嗅鞘细胞移植对脊髓损伤区勿动蛋白表达的影响

Effect of olfactory ensheathing cell transplantation on NogoA protein levels in the injured spinal cord region

背景：近年来人们认为只要能抑制脊髓损伤后神经再生的不利因素就能促进损伤脊髓的再生，抑制因子包括髓鞘相关抑制分子和胶质瘢痕。其中髓鞘相关抑制分子主要有勿动蛋白、髓磷脂相关糖蛋白及少突起胶质细胞髓鞘相关糖蛋白。目的：观察嗅鞘细胞移植前后脊髓损伤区勿动蛋白的动态变化。设计、时间及地点：开放性实验，于2006—09／2007—05在西安交大医学院教育部环境与基因重点实验室完成。材料：8周龄成年SD大鼠40只，体质量（2．50±0．25）kg，雌雄不拘，随机数字表法分为正常组、模型组、嗅鞘细胞组、DF12对照组，10只／组。另取30只健康成年雄性SD大鼠用于嗅鞘细胞的取材，体质量200～250g。方法：除正常组外，其余各组均建立全横切脊髓损伤模型。嗅鞘细胞组将原代培养12d的嗅鞘细胞悬液调整为1×10^11L^-1，在距损伤缘上下各1mm处分4点应用微量注射器注射，深度1．0mm，每处各注射1μL。DF12对照组同法每点注射等量DF12培养液，模型组、正常组不进行任何处理。主要观察指标：各组分别于移植后1，4，8周采用免疫组织化学技术动态检测脊髓损伤区勿动蛋白的变化。同时在移植后8周行嗜银染色检测组织形态学变化。结果：①勿动蛋白的变化：正常组勿动蛋白吸光度值明显低于其余3组（P〈0．05）。移植后1，4，8周，嗅鞘细胞组脊髓损伤区勿动蛋白均明显低于模型组和DF12对照组（P〈0．01），而模型组和DF12对照组差异无显著性意义（P〉0．01）。②组织形态学变化：嗅鞘细胞移植8周后，除正常组外，其余各组均可见明显的神经纤维再生，但模型组与DF12对照组大部分纤维排列紊乱，再生纤维方向性较差；嗅鞘细胞组可见明显的新生轴突，且神经纤维跨越损伤部位修复脊髓损伤，无论在数量还是质量上均优于模型组及DF12对照组。结论：嗅鞘细胞移植可能通过降低脊髓损伤区勿动蛋白水平促进损伤脊髓的修复。

BACKGROUND： Recently, people believed that if disadvantages of neural regeneration following spinal cord injury were inhibited, regeneration of injured spinal cord was promoted. Inhibitory factors included myelin-associated inhibitive molecule and glial scar. Myelin-associated inhibitive molecule mainly contained Nogo protein, myelin-associated glycoprotein and myelin associated glucoprotein oligodendrocyte. OBJECTIVE： To explore effect of olfactory ensheathing cells （OECs） transplantation on levels of NogoA protein in the injured spinal cord region. DESIGN, TIME AND SETTING： The opening experiment was enforced from September 2006 to May 2007 at the Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education of Xi＇an Jiaotong University. MATERIALS： A total of 40 adult Sprague Dawley rats, aged 8 years, weighing （2.50±0.25） kg, of both genders, were randomly divided into normal group, model group, OECs group and DF12 control group. In addition, 30 healthy male Sprague Dawley rats weighing 200-250 g were accepted as the material for cell culture. METHODS： All the animals apart from the normal group were established spinal cord injury model by cutting off T10 spinal cord. Then the primary culture OECs suspension （1×10^11/L） was injected into the region about 1 mm from the margin of injury （about 1.0 mm deep） in the OECs group （1 μ L every place） and DF12 culture fluid was injected into DF12 control group in a same dose and same method respectively. The normal group and model group were left intact. MAIN OUTCOME MEASURES： At 1, 4 and 8 weeks after transplantation, immunohistochemistry was used to evaluate NogoA protein changes in the injured spinal cord region. At 8 weeks, argyrophil staining was employed to measure histomorphological changes. RESULTS： Absorbance of NogoA protein in the normal group was significantly lower than model group, OECs group and DF12 control group （P 〈 0.05）. At 1, 4 and 8 weeks after transplantation, NogoA protein levels were significantly lower in the OECs group than in the model group and DF12 control group （P 〈 0.01）. No significant difference was detected between the model group and DF12 control group （P 〉 0.01）. At 8 weeks after transplantation, except the normal group, obvious regeneration of nerve fibers was presented in the model group, OECs group and DF12 control group, but the regenerated fiber in model and DF12 control group aligned in disorder. Significant new axons were shown and nerve fibers across injured region repaired injured spinal cord in the OECs group, which was better in quantity and quality compared with the model group and the DF12 control group. CONCLUSION： Olfactory ensheathing cell transplantation may enhance the repair of injured spinal cord by depressing the levels of NogoA protein.