神经干细胞移植治疗脊髓损伤大鼠的实验研究
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摘要
目的:探讨胎鼠脑源性神经干细胞(NSCs)移植对脊髓损伤大鼠的神经功能的修复作用。
方法:取孕13～15天SD大鼠胚胎的脑组织分离NSCs,在体外培养并鉴定。制作脊髓损伤(SCI)大鼠模型,伤后7天将BrdU标记好的NSCs局部移植在损伤脊髓处。SCI大鼠分为三组:A组,损伤组,SCI后自然恢复;B组,N.S移植组,SCI后7天局部移植10μL生理盐水;C组,NSCs移植组,SCI后7天局部移植10μLNSCs悬液(约1.0×10~6)。A.B.C三组损伤前及伤后6天、14天、21天、30天、60天时分别行后肢BBB行为学评分和躯体感觉诱发电位(SEP)、运动诱发电位(MEP)检测,伤后60天处死实验动物行病理学检查及免疫组织化学染色,观察移植NSCs在大鼠体内的存活、迁移及分化情况。
结果:
1.胚胎大鼠脑源性的NSCs可以在体外分离、培养及传代,体外培养的细胞nestin免疫荧光染色阳性,证实是NSCs。
2.后肢BBB运动功能评分:SCI后6天,A.B.C三组各组行为学评分无显著差异(P＞0.05);伤后14天,A组行为学评分高于B.C两组(P＜0.05),B组和C组比较无显著差异(P＞0.05);伤后21天,A组和C组行为学评分高于B组(P＜0.05),A组和C组比较无显著差异(P＞0.05);伤后30天,C组行为学评分最高,其次A组,B组最差,有统计学意义(P＜0.05);伤后60天,结果与伤后30天一致。表明NSCs移植组的行为学评分比其它两组有显著改善。
3.SEP检测:损伤前A.B.C三组SEP波潜伏期正常,各组比较无显著差异(P＞0.05);损伤后6天,各组SEP波均消失;伤后14天、21天、30天,各组SEP波逐渐恢复,但潜伏期延长;伤后60天,各组SEP波均出现,A组和B组的潜伏期比C组延长(P＜0.05),A组和B组比较无显著差异(P＞0.05),各组分别与损伤前相比,潜伏期均延长(P＜0.05)。表明NSCs移植组的神经传导功能比其它两组恢复的好。
4.MEP检测:损伤前A.B.C三组MEP潜伏期正常,各组比较无显著差异(P＞0.05);损伤后6天,各组MEP波均消失;伤后14天、21天、30天,各组MEP波逐渐恢复,但潜伏期延长;伤后60天,各组MEP波均出现,B组潜伏期最长,其次A组,C组最短(P＜0.05);各组分别与损伤前相比,潜伏期均延长(P＜0.05)。表明NSCs移植组的神经传导功能比其它两组恢复的好。
5.病理学和免疫组化双染发现,C组脊髓内可见BrdU染色阳性的细胞,表明NSCs可在宿主体内存活;在距离脊髓损伤中心1.5cm处可见BrdU染色阳性的细胞,表明NSCs在宿主脊髓内有迁移功能;C组脊髓损伤处发现BrdU+NSE、BrdU+GFAP免疫组化双染阳性细胞,表明NSCs在宿主体内可以分化为神经元和星形胶质细胞。
结论:胚胎大鼠脑源性的NSCs可以在体外分离、培养及鉴定。NSCs移植入SCI大鼠体内可以存活、迁移,并分化为神经元和神经胶质细胞。NSCs移植可以促进SCI大鼠的神经功能的恢复,在行为学评分和神经电生理检查方面得到明显改善。
Objective:To investigate the repairable effects of transplanting the neural stem cells (NSCs)derived from the fetal brain of rats on the neural function in rats with spinal cord injury(SCI).
Methods:The NSCs were isolated from fetal brain of rats(13～15 days during pregnancy),cultured and identified in vitro.The NSCs labeled by BrdU were locally transplanted into the injured spinal cord 7 days after the model rats with spinal cord injury were established.The rats with SCI were randomly divided into three groups. A group,injury group,the injury was recovered naturally after injury,B group,N.S group,10μL normal saline(N.S)was locally transplanted at 7 days after injury,C group,NSCs group,10μL NSCs suspension(approximately 1.0×10~6)was locally transplanted 7 days after injury.BBB function score,somatosensory evoked potential (SEP)and motet evoked potential(MEP)tests were performed before injury,6 days, 14 days,21 days,30 days and 60 days after injury,respectively.The model rats were killed 60 days after injury.The pathologic examination and immunohistochemical double stain were used to observe the survival,migration and differentiation of the transplanted NSCs in vivo.
Results:
1.The cultured cells in vitro showed positive nestin by immunofluorescence and were confirmed to be NSCs.
