【作者】 李哲；

【导师】 黄晓琳；

【作者基本信息】 华中科技大学 ， 康复医学与理疗学， 2010， 博士

【摘要】 第一部分不同强度磁刺激对星形胶质细胞迁移的影响及机制研究实验一：不同强度磁刺激对星形胶质细胞迁移的影响[摘要]目的研究不同强度磁刺激对星形胶质细胞迁移作用的量效关系。方法24只Sprague-Dawley大鼠按刺激强度被随机分为对照组(刺激强度：0Tesla)、0.76T组(刺激强度0.76Tesla)、1.52T组(刺激强度：1.52Tesla)、1.9T组(刺激强度：1.9Tesla),4组的刺激频率为1Hz,刺激量为30脉冲,均采用溴乙锭(EB)注射入脊髓左侧背索复制局灶性的脊髓损伤模型。刺激14天后,处死大鼠,Western-Blot检测GFAP、MAP-2,采用图像分析系统观察GFAP、Brdu和MAP-2的免疫组化表达及脊髓损伤空洞体积的变化。结果随着磁刺激强度的增加,在14天时空洞的体积逐渐缩小,组间差异具有显著性差异(P＜0.05)。在空洞缩小的区域中,可以观察到GFAP的阳性表达,而无Brdu、MAP-2的阳性表达。随着磁刺激强度的增加,GFAP的阳性表达显著增强(P<0.05)。Western-Blot显示：随着磁刺激强度的增加,GFAP的光密度值相应增加,而MAP-2的光密度值没有明显变化,与组化的变化趋势一致。结论刺激频率为1Hz,刺激强度不大于1.9Tesla的条件下,磁刺激可促进星形胶质细胞的迁移,随着刺激强度的增强,星形胶质细胞迁移的能力亦增强。实验二：不同强度磁刺激对星形胶质细胞迁移作用的机制研究[摘要]目的研究不同剂量ERK抑制剂U0126对1Hz磁刺激促进星形胶质细胞迁移作用的影响,以探讨不同强度磁刺激促进星形胶质细胞迁移作用和ERK信号通路的关系,并选择U0126合适的阻滞剂量。方法24只Sprague-Dawley大鼠按U0126剂量被随机分为对照组(Omg/kg U0126, n=6)、低剂量组(0.1mg/kgU0126,n=6)、中剂量组(0.2mg/kg U0126, n= 6)、高剂量组(0.4mg/kg U0126,n=6),4组的刺激参数都为频率1Hz,强度1.52Tesla,刺激量为30脉冲；均采用溴乙锭(EB)注射入脊髓左侧背索复制局灶性的脊髓损伤模型。刺激14天后,处死大鼠,Western-Blot检测GFAP、ERK1/2和MAP-2,采用图像分析系统观察GFAP、ERK1/2和MAP-2的免疫组化表达及脊髓损伤空洞体积的变化。结果随着U0126剂量的增加,在14天时空洞的体积逐渐增大,GFAP和ERK1／2的阳性表达逐渐减弱,与对照组相比具有显著性差异(P<0.05)；并且,GFAP和ERK1／2的阳性表达呈相同的趋势；U0126中剂量组和高剂量组作用则无明显差异(P>0.05)；而病灶区域MAP-2呈阴性表达。Western-Blot显示：随着U0126剂量的增加,ERK1／2逐渐被抑制,0.2mg/kg和0.4mg/kgU0126都可完全抑制ERK1／2的表达,随着ERK逐渐被抑制,GFAP的表达亦逐渐减弱。结论不同剂量的U0126可抑制1Hz磁刺激引起的星形胶质细胞的迁移,0.2mg/kg是较合适的剂量；1.52Tesla,1Hz磁刺激引起的星形胶质细胞迁移与ERK信号通路有关。第二部分不同频率磁刺激对星形胶质细胞迁移的影响及机制研究实验一：不同频率磁刺激对星形胶质细胞迁移的影响[摘要]目的研究不同频率磁刺激对星形胶质细胞迁移作用的量效关系。方法30只Sprague-Dawley大鼠按刺激频率被随机分为对照组(刺激频率：0Hz,强度：1.52Tesla)、1Hz组(刺激频率：1Hz,强度：1.52Tesla)、5Hz组(刺激频率：5Hz,强度：1.52Tesla)、10Hz组(刺激频率：10Hz,强度：1.52Tesla)和20Hz组(刺激频率：20Hz,强度：1.52Tesla),5组的刺激量均为30个脉冲,采用溴乙锭(EB)注射入脊髓左侧背索复制局灶性的脊髓损伤模型。刺激14天后,处死大鼠,Western-Blot检测GFAP、MAP-2,采用图像分析系统观察GFAP、Brdu和MAP-2的表达及脊髓损伤区空洞体积的变化。结果随着磁刺激频率的增加,在第14天时空洞的体积逐渐缩小,组间差异具有显著性差异(P<0.05)。在空洞缩小的区域中,可以观察到GFAP的阳性表达,而无Brdu和MAP-2的阳性表达。随着磁刺激频率的增加,GFAP的阳性表达亦显著增强(P＜0.05)。Western-Blot显示：随着磁刺激频率的增加,GFAP的光密度值相应增加,而MAP-2的光密度值没有明显变化,与组化变化趋势一致。结论刺激强度为1.52 Tesla,刺激频率不大于20Hz的条件下,磁刺激可促进星形胶质细胞的迁移,并随着刺激频率的提高,星形胶质细胞迁移的能力亦增强。实验二：不同频率磁刺激对星形胶质细胞迁移作用的机制研究[摘要]目的研究不同频率磁刺激促进星形胶质细胞迁移过程中ERK1／2所起的作用,以探讨不同频率磁刺激促进星形胶质细胞迁移作用的机制,并选择U0126合适的阻滞剂量。方法24只Sprague-Dawley大鼠按U0126剂量被随机分为对照组(0mg/kg U0126, n= 6)、低剂量组(0.1mg/kg U0126, n=6)、中剂量组(0.2mg/kg U0126, n=6)、高剂量组(0.4mg/kg U0126, n=6)，4组的磁刺激参数均为频率10Hz、强度1.52 Tesla,刺激量30脉冲,均采用溴乙锭(EB)注射入脊髓左侧背索复制局灶性的脊髓损伤模型。刺激14天后,处死大鼠,Western-Blot检测3FAP、ERK1/2和MAP-2,采用图像分析系统观察GFAP、ERK1/2和MAP-2的表达及脊髓损伤空洞体积的变化。结果随着U0126剂量的增加,在第14天时空洞的体积逐渐增大,GFAP和ERK1／2的阳性表达逐渐减弱,与对照组相比具有显著性差异(P<0.05),U0126中剂量组和高剂量组的作用相似无明显差异(P>0.05)。而病灶区域MAP-2呈阴性表达。Western-Blot显示：随着U0126剂量的增加,ERK1／2逐渐被抑制,0.2mg/kg和0.4mg/kgU0126都可完全抑制ERK1／2的表达,随着ERK逐渐被抑制,GFAP的表达亦逐渐减弱。结论不同剂量的U0126可抑制10Hz磁刺激引起的星形胶质细胞的迁移,0.2mg/kg是较合适的剂量；1.52 Tesla,10Hz磁刺激引起的星形胶质细胞迁移与ERK信号通路有关；更多还原

【Abstract】 Part I:Effects of magnetic stimulation with different intensities on astrocytes migration and underlying mechanismExp I:Effects of magnetic stimulation with different intensities on astrocytes migration[Abstract] Objective To investigate the effects of magnetic stimulation with different intensities on astrocytes migration.Methods Twenty-four adult healthy Sprague-Dawley rats were selected and injected with 0.5μl of 1% ethidium bromide (EB) in PBS into the dorsal spinal cord funiculus of the left side at T10-11 level to make located spinal cord injury models and were randomly divided into four groups.The rats of four groups were exposed to pulsed magnetic stimulation(1Hz,30 pulses) at the following intensities respectively:0 Tesla(control group),0.76 Tesla (Group 0.76T),1.52 Tesla(Group 1.52T),1.9 Tesla(Group 1.9T). On the day 14 after stimulation, the rats were sacrificed and the