电针对大鼠脊髓损伤后内源性神经干细胞分化与BMP-2表达的实验研究
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摘要
[目的]：1.建立可靠的急性脑动脉瘤夹钳夹型大鼠脊髓损伤模型；2.探讨电针对大鼠脊髓损伤后内源性神经干细胞的分化影响；3.探讨电针治疗脊髓损伤后局部脊髓BMP-2的表达变化；4.为电针治疗脊髓损伤提供实验依据。
[方法]：将SD大鼠随机分成假手术组、脊髓损伤组、电针组,动物模型采用医用脑动脉瘤临时夹钳夹的急性钳夹型大鼠脊髓损伤模型,分别于损伤后1d、3d、7d、14d、21d、28d进行取材。1.通过BBB评分、光镜、电镜病理学检查,SEP电生理检测、MRI影像学客观完整监测,对急性钳夹型大鼠脊髓损伤模型的稳定性进行评估；2.电针后采用BBB行为学评分进行损伤后的功能评定；3.取各组受损脊髓的组织行冰冻切片、染色后行病理分析、测定损伤后局部脊髓组织的内源性神经干细胞、少突胶质细胞及其前体细胞、星形胶质细胞的表达；4.取各时间段各组损伤局部脊髓组织,提取蛋白经电泳成像与数据分析,测定损伤后局部脊髓组织内的BMP-2的表达变化。
[结果]：
1.经两组数据纵向比较,BBB评分结果提示,假手术组明显高于损伤组；每组数据随着术后时间的增长逐渐增高；同一组内不同时间点差异有统计学意义(P<0.05)。同一时间点不同组间比较差异有统计学意义(P<0.05)。
2.假手术组SEP的波形正常,术后潜伏期略延长,波幅略降低,随后恢复正常。脊髓损伤组SEP测定：术后短期(7d内)未引出波形,7d以后SEP的潜伏期长,波幅低；随着时间的增长,潜伏期缓慢下降,波幅逐渐增高。假手术组各时间段的潜伏期、波幅对比差异无统计学意义(P>0.05)；脊髓损伤组各时间段的潜伏期、波幅对比差异有统计学意义(P<0.05)。
3.MRI结果：术后6h胸段脊髓创伤部位呈局限性点状T2WI高信号影；术后1d-3d,胸段脊髓肿胀,损伤处及尾端脊髓呈大片状T2高信号改变；术后7d-14d,胸段脊髓内片状T2WI高信号区范围缩小；术后21d-28d,胸段脊髓内仅有小片T2WI高信号影,脊髓远端直径较正常明显缩小。假手术组在各时间点脊髓MRI均无明显改变。
4.HE染色病理结果：损伤后6h-1d脊髓灰白质出血水肿、结构紊乱、灰质板层消失,3d-7d神经元结构逐渐破坏,神经细胞肿胀,核固缩；7d-14d炎性细胞大量浸润,神经细胞水肿减轻；21d-28d可见神经细胞水肿逐渐减轻,炎性细胞开始消退,胶质细胞增生；假手术组组织结构清晰。
5.电针治疗可提高BBB行为学评分,随电针治疗时间延长BBB评分逐渐升高。
6.同一时间点不同组之间,内源性神经干细胞数目比较,电针组高于其他组；组之间的差异有统计学意义(P<0.05)。电针组之间不同时间点对比,随着电针时间的增长,细胞数不断增长；不同时间点之间的差异有统计学意义(P<0.05)。
7.同一时间点不同组之间,少突胶质细胞数目比较,电针组明显高于其他组；组之间的对比有显著差异。电针组之间不同时间点对比,随着电针时间的增长,细胞数不断增长；不同时间点之间的差异有统计学意义(P<0.05)。
8.同一时间点不同组之间,OPCs数目比较,电针组明显高于其他组；组之间的差异有统计学意义(P<0.05)。电针组之间不同时间点对比,随着电针时间的增长,细胞数不断增多；不同时间点之间的差异有统计学意义(P<0.05)。电针组Brdu阳性细胞与NG2阳性细胞免疫荧光共表达细胞增多(P<0.05)。
9.同一时间点不同组之间,星形胶质细胞数目比较,电针组明显低于其他组；组之间的差异有统计学意义(P<0.05)。电针组之间不同时间点对比,随着电针时间的增长,细胞数不断减少；不同时间点之间的差异有统计学意义(P<0.05)。电针组Brdu阳性细胞与GFAP阳性细胞共免疫荧光共表达细胞减少(P<0.05)。
10.BMP-2的表达在大鼠脊髓损伤后7d-14d出现明显增强,高表达持续至28d以后,电针组BMP-2的表达低于脊髓损伤组,差异有统计学意义(P<0.05),这种差异随时间推移更加明显,差异有统计学意义(P<0.05)。
[结论]：1.急性钳夹型大鼠脊髓损伤模型制作简单,具有稳定性、可重复性和可控性；2.电针治疗可提高大鼠脊髓损伤后BBB行为学评分,从行为学角度上提示电针治疗对大鼠脊髓损伤有明确治疗效果。电针刺激对大鼠脊髓损伤后损伤远端脊髓内源性神经干细胞增殖,分化为少突胶质前体细胞、少突胶质细胞有促进作用,而对分化为胶质细胞有抑制作用；3.电针治疗脊髓损伤,促进脊髓功能恢复与抑制BMP-2有关；4.电针刺激治疗脊髓损伤有实验依据。
Objective1. To establish a reliable model of the acute clip-type spinal cord injury in rats;2. To explain the effect of electro-acupuncture on the spinal cord injury and the differentiation of the endogenous neural stem cells;3. To research the expression of BMP-2in SCI after the electro-acupuncture treatment.4. To provide experimental basis for electro-acupuncture treatment of spinal cord injury
Methods:SD rats were randomly divided into the SCI group, the SHAM group and the EA group. Aneurysm-clipping(temporary) methods were used to make up an acute spinal cord injury model and draw materials on the1st,3rd,7th,14th,21st and28th day respectively after the injury. The reliability of the acute clip-type SCI model was evaluated through BBB score, SEP, MRI, pathological section. The neural function was assessed after EA treatment with the use of the BBB scores. After the DAB staining to frozen sections, the proliferations of OPCs, APCs, eNSCs and ASs in the spinal cord anterior horn were viewed and the expression of the BMP-2in the injured spinal cord tissue in each group observed by WB.
