Glial cell line-derived neurotrophic factor (GDNF) 被認為是多巴胺神經以及脊髓運動神經的存活因子。GDNF的接受器(receptor)為一複合體，由屬於tyrosine kinase receptor的c-Ret 以及 GDNF family receptor (GFR)-? glycosylphosphatidyl -inositol (GPI)-linked receptor組成。這種GDNF接受器複合體廣泛表現於神經細胞與膠質細胞，然而GDNF對於膠質細胞的作用仍不清楚。
屬於膠質細胞之一的小膠質細胞 (microglia)，被認為是中樞神經系統 (CNS)的巨噬細胞，且在許多神經病變中扮演關鍵性的角色。當中樞神經系統損傷時，小膠質細胞將會被活化並移動到損傷區域。在先前研究中，我們已證實：GDNF可使初級培養的大白鼠小膠質細胞，增加細胞生長的速率與一氧化氮的產量。此結果指出GDNF對於小膠質細胞的活化具調節作用。本研究將探討GDNF對於小膠質細胞整合蛋白 (integrin)表現的影響。整合蛋白由 alpha與 beta subunits組成，可調控細胞的多種行為，包括細胞增生、移動與黏附等。本研究以初級培養的大白鼠小膠質細胞以及小鼠小膠質細胞株－BV2細胞為材料，證實GDNF可增加這兩種細胞整合蛋白alpha5與 beta1的表現，並具有隨著GDNF劑量升高而增加的現象。以固定劑量的10 ng/ml GDNF處理BV2細胞，發現整合蛋白alpha5 與 beta1的表現隨處理時間的增加而增加。進一步以RT-PCR分析這兩種整合蛋白 subunits的mRNA的表現，顯示整合蛋白 alpha5 mRNA於10 ng/ml GDNF處理後兩小時開始明顯的增加。相反的，整合蛋白 beta1 mRNA的表現量，只在處理後0.5到2小時之間有些微的上升。以U0126 (MEK的抑制劑) 與 LY294002 ( PI3 kinase 的抑制劑)前處理BV2細胞，發現這兩種抑制劑皆可完全抑制GDNF對整合蛋白alpha5與beta1的促進作用。此結果顯示，GDNF可能透過活化Erk1/2 與 PI3 kinase而造成整合蛋白alpha5與beta1的表現量增加。另外，觀察經GDNF處理的BV2細胞與初級培養的大白鼠小膠質細胞，顯示其相對於控制組，在型態上具黏附較好的特徵。進一步以”Adhesion Index”檢測，證實GDNF可增加BV2細胞的黏附能力。最後，利用免疫螢光染色法觀察損傷脊髓切片，再次測試GDNF對小膠質細胞整合蛋白alpha5表現的作用。 研究結果顯示，整合蛋白α5以及ED1－一種活化的小膠質細胞才會表現的細胞內物質，只在經GDNF處理的受傷後一星期的脊髓切片中發現。此結果顯示：於損傷的脊髓中，GDNF也許會誘導整合蛋白alpha5表現在活化的小膠質細胞。
基於以上的結果，我們證實GDNF可增加小膠質細胞整合蛋白α5的表現，且可能為透過調控轉錄層級所造成的結果。而GDNF誘導小膠質細胞整合蛋白β1表現量的增加，則可能是經由調節轉譯層級。整合蛋白 ?5與 ?1表現量的增加，可能緣於GDNF活化Erk1/2以及PI3 kinase所致。這兩種整合蛋白subunits表現量的上升，可能是進一步誘導BV2細胞黏附能力增加的原因。
當脊髓損傷發生時，活化的小膠質細胞藉由清除損傷細胞碎片及釋放神經滋養因子，而有益於神經細胞的再生。因此，我們的實驗結果暗示，GDNF促進脊髓損傷中小膠質細胞的增生和黏附能力增加，將有助於損傷神經細胞的再生。

Glial cell line-derived neurotrophic factor (GDNF) is characterized as a survival factor for dopaminergic neurons and spinal motor neurons. It utilizes a special receptor complex consisting of the tyrosine kinase receptor c-Ret and one of the GDNF family receptor (GFR) ? glycosylphosphatidylinositol (GPI)-linked receptors. The GDNF receptor complex is generally expressed in neurons and glia. However, the effect of GDNF on glia is still unclear.
Microglia, one of glial cell types, serve as macrophages of the central nervous system (CNS) and are believed to play the critical role in neuropathogenesis. After CNS injury, they are activated and migrate toward the injuried site. Previously, we found that GDNF increased the growth of rat primary microglia and microglial NO production, indicating the regulatory effect of GDNF on microglial activation. In this study, we attempted to understand the role of GDNF on the expression of microglial integrins, which basically comprise of α and β subunits and regulate a variety of cell behavior including proliferation, migration and adhesion. It was found that GDNF upregulated integrin α5 and β1 subunits in a dose-dependent manner on rat primary microglia and BV2 cells, a mouse microglial cell line. The upregulation of integrin α5 and β1 was observed on BV2 cells treated with 10 ng/ml GDNF in a time-dependent manner. Moreover, RT-PCR analyse revealed that integrin α5 mRNA expression in BV2 cells was increased 2 hr following GDNF treatment. In contrast, integrin β1 mRNA was only slightly increased from 0.5 hr to 2 hr after GDNF treatment. According to western blotting assays, we found that U0126 (a MEK inhibitor) and LY294002 (a PI3 kinase inhibitor) completely suppressed the upregulation of integrin α5 and β1 by GDNF, implying that GDNF enhanced integrin α5 and β1 expression through Erk1/2 or PI3 kinase activation. Furthermore, the morphological examination of GDNF-treated BV2 cells and primary microglia showed greater attachment when compared with control. Adhesion Index reconfirmed the result of GDNF enhancing the adhesion capacity of BV2 cells. Finally, the effects of GDNF on regulating microglial integrin α5 were examined in the injuried spinal cord by immunohistochemistry. The ED1, an intracellular marker for activated microglia, and integrin ?? positive cells were observed in GDNF-treated spinal cord, suggesting that GDNF might induce integrin α5 expression in activated microglia of the injured spinal cord.
Based on these observations, we demonstrated that GDNF may upregulate the expression of microglial integrin α5 through transcriptional control and integrin β1 through modulating translational level. The upregulation of integrin α5 and β1 may be due to the stimulation of Erk1/2 and PI3 kinase by GDNF, thereby improving adhesive ability of BV2 cells.
After SCI, activated microglia is beneficial for neuronal regeneration via scavenging cell debris and releasing neurotrophins. Therefore, our results suggest that GDNF-enhanced proliferation and adhesive ability of microglia in the SCI may improve spinal cord repair.

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