expressions of glial fibrillary acidic protein(GFAP),5-Bromo-2’-deoxyuridine (Brdu), microtubule associated protein-2 (MAP-2) and the volume of the holes of injuried area of spinal cord were detected with immunohistochemistry and Western-Blot technique. Quantitative analysis of the expression of GFAP, Brdu and MAP-2 were performed with the image analysis system.Results With the increase of magnetic stimulation intensity, the volume of the holes of injuried area of spinal cord discrease at day 14. In the reduced area of the holes,the expressions of GFAP could be seen,while Brdu and MAP-2 could not be seen. Significant differences were revealed in the expressions of GFAP among the four groups, which were significantly higher in magnetic stimulation groups than that in control group (P< 0.05). Conclusion Under the condition of 1Hz, no larger than 1.9Tesla magnetic stimulation, magnetic stimulation promote astrocytes to migrate. After magnetic stimulation, astrocytes migrate into the injuried spinal cord holes. the ability of astrocytes migration increase with the increase of magnetic stimulation intensities.ExpⅡ:The mechanism of different intensities magnetic stimulation promoting astrocytes migration.[Abstract] Objective To investigate effects of ERK inhibitor U0126 of different dose on the ability of 1Hz magnetic stimulation promoting astrocytes migration, then to select suitable dose of U0126, and study the mechanism of different intensity magnetic stimulation promoting astrocytes migration. Methods 24 adult healthy Sprague-Dawley rats were selected to inject 0.5ml of 1% ethidium bromide (EB) in PBS into the dorsal spinal cord funiculus on the left side at the T10-11 level to make located spinal cord injury models and be randomly devided into four groups.The four groups were exposed to magnetic stimulation(1Hz,1.52Tesla, 30pulses) at the following dose respectively:Omg/kg U0126 (control group), 0.1mg/kg U0126(low-dose group),0.2mg/kg U0126(middle-dose group),0.4mg/kg U0126(high-dose group). On the day 14 after stimulation, the rats were killed and the expression of glial fibrillary acidic protein(GFAP),microtubule associated protein-2 (MAP-2), extracellular signal-regulated kinase1/2 (ERK1/2) and the volume of holes were detected with immunohistochemistry and Western-Blot technique. Quantitative analysis of the expression of GFAP, MAP-2 and ERK1/2 were performed with the image analysis system.Results With the increase of U0126 dose,the volume of holes increased on day 14. In the lesion area, the positive expression of GFAP and ERK1/2 could be seen and had identical change tendency,while MAP-2 could not be seen. Significant difference was revealed in the expression of GFAP and ERK1/2 among the four groups, it was significantly lower in the U0126 groups than that in the control group (P< 0.05). while the middle-dose group had similar effect with the high-dose group (P> 0.05).Conclusion U0126 of different dose all could inhibit astrocytes migration that 1Hz,1.52Tesla magnetic stimulation caused,and 0.2mg/kg was the suitable dose.What’s more, the effect of 1 Hz,1.52 Tesla magnetic stimulation promoting astrocytes to migrate is relevant to ERK pathway.Part II:Effects of magnetic stimulation with different frequencies on astrocytes migration and underlying mechanismExp I:Effects of magnetic stimulation with different frequencies on astrocytes migration[Abstract] Objective To investigate the action tendency of magnetic stimulation with different frequencies on astrocytes migration. Methods Thirty adult healthy Sprague-Dawley rats were selected and injected with 0.5μl of 1% ethidium bromide (EB) in PBS into the dorsal spinal cord