嗅鞘细胞移植对脊髓损伤区RhoA表达的影响

Effect of olfactory ensheathing cells transplantation on RhoA expression in injured region after spinal cord injury

目的:轴突生长抑制分子须与其共同复合受体结合后才能激活下游抑制信号RhoA,从而导致细胞骨架塌陷,抑制神经再生。基础及临床研究均证实嗅鞘细胞能够促进脊髓功能的恢复,观察嗅鞘细胞移植前后脊髓损伤区RhoA的动态变化,探讨嗅鞘细胞移植治疗脊髓损伤的可能机制。方法:实验于2005-09/2006-05在西安交大医学院环境与基因重点实验室完成。①实验动物:成年SD大鼠40只,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大鼠作为嗅鞘细胞的来源。实验过程中对动物的处置符合动物伦理学标准。②实验方法:大鼠麻醉后处死,迅速取出头部两侧嗅球,除去软膜,剪取嗅球外面的嗅神经层和小球层,剪碎后离心,胰蛋白酶消化获取嗅鞘细胞悬液。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12 d的嗅鞘细胞悬液调整为1×10^(11)L^(-1),在距损伤缘上下各1 mm处分4点应用微量注射器注射,深度1.0 mm,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。③实验评估:各组分别于移植后1,2,4,6,8周采用免疫组化和Western blot免疫印迹技术动态检测脊髓损伤区RhoA表达的变化。同时在移植后8周行嗜银染色检测组织形态学变化。结果:①RhoA表达的变化:正常组RhoA表达的吸光度值明显低于其余3组(P<0.05)。嗅鞘细胞组于移植后1,2,4,6,8周脊髓损伤区RhoA的表达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RhoA蛋白的表达,从而促进损伤脊髓的修复。

AIM： Axonal growth inhibitor, combining with its receptor, can activate the signal RhoA and then result in cytoskeletal collapse and inhibit the neural regeneration. Basic and clinical researches have both proved that olfactory ensheathing cells （OECs） can improve the recovery of spinal function, we conducted a study to investigate the effect of OECs transplantation on level of RhoA in the injured region after spinal cord injury （SCI）, and explore the possible mechanism of OECs transplantation in the treatment of SCI. METHODS： The experiment was enforced from September 2005 to May 2006 in the Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, Xi＇an Jiaotong University. ①Forty adult SD rats were randomly divided into void group, model group, OECs group and DF12 control group, with 10 animals in each group. In addition, another 30 healthy male SD rats were served as the source for OECs. All the disposals in the experiment fit the ethical standard of animals. ②Rats were executed after anesthesia, which was followed by removing pia mater in the isolated bilateral olfactory bulb and cutting olfactory nerve layer as well as glomerular layer. OEC suspension was obtained after centrifugation and trypsin digestion. All the animals apart from void group were established SCI model by transection. Then the OECs were primarily cultured for 12 days, and the suspension （1×10^11 L^-1） was injected into four regions about 1 mm from the margin of injury （about 1.0 mm deep） of OECs group, 1 μL every point. DF12 culture fluid was injected into DF12 control group in the same dose and same method. The void group and model group were untreated. ③In the 1^st, 2^rd, 4^th, 6^th and 8^th weeks after injection, immunohistochemistry and Western blot assay were used to evaluate the level of RhoA in the injured region after OECs transplantation. And the silver staining was applied to detect the histomorphological changes in the 8^th week after injection. RESULTS： ①RhoA protein level in void group were obviously lower than that in other groups （P 〈 0.05）, and it in OECs group was distinctly lower than that in DF12 control group and model group after transplantation （P 〈 0.01 ）. There were no significant differences between model group and DF12 control group （P 〉 0.01）.②All the animals apart from the void＇s had an obvious regeneration of injured axons, but the regenerated fiber in model group and DF12 control group aligned in disorder and appeared bad directivity compared with that in OECs group after transplantation for 8 weeks; in OECs group, there were obvious regenerated axons and nerve fiber could repair SCI over the injured lesion. So the results were superior to that of model group and DF12 group. CONCLUSION： OECs transplantation may enhance the repair of injured spinal cord by depressing the level of RhoA in the injured region.