Results:
1. The Comparison of the BBB score data of the two groups vertically suggested that the data of the SHAM group were significantly higher than those of the SCI group. Each set of the data increased gradually with the prolongation of the postoperative time. There was significant difference as to the different time points within the same group (P<0.05) and the statistical comparison of the points at the same time between different groups was significant (P<0.05).
2. SEP waveform of the SHAM group was normal, with the latency prolonged slightly after the operation, the wave amplitude being reduced slightly and then being back to normal. As to SEP of the SCI group, no waveform could be detected short term after the operation (<7days). The latency was longer and the amplitude was after7days. However, the latency would decrease slowly and the wave amplitude would increase gradually with time. In comparison, there was no statistical significance as to the latency and amplitude in each time point of the SHAM group (P>0.05) and there was also obviously statistical significance as to the latency and amplitude in each time point of the SCI group (P<0.05).
3. MRI results:The injured region on the thoracic spinal cord showed a regional spot-like high T2WI signal6hrs after the operation, and with the swelling of the thoracic spinal cord, large areas of the injured region and caudal spinal cord presented high T2signal1-3days postoperatively. T2WI high signal area was narrowed7-14days after the operation and the T2WI high signal remained little and the diameter in distal spinal cord was reduced conspicuously21-28days after the operation. MRI of the SHAM group at various time points had no distinct change.
4. Pathological findings of the HE staining:Such signs as the hemorrhage, the edema, the structural disorder and the disappearance of gray matter layer were found with the examination of the spinal cord6hrs and lday postoperatively and the neuron structure was damaged along with the nerve cell swelling and nuclear pyknosis3-7days postoperatively. However,7-14days after the operation, the edema in nerve cells began to be relieved along with the infiltration of inflammatory cells.21-28days postoperatively, the edema in nerve cells significantly was alleviated, inflammatory cells began to fade away. As a result, glial cells began to proliferate and the tissue structure of the SHAM group was clear.
5. The electric-acupuncture treatment could obviously improve the BBB ethology score and higher scores could be gained with time.
