摘要
目的探讨嗅球源性嗅鞘细胞(olfactory ensheathing cells,OECs)移植联合鞘内重复注射VEGF对损伤脊髓功能恢复的促进作用,以及二者对神经元和神经纤维的协同保护作用。方法取新生2～3 d大鼠嗅球分离纯化培养OECs,于培养9 d用0.25%胰蛋白酶消化后收集细胞。将75只成年雌性Wistar大鼠(体重200～250 g)随机分为5组,A组为假手术组,仅行椎板切除术。B、C、D、E组采用重力打击法制作脊髓损伤模型后,B组术后1 d于脊髓损伤部位分4点共注射DMEM-F12无血清培养基10μL,并每天2次鞘内注射生理盐水10μL,持续1周;C组同上法分别注射DMEM-F12培养基10μL及重组大鼠VEGF165(recombined rat VEGF165,rrVEGF165)25 ng;D组注射含1×105个OECs的DMEM-F12培养基10μL及生理盐水10μL;E组注射含1×105个OECs的DMEM-F12培养基10μL及rrVEGF165 25 ng。术后1 d及术后1～8周每周采用Basso-Beattie-Bresnahan(BBB)评分标准评定大鼠后肢功能恢复情况;术后8周处死实验动物,取材行透射电镜、HE染色观察,并行p75NGF受体(p75 NGF receptor,p75NGFR)、半胱氨酸蛋白酶3(Caspases-3)、血管性假血友病因子(von Willebrand factor,vWF)免疫组织化学染色,评价损伤脊髓修复情况。结果术后E组大鼠后肢功能恢复较快,8周时BBB评分显著高于其余各组(P<0.05)。透射电镜和HE染色显示:B组有髓神经纤维和神经元受损严重;C、D组损伤轻于B组;E组受损最轻,断端间残存组织较多,有髓神经纤维髓鞘较完整,神经元呈多角形,有少量空泡化的线粒体。免疫组织化学染色示,B、C、D、E组Caspase-3阳性细胞数显著多于A组(P<0.05),B、D组明显多于C、E组(P<0.05),C、E组间差异无统计学意义(P>0.05)。C、E组vWF阳性血管数多于A、B、D组(P<0.05),但C、E组间差异无统计学意义(P>0.05)。D、E组均可见p75NGFR阳性OECs。结论 OECs移植联合鞘内重复注射VEGF可显著促进大鼠脊髓损伤修复,部分恢复损伤神经功能,对受损神经纤维和神经元的变性有保护作用,且二者有协同作用。
Objective To investigate the synergistic effect of a combination of grafted olfactory ensheathing cells（OECs） from the olfactory bulbs and intrathecal injection of vascular endothelial growth factor（VEGF） on repairing spinal cord injury,and to explore the neuroprotection on both neurons and nerve fibers.Methods OECs from neonatal rats were cultured,purified,and collected with 0.25% trypsin after 9 days.A total of 75 adult female Wistar rats（weighing 200-250 g） were randomly divided into 5 groups： group A was sham-surgery group receiving laminectomy;the spinal cord injury model was established with weight-dropped apparatus in the rats of groups B,C,D,and E.Then group B was injected with 10 μL DMEM-F12 medium without serum at injury site on the 1 day and was intrathecally administrated with 10 μL saline solution twice a day during the following 1 week;group C was injected with 10 μL DMEM-F12 medium and 25 ng recombined rat VEGF165（rrVEGF165）;group D was injected with 10 μL DMEM-F12 medium containing 1 × 105 OECs and 10 μL saline solution;group E was injected with 10 μL DMEM-F12 medium containing 1 × 105 OECs and 25 ng rrVEGF165.The functional recovery of hindlimb was evaluated by the Basso-Beattie-Bresnahan（BBB） score at 1 day and each week from 1 to 8 weeks.The histological changes and the changes of ultrastructure were observed at 8 weeks after operation by HE and electron microscope,and the immunohistochemistry staining was used for p75 nerve growth factor receptor（p75NGFR）,Caspase-3,and von Willebrand factor（vWF）.Results The function of hindlimb recovered rapidly in group E;the BBB score reached the peak at 8 weeks,and it was significantly higher than those in other groups（P 〈 0.05）.The histology and ultrastructure observation showed that nerve fibers and neurons were damaged seriously in group B,moderately in groups C and D,and slightly in group E.Numerous spared tissue between nerve stumps,fibers with regular myelination,and neurons with little vacuolar mitochondria were observed in group E.The immunohistochemistry staining revealed that Caspase-3 positive cells in groups B,C,D,and E were significantly more than that in group A（P 〈 0.05）;more Caspase-3 positive cells were found in groups B and D than in groups C and E（P 〈 0.05）,while no significant difference was found between groups C and E（P 〉 0.05）.And more vessels per high field were examined in groups C and E than in groups A,B,and D（P 〈 0.05）,while no significant difference was found between groups C and E（P 〉 0.05）.The p75NGFR positive results showed the survival of OECs in groups D and E at 8 weeks after OECs transplantation.Conclusion Grafted OECs combined with intrathecal injection of VEGF has significant promotive effects on restoration of spinal cord injury in rats,can improve part function of nerve fibers,and shows neuroprotection on damaged cells and fibers,which have a synergistic effect
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2011年第6期699-704,共6页
Chinese Journal of Reparative and Reconstructive Surgery
基金
山东省自然科学基金资助项目(Y2007C010)~~
关键词
嗅鞘细胞
VEGF
脊髓损伤
细胞移植
大鼠
Olfactory ensheathing cells Vascular endothelial growth factor Spinal cord injury Cell transplantation Rat
