【作者】 李在望；

【导师】 唐荣华；

【作者基本信息】 华中科技大学 ， 神经病学， 2010， 博士

【摘要】 第一部分表皮生长因子受体抑制剂AG1478对大鼠脊髓损伤后GAP-43的表达及运动功能缺损的影响目的：脊髓损伤后胶质瘢痕的形成及髓鞘相关抑制分子的产生是导致轴突不能有效再生的重要原因。而表皮生长因子受体的活化可导致静息星形胶质细胞反应性增生并形成胶质瘢痕,而且表皮生长因子受体抑制剂AG1478通过减轻髓鞘相关抑制分子及硫酸软骨素蛋白聚糖对轴突再生的抑制作用而促进了视神经轴突的再生,因此表皮生长因子受体抑制剂AG1478可能对脊髓损伤后轴突的再生有促进作用而改善脊髓损伤所导致的神经功能缺损。在本实验中我们通过在损伤脊髓局部给予表皮生长因子受体抑制剂AG1478以观察AG1478能否上调相关生长蛋白-43(GAP-43)的表达及其是否改善大鼠脊髓损伤后神经功能缺损症状。方法：应用重物坠落打击法建立大鼠脊髓损伤模型,假手术组仅切除T10椎板。48只健康雌性成年SD大鼠随机分为假手术组、损伤对照组和AG1478治疗组。AG1478治疗组大鼠在脊髓损伤后立即在局部给予含有0.2 ml2mM的AG1478的明胶海绵外敷治疗,而损伤对照组和假手术均在术后立即给予含溶剂(二甲基亚砜)的明胶海绵外敷作对照治疗。应用免疫荧光技术和western blot检测各组大鼠脊髓损伤后28天损伤区域生长相关蛋白-43(GAP-43)的表达情况。应用BBB评分法对大鼠脊髓损伤后第1d及以后每周(共8周)后肢运动功能进行评分。在损伤前及损伤后每周(共8周)测量各组大鼠体重。结果：免疫荧光检测显示在损伤后28天AG1478组GAP-43阳性细胞计数表达较对照组明显增多(P<0.01)。Western blot印迹分析也显示AG1478组GAP-43表达较对照组明显上调(P<0.01)。损伤对照组与AG1478治疗组大鼠在伤后第1天后肢均几乎完全瘫痪,表明两组大鼠损伤程度一致。经重复测量数据方差分析检验后显示AG1478组BBB评分明显高于对照组(P<0.01)。经重复测量数据方差检验分析显示AG1478组大鼠体重明显重于对照组大鼠体重P<0.05)。结论：脊髓损伤后立即在损伤局部给予表皮生长因子受体抑制剂AG1478干预,可以改善大鼠脊髓损伤后的神经功能缺损并明显上调与轴突生长相关的GAP-43。表皮生长因子受体抑制剂AG1478对脊髓损伤有确切的治疗作用,这提示其可能是中枢神经系统损伤的有效保护剂。第二部分表皮生长因子受体抑制剂AG1478对大鼠脊髓损伤后反应性星形胶质活化增生的影响目的：星形胶质增生被认为在脊髓损伤的病理生理改变中起着极其重要的作用,而在中枢神经系统损伤的病理生理改变中星形胶质细胞的增生与表皮生长因子受体的活化密切相关。因此我们试图应用表皮生长因子受体抑制剂AG1478对脊髓损伤进行干预以观察表皮生长因子受体抑制剂AG1478对脊髓损伤后星形胶质细胞上磷酸化表皮生长因子受体(pEGFR)的表达和反应性胶质增生及轴突生长抑制因子硫酸软骨素蛋白聚糖(CSPGs)产生的影响方法：应用重物坠落打击法建立大鼠脊髓损伤模型,假手术组仅切除T10椎板。45只健康雌性成年SD大鼠随机分为假手术组、损伤对照组和AG1478治疗组。AG1478治疗组大鼠在脊髓损伤后立即在局部给予含有0.2 ml2mM的AG1478的明胶海绵外敷治疗,而损伤对照组和假手术均在术后立即给予含溶剂(二甲基亚砜)的明胶海绵外敷作对照治疗。分别于损伤后14d、28d处死各组实验大鼠。应用免疫荧光技术和western blot技术检测GFAP在损伤后14天、28天的表达水平变化情况；采用免疫荧光双标技术检测脊髓损伤后14天各组大鼠星形胶质细胞表达磷酸化表皮生长因子受体(pEGFR)的情况并检测伤后28天时星形胶质细胞表达硫酸软骨素蛋白聚糖(CSPGs)的情况。结果：假手术组GFAP阳性细胞稀少,形态纤细；而损伤对照组在伤后14天GFAP表达明显上调,星形胶质细胞呈突触增多变大的活化状态,伤后28天典型胶质瘢痕出现；在AG1478治疗组GFAP的表达明显受到抑制,而伤后28天无明显胶质瘢痕形成。GFAP Western印迹分析也显示在损伤后14天和28天,AG1478组GFAP的表达均明显低于对照组假手术组(P<0.01)。假手术组pEGFR和GFAP表达均很弱,无荧光染色双标阳性区域,表明正常脊髓组织的星形胶质细胞不表达pEGFRo对照组pEGFR和GFAP均有强表达,同时损伤组脊髓组织病灶周围存在明显空洞和疤痕改变,且在空洞周围疤痕存在pEGFR和GFAP双标阳性区域,表明脊髓损伤后星形胶质细胞表达pEGFR,然而AG1478组pEGFR和GFAP的表达均较弱,其荧光强度较对照组明显较弱(P<0.01),且无pEGFR和GFAP双标阳性区域,表明AG1478对pEGFR和GFAP的表达均有明显抑制作用。假手术组脊髓组织CSPGs和GFAP表达均较弱；而对照组CSPGs和GFAP均有较强表达,且损伤中心CSPGs表达很强,在胶质瘢痕区域CSPGs和GFAP存在明显双标阳性区域,表明活化星形胶质细胞大量表达CSPGs;在AG1478组脊髓组织中CSPGs和GFAP的荧光强度均明显低于照组(P<0.01),这表明AG1478能明显抑制CSPGs和GFAP的表达。结论：脊髓损伤后星形胶质细胞明显上调pEGFR的表达,而表皮生长因子受体抑制剂AG1478通过对星形胶质细胞上pEGFR活化的抑制而导致其对脊髓损伤后反应性星形胶质增生及抑制因子CSPGs有明显抑制作用。第三部分表皮生长因子受体抑制剂AG1478对大鼠脊髓损伤后髓鞘脱失及髓鞘相关抑制因子表达的影响目的：外伤性脊髓损伤不仅造成神经元及轴突严重损伤而且导致少突胶质细胞受到严重伤害,而少突胶质细胞和轴突损害最终导致脊髓髓鞘的大量脱失。同时少突胶质细胞损伤后会释放大量髓鞘相关抑制分子,这些抑制分子对脊髓损伤后轴突的再生产生极其不利的影响。而表皮生长因子受体抑制剂AG1478对脊髓损伤有良好的治疗作用,故我们试图应用表皮生长因子受体抑制剂AG1478干预脊髓损伤以观察AG1478对大鼠脊髓损伤后髓鞘脱失及髓鞘相关抑制因子MAG、Nogo-A和OMgp mRNA表达的影响。方法：将33只健康雌性成年SD大鼠随机分为损伤对照组(重物坠落打击法建立脊髓损伤模型后立即在损伤局部给予含二甲基亚砜的明胶海绵外敷作对照治疗),AG1478治疗组(重物坠落打击法建立模型后立即在损伤局部给予含有0.2ml 2mM的AG1478的明胶海绵外敷治疗)和假手术组(只切除椎板并立即在损伤局部给予含二甲基亚砜的明胶海绵外敷作对照治疗)。各组大鼠均在脊髓损伤后14和28天处死。应用勒克司坚牢蓝染色(Luxol Fast Blue)方法分析各组大鼠脊髓损伤后28天髓鞘脱失情况。利用RT-PCR方法检测各组大鼠脊髓损伤后14天、28天髓鞘相关抑制因子MAG、Nogo-A和OMgp mRNA表达的情况。结果：在假手术组大鼠脊髓切片Luxol Fast Blue染色中,脊髓组织呈均匀的亮蓝色；而在对照组中损伤部位不能正常着色,这提示病灶中心髓鞘严重脱失；而在AG1478治疗组中脊髓组织切片不能正常着色的范围明显缩小,其髓鞘脱失面积明显小于对照组(P<0.01)。而髓鞘相关抑制分子MAG、Nogo-A和OMgp mRNA在假手术组有较弱的表达；在损伤对照组,脊髓损伤后14天及28天髓鞘相关抑制分子的表达均明显增加；而在AG1478组,脊髓损伤后14天及28天髓鞘相关抑制分子的表达均明显低于对照组(P<0.01)。结论：局部应用表皮生长因子受体抑制剂AG1478可改善大鼠脊髓损伤后髓鞘的脱失并能减少大鼠脊髓损伤后髓鞘相关抑制分子的MAG、Nogo-A和OMgp mRNA表达。更多还原