2.BBB function score:There were not significant differences among three groups 6 days after injury(P＞0.05).Behavioral score of A group was significantly higher than those of B and C group(P＜0.05)and there were not significant differences between B and C group(P＞0.05)14 days after injury.Behavioral score in both A and C group was higher than that in B group(P＜0.05)and there were not significant differences between A and C group(P＞0.05)21 days after injury.The behavioral score in C group was the highest,in A group second and in B group was the lowest(P＜0.05)30 days after injury,as well as 60 days after injury.It was showen that the behavioral score of model rats was significantly improved in other groups.
3.SEP examination:There were not significant differences among the normal SEP latent periods of three groups before injury(P＞0.05).All the SEP of three group disappeared 6 days after injury.The SEP of three groups gradually appeared,once more but the SEP latent periods were prolonged 14 days,21 days and 30 days after injury.All the SEP of three groups appeared 60 days after injury and the SEP latent periods both A and B group were prolonger than C group(P＜0.05). There were no significant differences between A and B group(P＞0.05).The SEP latent period of each group was prolonged compaired with each group before injury respectively(P＜0.05).
4.MEP examination:Before injury the normal MEP latent periods of three groups did not have significant difference(P＞0.05).All the MEP of three group disappeared 6 days after injury.The MEP Of three groups gradually appeared,but the latent periods of the MEP were prolonged 14 days,21 days and 30 days after injury.All the MEP of three groups appeared 60 days after injury and the latent period in B group was the longest,in A group second,in C group the shortest, respectively(P＜0.05).The MEP latent period of A.B and C group was prolonged in compairson with each group before injury,respectively(P＜0.05).It was shown that the neural conductive functions of NSCs group was better than the other group.
5.Pathology and immunohistochemical double stain:BrdU-positive cells could be detected in the spinal cord of rats in C group.It was shown that NSCs could survive in vivo.BrdU-positive cells could be noted in 1.5cm distance from the injured centre of spinal cord.It was exhibated that NSCs could migrate in vivo. BrdU+NSE and BrdU+GFAP double-positive cells could be observed in the injured spinal cord of rats in C group.It was suggested that NSCs could differentiate into neurons and astrocytes in vivo.
Conclusion:The NSCs derived from the brain of the rats can be isolated,cultured and reproduced in vitro.The NSCs transplanted into the injured spinal cord injury of rats can survive,migrate and differentiate into neurons and astrocytes.The treatment with transplanting NSCs for rats with SCI can markly promote the recovery of neural function and improve the recovery of behavioral score and neural electrophysiology.
方法:取孕13～15天SD大鼠胚胎的脑组织分离NSCs,在体外培养并鉴定。制作脊髓损伤(SCI)大鼠模型,伤后7天将BrdU标记好的NSCs局部移植在损伤脊髓处。SCI大鼠分为三组:A组,损伤组,SCI后自然恢复;B组,N.S移植组,SCI后7天局部移植10μL生理盐水;C组,NSCs移植组,SCI后7天局部移植10μLNSCs悬液(约1.0×10~6)。A.B.C三组损伤前及伤后6天、14天、21天、30天、60天时分别行后肢BBB行为学评分和躯体感觉诱发电位(SEP)、运动诱发电位(MEP)检测,伤后60天处死实验动物行病理学检查及免疫组织化学染色,观察移植NSCs在大鼠体内的存活、迁移及分化情况。
结果:
1.胚胎大鼠脑源性的NSCs可以在体外分离、培养及传代,体外培养的细胞nestin免疫荧光染色阳性,证实是NSCs。
2.后肢BBB运动功能评分:SCI后6天,A.B.C三组各组行为学评分无显著差异(P＞0.05);伤后14天,A组行为学评分高于B.C两组(P＜0.05),B组和C组比较无显著差异(P＞0.05);伤后21天,A组和C组行为学评分高于B组(P＜0.05),A组和C组比较无显著差异(P＞0.05);伤后30天,C组行为学评分最高,其次A组,B组最差,有统计学意义(P＜0.05);伤后60天,结果与伤后30天一致。表明NSCs移植组的行为学评分比其它两组有显著改善。
3.SEP检测:损伤前A.B.C三组SEP波潜伏期正常,各组比较无显著差异(P＞0.05);损伤后6天,各组SEP波均消失;伤后14天、21天、30天,各组SEP波逐渐恢复,但潜伏期延长;伤后60天,各组SEP波均出现,A组和B组的潜伏期比C组延长(P＜0.05),A组和B组比较无显著差异(P＞0.05),各组分别与损伤前相比,潜伏期均延长(P＜0.05)。表明NSCs移植组的神经传导功能比其它两组恢复的好。
4.MEP检测:损伤前A.B.C三组MEP潜伏期正常,各组比较无显著差异(P＞0.05);损伤后6天,各组MEP波均消失;伤后14天、21天、30天,各组MEP波逐渐恢复,但潜伏期延长;伤后60天,各组MEP波均出现,B组潜伏期最长,其次A组,C组最短(P＜0.05);各组分别与损伤前相比,潜伏期均延长(P＜0.05)。表明NSCs移植组的神经传导功能比其它两组恢复的好。
5.病理学和免疫组化双染发现,C组脊髓内可见BrdU染色阳性的细胞,表明NSCs可在宿主体内存活;在距离脊髓损伤中心1.5cm处可见BrdU染色阳性的细胞,表明NSCs在宿主脊髓内有迁移功能;C组脊髓损伤处发现BrdU+NSE、BrdU+GFAP免疫组化双染阳性细胞,表明NSCs在宿主体内可以分化为神经元和星形胶质细胞。
结论:胚胎大鼠脑源性的NSCs可以在体外分离、培养及鉴定。NSCs移植入SCI大鼠体内可以存活、迁移,并分化为神经元和神经胶质细胞。NSCs移植可以促进SCI大鼠的神经功能的恢复,在行为学评分和神经电生理检查方面得到明显改善。
Objective:To investigate the repairable effects of transplanting the neural stem cells (NSCs)derived from the fetal brain of rats on the neural function in rats with spinal cord injury(SCI).
