紅血球生成素影響 P19 分化神經元之神經纖維生長及生長相關基因表現研究

1. 紅血球生成素影響P19分化神經元之神經纖維生長及生長相關基因表現研究紅血球生成素影響P19分化神經元之神經纖維生長及生長相關基因表現研究 • 紅血球生成素(EPO)是一種具有神經保護性的細胞激素，其與紅血球生成素受體(EPO receptor)結合後，藉由活化JAK/STAT訊息傳遞路徑，調節抵抗細胞凋亡的下游基因表現，以促進細胞存活。然而，在神經細胞的發育過程中，EPO引發的訊息路徑，與其他細胞存活或細胞生長相關的訊息路徑之間的相互關連研究尚不明確。在本論文的研究中，我們利用畸胎瘤細胞株(P19 EC cells)分化而來的神經細胞，進行EPO對P19分化神經元細胞存活與生長相關基因的影響。在P19 EC cells分化的過程中，除了原本使用維生素A酸(retinoic acid)誘導神經分化之外， 另加入了纖維母細胞生長因子(FGF8)，使P19可以monolayer的細胞培養形式直接分化為神經細胞。螢光素酶活性分析(Luciferase activity assay)的結果可以得知，於P19分化神經元外加EPO，可促使生長相關蛋白(Growth-associated protein；GAP-43)的P2 promotor活性增加。除了以外加EPO進行實驗外，本論文研究中也利用了含有CMV promoter驅動epo基因表現的plasmid，轉染(transfect)進入P19分化神經元，使EPO基因過度表現。研究結果顯示，無論是外加的EPO，抑或是利用transfection使EPO基因過度表現，皆有助於P19神經纖維生長。GAP-43為神經纖維生長的marker；Bcl-xL功能為抵抗細胞計畫性死亡，此兩種基因皆受到EPO的調控，EPO可以促進GAP-43及Bcl-xL的基因表現。JAK抑制劑-AG490、PI3K抑制劑-LY294002、MAPK抑制劑-PD98059皆會阻斷EPO對P19分化神經元於神經纖維生長及維持細胞存活的促進現象，此結果意味了EPO的神經保護功能涉及了這三個訊息傳遞路徑。而EPO的神經保護性不會被NGF受體抑制劑-AG879所抑制，並且可以AG879抑制P19神經元細胞存活的情形被緩解。實驗結果進一步發現AG879會抑制P19神經元Bcl-xL的表現，但並不顯著抑制GAP-43的表現。epo基因在P19神經元的過度表現，可以回升被AG879抑制的Bcl-xL表現，同時也回升了GAP-43的表現量。因此，本論文研究結果，除了證明了EPO會透過JAK、PI3K、MAPK等訊息傳遞路徑促進神經纖維生長，並且發現EPO基因的過度表現，可以回升NGF/TrkA訊息傳遞受到抑制時所降低的Bcl-xL表現量，也促使GAP-43的基因表現, 可能藉由此一作用使得EPO可緩解因NGF/TrkA受到抑制所降低之神經存活。

2. The Effect of Erythropoietin on Growth and Survival-Related Gene Expressions in P19-Derived Neurons • Erythropoetin (EPO) is a neuroprotective cytokine that is known to mediate anti-apoptotic gene expression via EPO receptor and the JAK/STAT signaling pathway. However, the interplay of EPO signaling with other survival or growth-related signaling during neuronal development remains unclear. In the present study, we used pluripotent embryonal carcinoma (EC) P19 cell line-derived neurons as the experimental model to examine the effect of EPO on the survival and growth-related gene expressions. We modified the neural induction protocol of the P19 cells with addition of fibroblast growth factor 8 (FGF-8) in the monolayer culture. Luciferase activity assay shows that exogenous application of EPO could increase GAP-43 P2 promoter activity in P19-derived neurons. For the EPO effect, we applied exogenous EPO as well as established an epo gene overexpression system by transfection of CMV promoter-driven epo gene plasmid into differentiated P19 neurons. Our results show that both EPO gene overexpression and exogenous EPO administration enhanced neurite outgrowth. The growth-associated protein-43 (GAP-43), a marker for outgrowth and nerve regeneration, and an anti-apoptotic protein Bcl-xL, an EPO-mediated gene expression product, were both enhanced by the EPO treatment. The EPO enhancement of neurite outgrowth and neuronal survival was blocked by the JAK inhibitor AG490, PI3 kinase inhibitor LY294002, and MAP kinase inhibitor PD98059, three of the EPO receptor downstream signaling pathway inhibitors, suggesting that these pathways are involved in the EPO neuroprotection. However, the EPO neuroprotection was not blocked by the NGF receptor TrkA inhibitor AG879, and seems to reverse the AG879 impairment of neuronal survival. Furthermore, we found that AG879 significantly reduced Bcl-xL but not GAP-43 gene expression, and overexpression of EPO could reverse the AG879 downregulation of Bcl-xL as well as further up-regulate GAP-43 expression. Together, our results suggest that EPO-mediated neurite outgrowth depends on the JAK, PI3 kinase, and MAP kinase signaling pathways, and EPO overexpression seems capable of attenuating the NGF/TrkA hypofunction-reduced neuronal survival by upregulating Bcl-xL and GAP-43 gene expression.