【Abstract】 Part one The effects of epidermal growth factor receptor inhibitor AG1478 on GAP-43 expression and motor functional impairment after spinal cord injury in ratsObjective The formation of glial scar and production of myelin-associated inhibitory molecules are two main obstacles to axonal regeneration after spinal cord injury. Recent studies have indicated that epidermal growth factor receptor (EGFR) activation triggered quiescent astrocytes into becoming reactive astrocytes and forming glial scar. And epidermal growth factor receptor inhibitors AG1478 promoted the axonal regeneration post optic injury by attenuating the inhibition effects of myelin-associated inhibitor molecule and chondroitin sulfate proteoglycans of axon. Therefore epidermal growth factor receptor inhibitors may attenuate the function impairment by promoting the injury axon regeneration after spinal cord injury. In these experiments we would administer the epidermal growth factor receptor inhibitors AG1478 in the local lesion and then observed whether AG1478 could upregulate the expression of growth associated protein-43(GAP-43) and attenuate the function impairment post injury.Methods The rats spinal cord injury model were built by using the weight-drop method, and sham-operated animals underwent T10 laminectomy alone.48 SD health female adult rats were randomly divided into 3 equal groups:sham group, injury control group and AG1478-treated group. The rats in AG1478-treated group were locally administered 0.2 ml 2mM AG1478 using a Gelfoam as a carrier, while the sham group and control group were locally administered dimethylsulfoxid also using a Gelfoam as a carrier. Immunofluorescence staining and Western blotting was used to detect the expression of growth associated protein-43 (GAP-43) 28 day post injury. The BBB scores of the hindlimb locomotor were collected at the first day post injury and weekly afterward (8 weeks). The body weight of all rats was measured previous injury and weekly afterward post injury (8 weeks).Results Immunofluorescence staining results showed that the positive labeling cells of GAP-43 in AG1478 group were more than that of injury control group (P< 0.01). Western blot results showed that the level of GAP-43 protein expression was significant higher in AG1478 group than that in the control group or sham group (P< 0.01). Hindlimb of rats in injury control group and AG1478-treated group were almost totally paralysed at first day post injury, which indicated that the two group rats underwent the same degree injury. Statistical analysis by repeated-measurement ANOVA indicated that the BBB scores of AG1478 group rats were significantly higher than that of control group rats. And statistical analysis by repeated-measurement ANOVA indicated that the body weight of AG1478 group rats were significantly higher than that of control group rats. (P<0.05).Conclusion Acute local administration of epidermal growth factory receptor inhibitors AG1478 in the contusion site could attenuate locomotor function impairment in spinal cord injured rats and significantly upregulated the level of GAP-43 expression. The robust effects of epidermal growth factor receptor inhibitors AG1478 on spinal cord injury indicated that epidermal growth factor receptor inhibitors could be efficient protective agent on central nervous system injury. Part two Effects of epidermal growth factor receptor inhibitor AG1478 on reactive astrogliosis after spinal cord injury in ratsObjective Astrogliosis are thought to play a key role in the pathophysiology of spinal cord injury (SCI), and there are a close relationship between astrogliosis and the activation of epidermal growth factor receptor in the pathophysiology of central nervous system injury. We sought to administer epidermal growth factor recepter AG1478 to treat spinal cord injury in order to investigate the effects of epidermal growth factor receptor inhibitor AG1478 on the expression of pEGFR in astrocytes and reactive astrogliosis and production of axon growth inhibitory factor chondroitin sulphate proteoglycans after rat spinal cord injury.Methods The rats spinal cord injury model were built by using the weight-drop method, and sham-operated animals underwent T10 laminectomy alone. Forty five SD health female adult rats were randomly divided into 3 equal groups:sham group, injury control group and AG1478-treated group. The rats in AG1478-treated group were locally administered 0.2 ml 2mM AG 1478 using a Gelfoam as a carrier, while the sham group and control group were locally administered dimethylsulfoxid also using a Gelfoam as a carrier. All rats were sacrificed at 14d and 28d. The expression of GFAP at day 14 and day 28 after spinal cord injury were detected by immunofluorescence and western blot. The expression of phosphorylated epidermal growth factor receptor inhibitors in astrocytes at day 14 after spinal cord injury was observed by double-immunofluorescence and the expression of chondroitin sulphate proteoglycans in astrocytes at day 28 after spinal cord injury were observed by double-immunofluorescence.Results GFAP-positive cells were sparse and slim in the sham group. In contrast, activated astrocytes characterized by the hypertrophy of astrocytic processes and up-regulation of GFAP were present at 2 weeks after SCI and the typical glial scar became evident at 4 weeks after SCI in the control group. While the up-regulation GFAP was significantly suppressed at 2 weeks after SCI and the dense glial scar was not observed at 4 weeks after SCI in the AG1478 group. Western blot analysis also showed that the GFAP expression of AG1478-treated group at 2 and 4 weeks after SCI was significant lower than that of control group (P< 0.01). By double-immunofluorescence, positive labeling of pEGFR and GFAP was weakly present or absent in sham group and no colozalition of pEGFR and GFAP appeared in astrocytes in sham group, which indicated that the astrocytes in sham group did not expressed pEGFR. While positive labeling of pEGFR and GFAP was strongly present in control group. The cyst and glial scar were formed at the lesion center in control group, and there was significant colozalition of pEGFR and GFAP in the glia scar around the cyst. This indicated that astrocytes after spinal cord injury expressed pEGFR. The immunofluorescence intensity of pEGFR and GFAP in AG1478-treated group was significantly weaker compared with that in control group(P<0.01), which indicated that the expression of pEGFR and GFAP was suppressed by AG1478. No colozalition of pEGFR and GFAP appeared in AG1478-treated group. Positive labeling of CSPGs and GFAP was weakly present in sham group, while positive labeling of CSPGs and GFAP was strongly present in control group. Moreover, there was significant colozalition of CSPGs and GFAP in the glial scar, which indicated that active astrocytes significanlty expressed CSPGs. The immunofluorescence intensity of CSPGs and GFAP in AG1478-treated group was significantly weaker