funiculus of the left side at T10-11 level to make located spinal cord injury models and were randomly divided into five groups.The rats of five groups were exposed to pulsed magnetic stimulation (1.52Tesla,30 pulses) at the following frequencies respectively:0Hz(control group, 1Hz(Group 1HzT),5Hz(Group 5Hz),10Hz(Group 10Hz) and 20Hz(20Hz Group). On the day 14 after stimulation, the rats were sacrificed and the expressions of glial fibrillary acidic protein(GFAP),5-Bromo-2’-deoxyuridine(Brdu), microtubule associated protein-2 (MAP-2) and the volume of the holes of injuried area of spinal cord were detected with immunohistochemistry and Western-Blot technique. Quantitative analysis of the expression of GFAP, Brdu and MAP-2 were performed with the image analysis system. Results With the increase of magnetic stimulation frequency, the volume of the holes of injuried area of spinal cord discrease at day 14. In the reduced area of the holes, the positeve expressions of GFAP could be seen, while Brdu and MAP-2 could not be seen. Significant differences were revealed in the expressions of GFAP among the five groups, which were significantly higher in magnetic stimulation groups than that in control group (P< 0.05). Conclusion Under the condition of 1.52 Tesla, no larger than 20Hz magnetic stimulation, Magnetic stimulation promote astrocytes to migrate. After magnetic stimulation, astrocytes migrate into the injuried spinal cord holes. the ability of astrocytes migration increase with the increase of magnetic stimulation frequencies.Exp II:The mechanism of different frequencies magnetic stimulation promoting astrocytes migration.[Abstract] Objective To investigate the effects of ERK inhibitor U0126 of different doses on the ability of lOHz magnetic stimulation promoting astrocyte migration, then to select suitable dose of U0126, and study the mechanism of different frequencies magnetic stimulation promoting astrocyte migration. Methods Twenty-four adult healthy Sprague-Dawley rats were selected to inject 0.5ml of 1% ethidium bromide (EB) in PBS into spinal cord dorsal funiculus on the left side at T10-11 level to make located spinal cord injury models and randomly divided into four groups.The four groups were exposed to magnetic stimulation (10Hz,1.52Tesla, 30 pulses) at the following doses respectively:Omg/kg U0126(control group), 0.1mg/kg U0126(low-dose group),0.2mg/kg U0126 (middle-dose group),0.4mg/kg U0126(high-dose group). On the 14 after stimulation, the rats were sacrificed and the expression of glial fibrillary acidic protein(GFAP), microtubule associated protein-2 (MAP-2), extracellular signal-regulated kinasel/2 (ERK1/2) and the volume of spinal cord injuried holes were detected with immunohistochemistry and Western-Blot technique. Quantitative analysis of the expression of GFAP, MAP-2 and ERK1/2 were performed with the image analysis system. Results With the increase of U0126 dose, the volume of spinal cord injuried hole increase on 14(P< 0.05). In the lesion area, the positive expression of GFAP and ERK1/2 could be seen and had identical change tendency,while MAP2 could not be found. Significant difference was revealed in the expression of GFAP and ERK1/2 among the four groups, and it was significantly lower in U0126 groups than that in control group (P< 0.05). while the middle-dose group had similar effect with the high-dose group (P> 0.05) Conclusion U0126 of different dose all could inhibit astrocytes migration that 1.52Tesla,10Hz magnetic stimulation caused, and 0.2mg/kg was the suitable dose. What’s more, the effect of 1.52Tesla,10Hz magnetic stimulation promoting astrocytes migrate is relevant to ERK pathway.更多还原