甲基去氫可體醇處理對神經膠細胞 Nogo-A 基因表現調節之研究

1. 甲基去氫可體醇處理對神經膠細胞Nogo-A基因表現調節之研究甲基去氫可體醇處理對神經膠細胞Nogo-A基因表現調節之研究 • 哺乳類動物的神經系統研究中發現，當神經發生損傷時，其周邊神經系統具有再生的能力，而在成熟的中樞神經系統損傷時卻缺乏了修復及再生的功能。近年來的研究指出，在中樞神經系統中存在了一些抑制物質（例如 Nogo-A蛋白和 proteoglycans等）會影響神經的生長及再生，這些抑制性物質目前已經被鑑定出來，它們是一群存在於中樞神經髓鞘的蛋白質：Nogo-A、MAG以及OMgp，而Nogo-A則是其中最重要的抑制蛋白。在臨床上對於中樞神經系統損傷的患者通常會立即給予高劑量人工合成的糖質皮質固醇（methylprednisolone；MP) 來治療，對於神經復原具有一定程度的療效。因此MP透過何種途徑，來達到治療效果是一個值得探討的研究主題，由於目前的研究指出影響神經再生的抑制蛋白主要是 Nogo-A；MP 作用是否透過影響Nogo-A上游啟動子的調節，進而影響其基因的表現，而對神經損傷有良好的復原效果為本論文主要探討的主題。本實驗利用大白鼠的星狀細胞和寡突膠細胞為離體實驗系統，給予MP（1μM）處理後再分析 2.4kb、1.2kb以及 2.1kb等不同長度 Nogo-A 啟動子的表現。結果顯示當MP處理18小時後會影響Nogo-A 2.4kb的啟動子片段活性，使報導基因表現下降；而deletion 400bp的rNogo-A 2.1kb啟動子片段，則沒有前述的影響。當利用AMPA處理上述細胞，使產生興奮性毒性物質來模擬神經細胞受到傷害時的狀態，結果也發現 Nogo-A表現量並未如預期顯著增加；反而在AMPA 投予之下發現Nogo-A基因表現有下降的現象（在寡突膠細胞中最為明顯）。然而給予AMPA (星狀細胞200μM；寡突膠細胞100μM) 後再合併給予MP處理，可發現Nogo-A基因表現有更加明顯的抑制現象。而在脊髓損傷的動物模式中也有同樣的情形；Nogo-A基因在損傷部位並沒有顯著的增加，反而在損傷部位鄰近組織發現Nogo-A的大量表現，而當予以MP治療後可觀察到Nogo-A表現受到抑制而表現量下降。在活體以及離體的實驗皆可證明MP對Nogo-A所具有的影響。另外從斑馬魚的動物實驗系統也觀察到，當同時給予MP 和AMPA處理的魚，其神經生長相較於對照組以及其他單獨處理藥物的組別有明顯神經樹突增加的現象；此結果顯示出MP 和AMPA同時處理，可能具有促進神經生長的協同作用存在。綜合本實驗結果可知，給予MP處理對於Nogo-A基因表現確實具有抑制的作用，而且deletion 400bp的這段啟動子序列中可能含有一些重要的轉錄因子調控著MP對Nogo-A的影響。此外，在MP與AMPA的協同作用之下確實具有助於降低Nogo-A的表現並且促進神經生長的結果，然而其中機轉為何，值得未來更進一步的探討。

2. Regulation of Nogo-A gene expression upon Methylprednisolone treatment in neuroglia • In mammalian nervous system, axons in the peripheral nervous system (PNS) can be regenerated after injury, whereas axons in the central nervous system (CNS) can not. The lack of ability of mammalian CNS axons to regenerate after spinal cord injury (SCI) maybe due to the glial scar formation will result in the generation of a number of proteins (e.g., Nogo proteins) and proteoglycans (e.g., chondroitin sulfate proteoglycans) then inhibit neurite outgrowth. Several of these inhibitory proteins are associated with CNS myelin have been characterized, including Nogo, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), Nogo-A is the most important one that interferes with axon regrowth in the mammalian CNS and stimulates growth cone collapse in vitro. Methylpredinisolone (MP), a synthetic glucocorticoid (GC), is the known as the major therapeutic agent for acute spinal cord injury (SCI). MP, like other glucocorticoids, has broad effects on transcription factors including influencing cell viability. In this study, we investigated the regulation of the upstream element of Nogo-A gene promoter to understanding essential mechanism of MP mediated of Nogo-A gene expression after CNS injury. We have established rat Nogo-A 2.4 kb, 1.2 kb and 2.1 kb promoter sequences-driven reporter genes for investigating the transcriptional activation process of the Nogo-A expression. The pGL3-Nogo-A- 2.4 and pGL3-Nogo-A-1.2 constructs was delivered into astrocytes or oligodendrocytes to investigate the possible mechanism of MP mediated Nogo-A gene expression.The result shows that MP (1μM) down-regulates Nogo-A promoter activity but the deletion type of Nogo-A promoter 2.1kb has no significant effect on Nogo-A promoter activity. Furthermore, we also found that AMPA, which will induced neurotoxicity, did not enhance Nogo-A gene expression. On the contrary, AMPA will down-regulate Nogo-A expression (especially in oligodendrocytes). These findings suggest that the effects of MP and AMPA (200μM in astrocytes and 100μM in oligodendrocytes) combined treatment have higher ability to reduce the expression of Nogo-A in vitro and in vivo. Similar results could be found in spinal cord injury animal mode. We failed to detect the up-regulation of Nogo-A on the injury site but on adjacent site. Nogo-A expression could be inhibited after MP treatment. Furthermore, MP and AMPA combined treatment could enhance neurite outgrowth in zebrafish system. These results showed the synergistic effect of MP and AMPA, in promoting neurite outgrowth. Taken together, these results suggest that Nogo-A expression was down-regulated by MP treatment. There may exist certain transcription factors in the Nogo-A promoter of deleted 400bp that modulate MP function on Nogo-A. Moreover, MP and AMPA have synergistic effect on inhibiting Nogo-A expression and promoting neurite growth. The underlining mechanism needs to be further investigated in the future.