compared with that in control group(P＜0.01), which indicated that the expression of CSPGs and GFAP was suppressed by AG1478.Conclusion Astrocytes were significantly up-regulated the expression of pEGFR after SCI. Epidermal growth factor receptor inhibitor AG1478 can attenuate the reactive astrogliosis and the prodution of inhibitory factor CSPGs after SCI by inhibiting the active pEGFR in astrocytes after spinal cord injury. Part three Effects of epidermal growth factor receptor inhibitor AG1478 on demyelination and the expression of myelin-associated inhibitory molecules after spinal cord injury in ratsObjective Traumatic spinal cord injury (SCI) not only cause damage to neuron and axon but also induces harmful effects on the oligodendrocytes, which contribute to severe demyelination. At the same time, the damage oligodendrocytes release lots of myelin-associated inhibitory molecules, which induce an harmful influence on the axonal degeneration after spinal cord injury. Since epidermal growth factor receptor inhibitor AG1478 has a robust effects on the function recovery of spinal cord injury, we sought to administrate AG1478 to treat spinal cord injury in order to investigate the effects of AG1478 on demyelination and the mRNA expression of myelin-associated inhibitory molecules MAG、Nogo-A and OMgp after rat spinal cord injury.Methods Thirty three health female adult SD rats were randomly divided into 3 equal groups:control group undergoing SCI by weight drop method and acute local administration dimethylsulfoxide using a Gelfoam as a carrier after SCI, AG1478-treated group undergoing SCI by weight drop method and acute local administration 0.2 ml 2mM AG1478 using a Gelfoam as a carrier after SCI, and sham group undergoing T10 laminectomy alone and acute local administration dimethylsulfoxide using a Gelfoam as a carrier postoperation. All rats were sacrificed at 14d and 28d post injury. The demyelination of all group rats at day 28 after spinal cord injury was observed by Luxol Fast Blue staining. The mRNA expression of MAG、Nogo-A and OMgp in all group rats at day 14 and day 28 after spinal cord injury were detected by RT-PCR.Result By Luxol Fast Blue staining, sham-operated spinal cord tissue was characterized by light blue equably across the entire spinal cords, while there was normal coloring deletion area in the lesion center of injury spinal cord tissue, which indicated that demyelination was severe in the lesion center. And the normal coloring deletion area was significantly reduced in the lesion center of AG1478-treated group spinal cord tissues. The demyelination area of AG1478-treated group was significant smaller compared with that of control group(P<0.01). The mRNA expression of myelin-associated inhibitory molecules MAG、Nogo-A and OMgp of sham group was weak post injury, while the mRNA expression of myelin-associated inhibitory molecules MAG、Nogo-A and OMgp of control group significantly increased at day 14 and day 28 after spinal cord injury. The mRNA expression level of myelin-associated inhibitory molecules MAG、Nogo-A and OMgp of AG1478-treated group was significant lower compared to that of control group (P<0.01).Conclusion Local administration of epidermal growth factor receptor inhibitor AG1478 can attenuate the demyelination after spinal cord injury and reduces the mRNA expression level of myelin-associated inhibitory molecules MAG、Nogo-A and OMgp post injury.更多还原