Methods:The NSCs were isolated from fetal brain of rats(13～15 days during pregnancy),cultured and identified in vitro.The NSCs labeled by BrdU were locally transplanted into the injured spinal cord 7 days after the model rats with spinal cord injury were established.The rats with SCI were randomly divided into three groups. A group,injury group,the injury was recovered naturally after injury,B group,N.S group,10μL normal saline(N.S)was locally transplanted at 7 days after injury,C group,NSCs group,10μL NSCs suspension(approximately 1.0×10~6)was locally transplanted 7 days after injury.BBB function score,somatosensory evoked potential (SEP)and motet evoked potential(MEP)tests were performed before injury,6 days, 14 days,21 days,30 days and 60 days after injury,respectively.The model rats were killed 60 days after injury.The pathologic examination and immunohistochemical double stain were used to observe the survival,migration and differentiation of the transplanted NSCs in vivo.
Results:
1.The cultured cells in vitro showed positive nestin by immunofluorescence and were confirmed to be NSCs.
2.BBB function score:There were not significant differences among three groups 6 days after injury(P＞0.05).Behavioral score of A group was significantly higher than those of B and C group(P＜0.05)and there were not significant differences between B and C group(P＞0.05)14 days after injury.Behavioral score in both A and C group was higher than that in B group(P＜0.05)and there were not significant differences between A and C group(P＞0.05)21 days after injury.The behavioral score in C group was the highest,in A group second and in B group was the lowest(P＜0.05)30 days after injury,as well as 60 days after injury.It was showen that the behavioral score of model rats was significantly improved in other groups.
3.SEP examination:There were not significant differences among the normal SEP latent periods of three groups before injury(P＞0.05).All the SEP of three group disappeared 6 days after injury.The SEP of three groups gradually appeared,once more but the SEP latent periods were prolonged 14 days,21 days and 30 days after injury.All the SEP of three groups appeared 60 days after injury and the SEP latent periods both A and B group were prolonger than C group(P＜0.05). There were no significant differences between A and B group(P＞0.05).The SEP latent period of each group was prolonged compaired with each group before injury respectively(P＜0.05).
4.MEP examination:Before injury the normal MEP latent periods of three groups did not have significant difference(P＞0.05).All the MEP of three group disappeared 6 days after injury.The MEP Of three groups gradually appeared,but the latent periods of the MEP were prolonged 14 days,21 days and 30 days after injury.All the MEP of three groups appeared 60 days after injury and the latent period in B group was the longest,in A group second,in C group the shortest, respectively(P＜0.05).The MEP latent period of A.B and C group was prolonged in compairson with each group before injury,respectively(P＜0.05).It was shown that the neural conductive functions of NSCs group was better than the other group.
5.Pathology and immunohistochemical double stain:BrdU-positive cells could be detected in the spinal cord of rats in C group.It was shown that NSCs could survive in vivo.BrdU-positive cells could be noted in 1.5cm distance from the injured centre of spinal cord.It was exhibated that NSCs could migrate in vivo. BrdU+NSE and BrdU+GFAP double-positive cells could be observed in the injured spinal cord of rats in C group.It was suggested that NSCs could differentiate into neurons and astrocytes in vivo.
Conclusion:The NSCs derived from the brain of the rats can be isolated,cultured and reproduced in vitro.The NSCs transplanted into the injured spinal cord injury of rats can survive,migrate and differentiate into neurons and astrocytes.The treatment with transplanting NSCs for rats with SCI can markly promote the recovery of neural function and improve the recovery of behavioral score and neural electrophysiology.
引文
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