同种异体骨髓间充质干细胞移植治疗大鼠脊髓损伤的时效性分析

Analysis of chronergy for treatment of spinal cord injury with the allogeneic bone mesenchymal stem cells (BMSCs) transplantation in rats

目的:观察脊髓损伤后不同时间点骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)移植治疗后大鼠行为学变化、脊髓的病理改变及脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)和神经生长因子(nerve growth factor,NGF)表达变化,探讨BMSCs的最佳移植时间。方法:80只健康成年SD大鼠随机分为8组,每组10只。A组为假损伤组,暴露胸10段脊髓但不造成冲击伤,B、C、D、E、F、G、H组以改良Allen法建立脊髓损伤模型。造模成功后,C、D、E、F、G、H组分别于损伤后0 h、6 h、24 h、3 d、5 d和7 d,将1×106体外培养的BMSCs用微量注射器注入于脊髓损伤局部,B组为单纯造模组,用等量细胞培养液代替。各组分别于损伤后1、2、4周进行脊髓运动功能BBB(Basso,Beattie,Bresnahan)运动学评分;分别取各组脊髓损伤组织0.5 cm,制作HE染色病理切片,观察其形态学改变;Elisa法检测各组大鼠脊髓BDNF和NGF表达情况。结果:假手术组1、2、4周大鼠脊髓功能BBB评分均明显高于其余7组(P<0.01);术后1周细胞移植各组评分与单纯造模组相比差异无统计学意义(P>0.05);术后2周和4周细胞移植各组评分高于单纯造模组(P<0.05);损伤后2周BBB评分从高到低依次为F、E、G、D、H、C组,但6组组间比较差异无统计学意义(P>0.05);损伤后4周BBB评分,F组与其余5组(C,D,E,G,H组)比较差异有统计学意义(P<0.05),但其余5组组间差异无统计学意义(P>0.05)。ELISA检测结果显示,F组BDNF和NGF含量均高于其他7组(P<0.05)。大鼠脊髓标本切片HE染色,假手术组脊髓组织结构完整清楚,无中性粒细胞浸润;其余7组局部组织水肿明显,灰白质交界处模糊,周围可见不同程度胶质细胞增生及炎细胞浸润。结论:大鼠脊髓损伤后同种异体BMSCs移植对脊髓功能恢复有一定疗效,损伤后3 d可能是BMSCs最佳移植时间。

Objective： To observe the change of behavior, pathological change of the spinal cord, and expression of brain- derived neurotrophic factor （BDNF） and brain-derived neurotrophic factor （NGF） on rats with spinal cord injury in order to explore the optimal time of BMSCs transplantation. Methods：Eighty health SD rats were randomly divided into 8 groups （group A, B, C, D, E, F, G, H ）, 10 rats in each group. According to the modified Allen method, the rat model of spinal cord in- jury was built. Group A as non-injured group only exposed the spinal cord but not resuh in blast injury. BMSCs of vitro cuhure were respectively infunded the region of spinal cord injury in group C, D, E, F, G, H （as transplantation groups） at the 0 h, 6 h, 24 h, 3 d, 5 d, 7 d after model made. Group B as single model group was infunded the equal cell culture fluid. BBB score was used to evaluate the function of spinal cord at the lst,2nd and 4th weeks after injury. The morphological changes of the tissue of spinal cord injury were observed by HE stain and the expression of BDNF and NGF were detected by Elisa method at the 4th weeks after BMSCs transplantation. Results ： In non-injured group, BBB score was highest than that of other 7 groups at the 1st, 2nd and 4th weeks after injury （P〈0.01）. There was no significant difference in BBB score between single model group and transplantation groups at the 1st week after BMSCs transplantation （P〉0.05）. BBB score in transplantation groups were higher than that of single model group at the 2nd and 4th weeks after BMSCs transplantation （P〈0.05）. At the 2nd week after injury, BBB score from high to low was group F, E, G, D, H, C, but there was no significant difference among the groups （P〉0.05）. At the 4th week after injury,there was significant differences in BBB score between group F and other transplantation groups（group C,D,E,G,H） （P〈0.05）,but there was no significant difference among 5 groups （P〉0.05）. The levels of BDNF and NGF in group F was highest at the 4th week after BMSCs transplantation （P〈0.05）. In non-injured group, HE staining showed the struction of spinal cord was clear and complete, no neutrophil was found. But in other 7 groups, HE staining showed obvi- ously oedema at local tissue,juncture of gray and white matter was undefined, and different gliocyte proliferation and inflam- matory cell infiltrate cound be found. Conelusion：Allogeneic BMSCs transplantation is effective to stimulate the recovery of spinal cord function in rats with spinal cord injury, and the optimal time of BMSCs transplantation maybe at the 3 d after injury.