硫酸軟骨素蛋白多醣對中樞神經損傷後神經纖維生長調節之分子機制研究

Molecular Mechanisms Underlying Chondroitin Sulfate Proteoglycan Regulation of Neurite Outgrowth After CNS Injury

劉為麟(Wei-Lin Liu)

指導教授 ： 楊維中

共同指導教授 ： 李怡萱

臺北醫學大學/醫學院/醫學科學研究所/博士(2011年)

摘要

中樞神經系統在受傷後，再活化之星狀膠質細胞會表現出大量抑制神經纖維再生的硫酸軟骨素蛋白多醣 (chondroitin sulfate proteoglycans, CSPGs)。糖皮質固醇藥物 methylprednisolone (MP)，具有神經保護及抑制發炎反應的作用，但此類藥物是否會影響星狀膠質細胞分泌硫酸軟骨素蛋白多醣的能力並不是很清楚。本研究利用體外培養初代星狀膠質細胞並給予興奮性氨基酸麩胺酸相似物AMPA，以模擬星狀膠質細胞在脊髓損傷後興奮性胺基酸刺激的情況，結果顯示MP的加入的確可以有效抑制膠質纖維酸性蛋白(glial fibrillary acidic protein, GFAP)、硫酸軟骨素氨基葡聚醣 (CS-glycosaminoglycan, CS-GAG)及CSPGs在受傷模式下的表現。進一步的分子基因調控研究顯示MP對於CSPGs之抑制作用可能是透過糖皮質固醇接受器 (glucocorticoid receptors) 的作用，在星狀膠質細胞培養基中加入MP可以促進神經軸突的生長，推測是透過抑制CSPGs而達成。給予脊髓損傷老鼠MP處理則會抑制GFAP及CSPGs的表現，這些結果可以推測MP類藥物參與調控中樞神經受傷後細胞外間質的重組，並藉以影響神經軸突的生長。許多研究已證實，硫酸軟骨素氨基葡聚醣(CS-GAG)，是中樞神經受傷後產生的神經膠疤中抑制神經纖維生長的主要成分。然而，CS-GAG抑制神經纖維生長的相對應細胞表面受器還不清楚，本研究第二部分則是探討CS-GAG相對應細胞表面的受器。結果顯示，脊髓、大腦損傷老鼠中膜聯蛋白VI (annexin VI)表現位置與硫酸軟骨素蛋白多醣, neurocan重疊。且CS-GAG會造成PC12及背根神經節神經細胞中annexin VI由細胞質轉移至生長錐中。當給予神經細胞CSGAG前，以siRAN抑制內生性annexin VI 的表現則會促進神經纖維的生長。在細胞免疫染色實驗結果亦顯示annexin VI與CSGAG結合導致annexin VI 與actin及protein kinase C分離並造成F-actin及生長錐的瓦解。酵素連結免疫分析法結果顯示，annexin VI與CS-GAG有直接結合的關係。本研究發現annexin VI可能是CS-GAG抑制神經纖維生長的受器，可調控硫酸軟骨素氨基葡聚醣引發神經細胞內訊號傳遞。以上研究有助於發展新穎的中樞神經系統損傷治療方式以促進受損神經纖維再生及神經系統功能的回復。

關鍵字

糖皮質固醇 ； 膜聯蛋白VI ； 硫酸軟骨素氨基葡聚醣 ； 神經纖維生長 ； 脊髓損傷

並列摘要

Reactive gliosis caused by post-traumatic injury often results in marked expression of chondroitin sulfate proteoglycan (CSPG), which inhibits neurite outgrowth and regeneration. Methylprednisolone (MP), a synthetic glucocorticoid, has been shown to have neuroprotective and anti-inflammatory effects for the treatment of acute spinal cord injury (SCI). However, the effect of MP on CSPG expression in reactive glial cells remains unclear. In the present study, astrocyte was activated by using α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and cyclothiazide to mimic the excitotoxic stimuli of SCI. The expression of glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivation, and CSPG neurocan and phosphacan were significantly elevated by AMPA treatment. The conditioned media from AMPA treated astrocytes strongly inhibited neurite outgrowth of rat dorsal root ganglion neurons, and this effect was reversed by pretreatment with MP. Furthermore, MP downregulated GFAP and CSPG expression in adult rats with SCI. Additionally, both the glucocorticoid receptor (GR) antagonist RU486 and GR siRNA reversed the inhibitory effects of MP on GFAP and neurocan expression. Taken together, these results suggest that MP may improve neuronal repair and promote neurite outgrowth after excitotoxic insult via GR-mediated downregulation of astrocyte reactivation and inhibition of CSPG expression.
The cell surface receptor that responds to chondroitin sulfate glycosaminoglycan (CS-GAG)-induced neurite outgrowth inhibition remains unclear. The second part of this study is to search the possible receptor that responses to the CSPG regulating neurite outgrowth signals. The data demonstrated that annexin VI mediates the CS-GAG signal cascade in neurite outgrowth. Colocalization of annexin VI with upregulated neurocan was found in rats with spinal cord injury or traumatic brain injury. Observations in PC12 cells and dorsal root ganglion (DRG) neurons showed that CS-GAG causes the translocation of annexin VI from cytoplasm to growth cones and siRNA-induced knockdown of annexin VI expression improves neurite outgrowth. Immunocytochemistry revealed that annexin VI binding with CS-GAG causes its dissociation with actin and protein kinase C, resulting in F-actin disorganization and growth cone collapse. An ELISA binding assay demonstrates specific binding between annexin VI and CS-GAG. Therefore, after CNS injury, increased CSPG may induce the translocation of annexin VI from the ctytoplasm to the membrane; the membrane annexin VI mediates the CS-GAG–induced inhibition of neurite outgrowth. These studies may lead to the development of novel treatment to improve neurite regeneration and functional recovery after neurotrauma.

並列關鍵字

methylprednisolone ； annexin VI ； chondroitin sulfate glycosaminoglycan ； neurite outgrowth ； spinal cord injury

參考文獻

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