过氧化物酶6作用下神经干细胞对面神经损伤的修复研究

Facial nerve injury repair of neural stem cells in peroxidase 6 action

目的探索过氧化物酶6(Prdx6)作用下神经干细胞(NSC)对面神经离断性损伤的修复效果。方法选择新生6 h内SD大鼠6只,雌雄不限,体质量100 g左右。选择清洁级雄性成年SD大鼠30只,体质量(220±50)g,鼠龄6~8周。从新生6 h内SD大鼠取脑内分离NSC,采用悬浮的方法体外培养NSC,并进行NSC特异性标记蛋白Nestin的免疫荧光染色鉴定。将清洁级雄性成年SD大鼠分为A组(假手术组)、B组(溶媒组)、C组(Prdx6组)、D组(NSC组)、E组(NSC联合Prdx6组),每组6只。除A组外,其余各组采用手术离断方法对大鼠左侧面神经颊支离断形成5 mm缺损,制作SD大鼠面神经缺损模型,使用大鼠同种自体脱细胞动脉导管作为移植支架进行NSC移植治疗或其他处理。分别在第2、4、6、8周对各组大鼠进行面神经麻痹评分;术后8周利用尼氏染色观察各组大鼠面神经核团内的神经元情况;术后8周利用透射电子显微镜观察再生神经的恢复情况。结果取第3代神经球,用激光共聚焦电子显微镜检测到NSC特异性标记蛋白Nestin阳性表现。获得无细胞成分的脱细胞动脉导管。除A组外,术后第2、4、6、8周E组大鼠面神经恢复情况最佳,D组次之。术后8周,除A组(29.83个±2.64个)外,E组神经元数量(27.83个±1.47个)最多,其次为D组(24.17个±0.98个),差异都有统计学意义(P<0.05)。各组大鼠面神经再生后通过透射电子显微镜观察,A组神经髓鞘的厚度与成熟度最好,神经外膜完整,束膜清晰,轴突横切面近圆形;其次为E组,细胞器丰富,结构清晰,形态规则,但未达到正常,且E组面神经恢复效果优于D组。结论Prdx6可以促进NSC对面神经损伤的修复效果。

Objective To explore the repair effect of neural stem cells(NSC)on facial nerve injury induced by peroxiredoxin 6(Prdx6).Methods Six 6-hour-old newborn SD rats,male and female,with body weight about 100 g were selected.Thirty clean grade male SD rats with body weight(220±50)g,aged 6-8 weeks were selected.NSCwas isolated from brains of the newborn SD rats and cultured in vitro by suspension,and then identified by immunofluorescence staining of NSC-specific labeled protein Nestin.The clean grade male SD rats were divided into group A(sham operation group),group B(solvent group),group C(Prdx6),group D(NSC)and group E(NSCcombined with Prdx6),with 6 rats in each group.Except group A,the 5 mm defect of the left buccal branch of rats was formed by surgical dissection,and facial nerve defect model of SD rats was estab-lished.The rat autologous acellular devascularization catheter was used as graft stent for NSCtransplantation or performed oth-er treatments.The rats in each group were scored for facial nerve palsy at the 2nd,4th,6th,and 8th week after surgery.The neurons in facial nucleus of each group were observed by Nissl staining and transmission electron microscopy was used to ob-serve the recovery of regenerative nerves at 8th week after surgery.Result The Nestin positive performance in the third generation of neurospheres was detected by laser confocal electron microscopy,and cellular-free acellular arterial catheter was obtained.Except group A,the facial nerve recovery at the 2th,4th,6th and 8th week after surgery of group E was the best a-mong the tested groups,followed by group D.At the 8 th week after surgery,except group A(29.83±2.64),the neuron number of group E(27.83±1.47)was the most,followed by group D(24.17±0.98),and the difference was statistically significant(P<0.05).Observation results by transmission electron microscope after facial nerve regeneration in rats of each group showed that the thickness and maturity of nerve myelin sheath in group A was the best,the outer nerve membrane was complete with clear bundle membrane,and transverse section of axon was nearly round.The second best results were found in group E,with abun-dant organelles,clear structure and regular morphology but not up to normal,while recovery effect of facial nerve in group E was better than that in group D.Conclusion It is demonstrated that Prdx6 could promote the repair effect of NSC on facial nerve injury.