6. Compared to the other two groups at the same time, the number of eNSC in EA group was significantly higher and there was statistical significance between the different groups (P<0.05). The quantity of cells between the different time points in EA group increased gradually as the time of the treatment went on, indicating that there was statistical significance between the different time (P<0.05).
7. In comparison to the number of oligodendrocyte between the different groups at the same time, the number in EA group was significantly higher than that in the other two groups and there was statistical significance between the different groups (P<0.05). Also the amount of cells between the different time points in EA group increased gradually as the time of the treatment went on. The contrast between the different time points was significant statistically (P<0.05).
8. Compared with the other two groups at the same time, the number of OPCs in EA group was significantly higher than that of the other two groups, which indicated that there was statistical significance between the different groups (P<0.05). In comparison between the different time points in EA group, the number of cells increased gradually as the time fo the treatment went on. The contrast between the different time points was significant statistically (P<0.05). Brdu positive cells and NG2positive cells were increased express with immunofluorescence in EA group (P <0.05).
9. As contrasted with the other two groups at the same time, the number of astroglia in EA group was significantly lower and there was statistical significance between the different groups (P<0.05). Compared between different time points in EA group, the number of cells decreased gradually with the time going. The contrast between the different time points was significant statistically (P<0.05). Brdu positive cells and GFAP positive cells were decreased express with immunofluorescence in EA group (P <0.05).
10.7-14days after the injury, the expression of BMP-2appeared obviously enhanced and by the time this high-level expression continued up to28days, the expression of BMP-2in the EA group was lower than that in the SCI group. The difference was highly significant statistically with the passage of time (P<0.05).
Conclusion:
1. It is simple to make the model of the acute clip-type spinal cord injury in rats and it is stable, can be used repeatedly and can be controlled
2. Electric-acupuncture can significantly improve the BBB behavior score of rats in spinal cord injury and from the perspective of ethology it obviously has a therapeutic effect on the spinal cord injury. Electric-acupuncture could induce the proliferation and differentiation of eSCs,OLs and its precursor cells in the remote segment behind the injury point. Also, it could inhibit the proliferation and differentiation of astroglia in the remote segment behind the injury point.
3. Electric-acupuncture might promote the functional recovery after the spinal cord injury by inhibiting the expression of BMP-2.
