D).After 14 days in culture,the SF/collagen conduit with SCs-NSCs was observed under a scanning electron microscope.At 12th week after the three different grafts were transplanted to bridge a 10 mm rat sciatic nerve gap,the morphological analysis and electrophysiological examination were done to evaluate the effect of nerve regeneration.Results At 12th week after surgery,the bridging groups were repaired and no obvious rejection and inflammation occurred in the animals.The amplitude of electrophysiological CMAPs were uninjured side of animals (21.00 ± 1.83),(15.00 ± 1.12),(13.00 ± 1.06) and (6.00 ± 0.58) mV in groups A,B,and C respectigvely.Myelin sheath thickness of regenerated nerve fibers under transmission electron microscopy were (0.80 ± 0.15),(0.70 ± 0.11),(0.30 ± 0.07) and (0.25 ± 0.06) μm in groups A,B,C and D respectively.Statistical results showed that myelin sheath thickness of regenerated nerve fibers showed no significant difference between groups A and B (P ＞ 0.05),while it was significantly greater in either group A or B than that in groups C and D (P ＜ 0.05).Conclusion SCs and NSCs have good compatibility with collagen/silk fibroin conduit.The tissue engineered nerve graft constructed by collagen/silk fibroin conduit combined with the co-culture of SCs and NSCs could repair the defect of sciatic nerve and promote nerve regeneration.