嗅鞘细胞联合神经干细胞对大鼠急性脊髓损伤及神经营养素-3表达的影响

Impact of Olfactory Ensheathing Cells Combined Neural Stem Cells in Rats with Acute Spinal Cord Injury and Neurotrophin-3 Expression

目的:观察嗅鞘细胞(OECs)联合神经干细胞(NSCs)对大鼠急性脊髓损伤及神经营养素-3(NT-3)表达的影响。方法:SD大鼠80只,Allen’s法行急性脊髓损伤造模后,将大鼠按随机数字表法分为对照组(假手术组)、实验1组(NSCs移植组)、实验2组(OECs移植组)和实验3组(NSCs+OECs联合移植组)。局部注射法进行细胞移植。于术前和术后1、3、5、7、14、21、28 d,采用BBB评分法、改良的Tralov评分法和斜板实验,对大鼠后肢运动功能状况进行评分,进行运动诱发电位(MEP N1波)潜伏期的检测。最后,各组随机抽取一只大鼠进行灌注、取材与固定,免疫组织化学染色的方法观察各组NT-3在局部受损脊髓组织中的表达情况。结果:(1)造模及细胞移植术后,24 h时内各组大鼠BBB评分及改良的Tralov评分均为0分;斜板实验角度下降非常明显,由实验前(82.00±3.45)°降为(12.73±1.34)°,差异有统计学意义(P<0.05);各组MEP(N1波)潜伏期明显延长。(2)随着实验的进行,各组均表现出不同程度的恢复情况:各实验组BBB评分及改良的Tralov评分与对照组比较增高显著(P<0.05);1周后各组各时间点比较差异均有统计学意义(P<0.05),且各实验组两两比较差异均有统计学意义(P<0.05)。(3)斜板实验角度也逐渐增加,1周后,实验组增幅大于对照组(P<0.05);1周后各组各时间点比较差异均有统计学意义(P<0.05),且各实验组两两比较差异有统计学意义(P<0.05)。(4)各组MEP(N1波)潜伏期均逐渐缩短,3 d后,各实验组潜伏期缩短幅度均大于对照组(P<0.05);3 d后各组各时间点比较差异均有统计学意义(P<0.05),且各实验组两两比较差异有统计学意义(P<0.05)。(5)各组大鼠受损脊髓组织中NT-3均有明显增高,7 d内维持在较高的水平,此后逐渐减少。其中从第5天开始,各实验组与对照组比较差异有统计学意义(P<0.05);各组各时间点比较差异均有统计学意义(P<0.05),且各实验组两两比较差异有统计学意义(P<0.05)。结论:(1)细胞移植治疗大鼠急性脊髓损伤时,单种细胞单独移植,OECs的治疗修复效果要强于NSCs的治疗修复效果,OECs与NSCs联合移植取得的治疗修复效果最好。(2)NT-3在OECs与NSCs细胞联合移植组中表达最高。

To observe the impact of olfactory ensheathing cells combined neural stem cells in rats with acute spinal cord injury and neurotrophin-3 Expression.Method：80 SD rats were selected,after acute spinal cord injury modeling by Allen＆#39;s method,the rats were randomly divided into the control group（sham group）,the experimental 1 group（NSCs transplantation group）,the experimental 2 group（OECs transplantation group）and the experimental 3 group（NSCs＋OECs transplantation group）. Cells were used local injection method to transplant. In the preoperative and postoperative 1,3,5,7,14,21,28 days,adopt BBB score,improved Tralov score and inclined plane experiments were used to rate the status of rat’s hindlimb motor function,tested motor evoked potential（MEP N1 wave）preclinical. Finally,one rat in each group were selected,randomly,then perfused,drawn and fixed. Immunohistochemical staining were used to observe neurotrophin-3（NT-3）in the damaged spinal cord tissue localized expression in each group. Result：（1）After modeling and cell transplanting,the rats’BBB rating and improved Tralov score was 0 points within 24 hours,inclined plane experiments’angle decreased very significantly,from the previous experiment（82.00±3.45）° reduced to（12.73±1.34）°（P〈0.05）.Each group’s MEP（N1 wave）latency was prolonged.（2）As the experiment progresses,the groups had shown varying degrees of recovery. Compared with the control group,the BBB score and improved Tralov score of the experimental group were increased significantly（P〈0.05）;after 1 week,the differences of each group in each time point were statistically significant（P〈0.05）,and the differences in each two of the experimental group were statistically significant（P〈0.05）.（3）Oblique plate experiment angle also gradually increased,after 1 week,the experimental group was increased than the control group（P〈0.05）;after 1 week,the differences of each group in each time point were statistically significant（P〈0.05）,and the differences in each two of the experimental group were statistically significant（P〈0.05）.（4）MEP（N1 wave）of each groups were gradually shortened in the incubation period;after 3 d,the incubation period shorten amplitude of the experimental group were greater than the control group（P〈0.05）;after 3 d,the differences of each group in each time point were statistically significant（P〈0.05）,and the differences in each two of the experimental group were statistically significant（P〈0.05）.（5）The NT-3 of damaged spinal cord tissue rats in each groups were significantly higher,7 d maintain at a high level,then gradually reduced. Since 5 days, the difference of the experimental group and the control group was statistically significant（P〈0.05）;the differences of each group in each time point were statistically significant（P〈0.05）,and the differences in each two of the experimental group were statistically significant（P〈0.05）.Conclusion：（1）When a single cell specie transplant to treat acute spinal cord injury alone,OECs transplantation has better restoration treatment effect than NSCs transplantation. OECs and NSCs transplantation group achieve the best results.（2）NT-3 expresses highest in the OECs and NSCs cell transplantation group.