4. Providing experimental basis for electro-acupuncture treatment of spinal cord injury
[方法]：将SD大鼠随机分成假手术组、脊髓损伤组、电针组,动物模型采用医用脑动脉瘤临时夹钳夹的急性钳夹型大鼠脊髓损伤模型,分别于损伤后1d、3d、7d、14d、21d、28d进行取材。1.通过BBB评分、光镜、电镜病理学检查,SEP电生理检测、MRI影像学客观完整监测,对急性钳夹型大鼠脊髓损伤模型的稳定性进行评估；2.电针后采用BBB行为学评分进行损伤后的功能评定；3.取各组受损脊髓的组织行冰冻切片、染色后行病理分析、测定损伤后局部脊髓组织的内源性神经干细胞、少突胶质细胞及其前体细胞、星形胶质细胞的表达；4.取各时间段各组损伤局部脊髓组织,提取蛋白经电泳成像与数据分析,测定损伤后局部脊髓组织内的BMP-2的表达变化。
[结果]：
1.经两组数据纵向比较,BBB评分结果提示,假手术组明显高于损伤组；每组数据随着术后时间的增长逐渐增高；同一组内不同时间点差异有统计学意义(P<0.05)。同一时间点不同组间比较差异有统计学意义(P<0.05)。
2.假手术组SEP的波形正常,术后潜伏期略延长,波幅略降低,随后恢复正常。脊髓损伤组SEP测定：术后短期(7d内)未引出波形,7d以后SEP的潜伏期长,波幅低；随着时间的增长,潜伏期缓慢下降,波幅逐渐增高。假手术组各时间段的潜伏期、波幅对比差异无统计学意义(P>0.05)；脊髓损伤组各时间段的潜伏期、波幅对比差异有统计学意义(P<0.05)。
3.MRI结果：术后6h胸段脊髓创伤部位呈局限性点状T2WI高信号影；术后1d-3d,胸段脊髓肿胀,损伤处及尾端脊髓呈大片状T2高信号改变；术后7d-14d,胸段脊髓内片状T2WI高信号区范围缩小；术后21d-28d,胸段脊髓内仅有小片T2WI高信号影,脊髓远端直径较正常明显缩小。假手术组在各时间点脊髓MRI均无明显改变。
4.HE染色病理结果：损伤后6h-1d脊髓灰白质出血水肿、结构紊乱、灰质板层消失,3d-7d神经元结构逐渐破坏,神经细胞肿胀,核固缩；7d-14d炎性细胞大量浸润,神经细胞水肿减轻；21d-28d可见神经细胞水肿逐渐减轻,炎性细胞开始消退,胶质细胞增生；假手术组组织结构清晰。
5.电针治疗可提高BBB行为学评分,随电针治疗时间延长BBB评分逐渐升高。
6.同一时间点不同组之间,内源性神经干细胞数目比较,电针组高于其他组；组之间的差异有统计学意义(P<0.05)。电针组之间不同时间点对比,随着电针时间的增长,细胞数不断增长；不同时间点之间的差异有统计学意义(P<0.05)。
7.同一时间点不同组之间,少突胶质细胞数目比较,电针组明显高于其他组；组之间的对比有显著差异。电针组之间不同时间点对比,随着电针时间的增长,细胞数不断增长；不同时间点之间的差异有统计学意义(P<0.05)。
8.同一时间点不同组之间,OPCs数目比较,电针组明显高于其他组；组之间的差异有统计学意义(P<0.05)。电针组之间不同时间点对比,随着电针时间的增长,细胞数不断增多；不同时间点之间的差异有统计学意义(P<0.05)。电针组Brdu阳性细胞与NG2阳性细胞免疫荧光共表达细胞增多(P<0.05)。
9.同一时间点不同组之间,星形胶质细胞数目比较,电针组明显低于其他组；组之间的差异有统计学意义(P<0.05)。电针组之间不同时间点对比,随着电针时间的增长,细胞数不断减少；不同时间点之间的差异有统计学意义(P<0.05)。电针组Brdu阳性细胞与GFAP阳性细胞共免疫荧光共表达细胞减少(P<0.05)。
10.BMP-2的表达在大鼠脊髓损伤后7d-14d出现明显增强,高表达持续至28d以后,电针组BMP-2的表达低于脊髓损伤组,差异有统计学意义(P<0.05),这种差异随时间推移更加明显,差异有统计学意义(P<0.05)。
[结论]：1.急性钳夹型大鼠脊髓损伤模型制作简单,具有稳定性、可重复性和可控性；2.电针治疗可提高大鼠脊髓损伤后BBB行为学评分,从行为学角度上提示电针治疗对大鼠脊髓损伤有明确治疗效果。电针刺激对大鼠脊髓损伤后损伤远端脊髓内源性神经干细胞增殖,分化为少突胶质前体细胞、少突胶质细胞有促进作用,而对分化为胶质细胞有抑制作用；3.电针治疗脊髓损伤,促进脊髓功能恢复与抑制BMP-2有关；4.电针刺激治疗脊髓损伤有实验依据。
Objective1. To establish a reliable model of the acute clip-type spinal cord injury in rats;2. To explain the effect of electro-acupuncture on the spinal cord injury and the differentiation of the endogenous neural stem cells;3. To research the expression of BMP-2in SCI after the electro-acupuncture treatment.4. To provide experimental basis for electro-acupuncture treatment of spinal cord injury
Methods:SD rats were randomly divided into the SCI group, the SHAM group and the EA group. Aneurysm-clipping(temporary) methods were used to make up an acute spinal cord injury model and draw materials on the1st,3rd,7th,14th,21st and28th day respectively after the injury. The reliability of the acute clip-type SCI model was evaluated through BBB score, SEP, MRI, pathological section. The neural function was assessed after EA treatment with the use of the BBB scores. After the DAB staining to frozen sections, the proliferations of OPCs, APCs, eNSCs and ASs in the spinal cord anterior horn were viewed and the expression of the BMP-2in the injured spinal cord tissue in each group observed by WB.
