嗅鞘细胞移植结合步行训练对大鼠脊髓挫伤部位神经递质水平和神经营养因子表达的影响

The effect of olfactory ensheathing cells transplantation combining with step training on neurotransmitter level and neurotrophic factor protein expression in spinal cord injury site in rats with spinal cord contusion

目的:初步研究嗅鞘细胞移植、步行训练、嗅鞘细胞移植结合步行训练后脊髓损伤部位神经递质含量和神经营养因子蛋白水平的变化,探索嗅鞘细胞移植结合步行训练促进大鼠神经功能恢复作用的可能机制。方法:鼠龄(75±1)d的雌性SD大鼠32只,分为A、B、C、D四组,制作脊髓挫伤模型后,A组行嗅鞘细胞移植结合步行训练,B组行嗅鞘细胞移植,C组行步行训练结合改良Eagle培养基(DMEM)注射,D组行DMEM注射。细胞移植和DMEM注射在造模后2周进行,步行训练利用大鼠活动平板进行,自伤后1周开始,每周5d,每天1次,每次15—30min,共训练10周。伤后11周取材,反相高效液相色谱—荧光检测法测定脊髓损伤部位5羟色胺(5-HT)、多巴胺(DA)、去甲肾上腺素(NE)等几种神经递质含量,Western Blot检测脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养因子3(NT-3)蛋白表达。结果:1各组损伤部位DA和DOPA均未测出,C组5-HT、NE含量较其他各组出现增高趋势,A组较B、D组出现增高趋势,却较C组出现降低趋势;2B组BDNF、NGF、NT-3蛋白水平较其他各组出现增高趋势,A组BDNF、NT-3水平较C、D组出现增高趋势,但却较B组出现降低趋势;但上述差异不具有显著性意义。结论:伤后1周开始步行训练、2周进行嗅鞘细胞移植的结合型策略不能促进脊髓损伤部位神经营养因子表达和脊髓神经递质分泌。

Objective： To explore the influence of step training on neurotransmitter level rats with spinal cord contusion. olfactory ensheathing cell（OECs） transplantation combined with treadmill and neurotrophic factor protein expression in spinal cord injury site in Method： Thirty-two adult fhmale Sprague Dawley rats aged （75±1）d were subjected to experimental spinal cord contusion at T10 level using a New York University impactor at the height of 25 mm. Rats were then randomly divided into 4 groups： group A, OECs transplantation combined with step training （OECs-step training） group; Group B, an OECs transplantation（OECs） group; Group C, step training combined with Dulbecco＇s modification of Eagle＇s medium（DMEM） injection （steo training-DMEM） group;Group D,DMEM injection（SCI-DMEM） group. The OECs transplantation and DMEM injection were performed at 2 weeks post-injury. Step training began at the 7th d post-injury and consisted of daily sessions （once daily, 5d a week, for 10 weeks） of quadrupedal treadmill training, starting from 15 min and gradually increasing to 30 min daily, at speeds starting from 3m/min and gradually increasing in accordance to the condition of rats. At the llth week postinjury, all the rats were executed and the spinal cord encompassing the injury site were removed. The levels of 5-hydroxytryptamine（5-HT）, dopamine（DA）, 3,4-dihydroxyphenyl alanine（DOPA） and norepinephrine （NE） of spinal cord lesion site were assessed by reversed-phase high performance liquid chromatographic methodolo- gies（RP-HPLC）, and the protein expressions of nerve growth factor（NGF）, brain derived neurotrophic factor （BDNF） and neurotrophin-3（NT-3） in spinal cord lesion site were analyzed by Western blotting. Result： At the end of the llth week post-injury, the DA and DOPA were not founded by RP-HPLC. An increased trend of 5-HT and DA levels in step training group, and expressions of BDNF, NGF and NT-3 protein in OECs group were observed. However, no synthetic effect on 5-HT, DA levels and BDNF, NGF, NT-3 protein expressions were demonstrated in OECs-step training group. Conclusion： OECs transplantation combined with step training fail to promote the secretion of 5-HT, DA and expressions of BDNF, NGF, NT-3 protein in spinal cord injury site. Author＇s address Orthopaedic Department, The Beijing Army General Hospital, Beijing, 100700