Results:
1. The Comparison of the BBB score data of the two groups vertically suggested that the data of the SHAM group were significantly higher than those of the SCI group. Each set of the data increased gradually with the prolongation of the postoperative time. There was significant difference as to the different time points within the same group (P<0.05) and the statistical comparison of the points at the same time between different groups was significant (P<0.05).
2. SEP waveform of the SHAM group was normal, with the latency prolonged slightly after the operation, the wave amplitude being reduced slightly and then being back to normal. As to SEP of the SCI group, no waveform could be detected short term after the operation (<7days). The latency was longer and the amplitude was after7days. However, the latency would decrease slowly and the wave amplitude would increase gradually with time. In comparison, there was no statistical significance as to the latency and amplitude in each time point of the SHAM group (P>0.05) and there was also obviously statistical significance as to the latency and amplitude in each time point of the SCI group (P<0.05).
3. MRI results:The injured region on the thoracic spinal cord showed a regional spot-like high T2WI signal6hrs after the operation, and with the swelling of the thoracic spinal cord, large areas of the injured region and caudal spinal cord presented high T2signal1-3days postoperatively. T2WI high signal area was narrowed7-14days after the operation and the T2WI high signal remained little and the diameter in distal spinal cord was reduced conspicuously21-28days after the operation. MRI of the SHAM group at various time points had no distinct change.
4. Pathological findings of the HE staining:Such signs as the hemorrhage, the edema, the structural disorder and the disappearance of gray matter layer were found with the examination of the spinal cord6hrs and lday postoperatively and the neuron structure was damaged along with the nerve cell swelling and nuclear pyknosis3-7days postoperatively. However,7-14days after the operation, the edema in nerve cells began to be relieved along with the infiltration of inflammatory cells.21-28days postoperatively, the edema in nerve cells significantly was alleviated, inflammatory cells began to fade away. As a result, glial cells began to proliferate and the tissue structure of the SHAM group was clear.
5. The electric-acupuncture treatment could obviously improve the BBB ethology score and higher scores could be gained with time.
6. Compared to the other two groups at the same time, the number of eNSC in EA group was significantly higher and there was statistical significance between the different groups (P<0.05). The quantity of cells between the different time points in EA group increased gradually as the time of the treatment went on, indicating that there was statistical significance between the different time (P<0.05).
7. In comparison to the number of oligodendrocyte between the different groups at the same time, the number in EA group was significantly higher than that in the other two groups and there was statistical significance between the different groups (P<0.05). Also the amount of cells between the different time points in EA group increased gradually as the time of the treatment went on. The contrast between the different time points was significant statistically (P<0.05).
8. Compared with the other two groups at the same time, the number of OPCs in EA group was significantly higher than that of the other two groups, which indicated that there was statistical significance between the different groups (P<0.05). In comparison between the different time points in EA group, the number of cells increased gradually as the time fo the treatment went on. The contrast between the different time points was significant statistically (P<0.05). Brdu positive cells and NG2positive cells were increased express with immunofluorescence in EA group (P <0.05).
9. As contrasted with the other two groups at the same time, the number of astroglia in EA group was significantly lower and there was statistical significance between the different groups (P<0.05). Compared between different time points in EA group, the number of cells decreased gradually with the time going. The contrast between the different time points was significant statistically (P<0.05). Brdu positive cells and GFAP positive cells were decreased express with immunofluorescence in EA group (P <0.05).
10.7-14days after the injury, the expression of BMP-2appeared obviously enhanced and by the time this high-level expression continued up to28days, the expression of BMP-2in the EA group was lower than that in the SCI group. The difference was highly significant statistically with the passage of time (P<0.05).
Conclusion:
1. It is simple to make the model of the acute clip-type spinal cord injury in rats and it is stable, can be used repeatedly and can be controlled
2. Electric-acupuncture can significantly improve the BBB behavior score of rats in spinal cord injury and from the perspective of ethology it obviously has a therapeutic effect on the spinal cord injury. Electric-acupuncture could induce the proliferation and differentiation of eSCs,OLs and its precursor cells in the remote segment behind the injury point. Also, it could inhibit the proliferation and differentiation of astroglia in the remote segment behind the injury point.
3. Electric-acupuncture might promote the functional recovery after the spinal cord injury by inhibiting the expression of BMP-2.
4. Providing experimental basis for electro-acupuncture treatment of spinal cord injury
引文
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