SDF-1調控神經幹細胞之生長及分化機轉之研究

背景：神經幹細胞(NSC)是一種具備新生與分化能力的細胞，可以進行細胞再生甚至組織再造，所以神經幹細胞的生長與分化是神經新生的重要關鍵，因此常被視為細胞治療的重要來源。近年來發現許多腦部發炎性疾病中，體內Stromal derived factor-1 (SDF-1)會被大量增加，並且同時出現大量神經幹細胞的新生與遷移。因此SDF-1極為可能是神經新生驅動活化的重要因子。目標: 探討SDF- 1對小鼠神經幹細胞之特性維持與生長分化能力的影響以及其相關機轉的研究研究方法：本篇論文利用體外培養小鼠初代神經幹細胞球，使用CXCR4拮抗劑, AMD3100競爭阻斷SDF-1活化神經幹細胞中CXCR4的訊息路徑。並且藉由分析形成之神經幹細胞球數量大小、形態特徵及細胞特性來評估缺少SDF-1作用時對於神經幹細胞球所造成之影響結果：我們成功的從小鼠大腦組織培養出神經幹細胞並且藉由給予AMD3100阻斷神經幹細胞中SDF-1/CXCR4的訊息傳遞。研究結果發現AMD3100不會造成細胞死亡也不會影響神經幹細胞球之形成比率。但是AMD3100會顯著降低神經幹細胞球之平均大小，而且與劑量呈現正相關(控制組為1818.07±84.9 pixel2；AMD3100 0.1μg/ml為1411.01±107.7 pixel2；AMD3100 0.5μg/ml為906.20±97.8 pixel2；AMD3100 1.0μg/ml為626.60±66.3 pixel2)。同時也發現AMD3100會明顯減少神經幹細胞球中表現Ki67之細胞比例(控制組為52.25±8.45%；而AMD3100 1.0μg/ml為32.67±3.82%)，表示當神經幹細胞之CXCR4訊息路徑受阻斷時可能會因此抑制其增殖能力。此外，從流式細胞分析結果顯示AMD3100阻斷CXCR4後會降低神經幹細胞球中表現nestin之神經幹細胞比例(控制組為96.87±0.7%；AMD3100 1.0 μg/ml為90.6±2.6%)，但是會增加表現β III tubulin之神經細胞比例(控制組為52±1.4%；AMD3100 1.0 μg/ml為67.7±5.1%)，表示CXCR4訊息路徑的阻斷可能會因此導致神經幹細胞趨向神經分化而降低神經幹細胞之特性。西方墨漬法結果發現受到AMD3100作用後的神經幹細胞球，其REST和Notch的表現量明顯減低，表示神經幹細胞特性可能發生變化且趨向神經分化。同時也發現神經幹細胞球之pAkt和pS6蛋白表現量有顯著增加，因此推測Akt-mTOR訊息路徑可能是AMD3100影響神經幹細胞之增殖與分化活性的可能路徑。此外，增殖時期受AMD3100影響過之神經幹細胞球在誘發分化後，其分化神經的軸突長度較長(控制組為153.21±88.3μm；AMD3100 1.0 μg/ml 385.93±47.6 μm)和分支程度也會增加(控制組為1.08±0.12級；AMD3100 1.0 μg/ml為2.12±0.17級)，表示AMD3100可能潛在具備促進新生神經成熟的作用。另一方面，也發現神經幹細胞在增殖時期缺少SDF-1作用不僅會改變細胞增殖活性，也可能因此影響神經幹細胞球最終分化細胞之蛋白表現。結論：本篇論文結果發現，當SDF-1/CXCR4訊息功能受到AMD3100的阻斷時，神經幹細胞之特性會降低並且促使神經幹細胞趨向神經分化，而這其中Akt-mTOR之訊息傳遞路徑可能參與其中。這代表SDF-1/CXCR4訊息路徑對神經幹細胞之特性維持可能扮演著十分重要的角色，也可能成為未來誘導神經幹細胞特性的重要標的。

關鍵字

神經幹細胞；神經新生； SDF-1(Stromal derived factor-1) ；神經幹細胞球； CXCR4拮抗劑； Akt-mTOR

並列摘要

Background：Neural stem cell (NSC) is a kind of cell has the ability for self-renewal and differentiation. Due to NSC is able to generate new-born cells and repopulate tissues, so the growth and development of NSC is the key of the neurogenesis. Thus, NSCs have been considered as an attractive source of cell-based therapy. Recently, it’s been reported that neuronal generation and migration occurred with a burst of SDF-1 elevation. Therefore, SDF-1 probably is the main regulator of driving neurogenesis. Aim : To study the effects of SDF-1on NSC maintenance and its develo[1]ping ability and to further investigate their related mechanisms Methods : In this thesis, using CXCR4 antagonist, AMD3100 blocked the activation of CXCR4 signaling pathway by SDF-1 in the mice primary neurosphere culture. We analyzed the size and population of neurospheres, cell morphologic features, and cell expression markers to investigate the effects on neurospheres without SDF-1. Results : We successfully cultured primary neural stem cells to form neurospheres from postnatal mice brain and blocked the SDF-1/CXCR4 signaling pathway by AMD3100. We found that CXCR4 blockage by AMD3100 had no effects on the cell death and the neurosphere formation. However, AMD3100 significantly reduced the mean size of neurospheres, and it’s in a dose-dependent manner. (Control:1818.07±84.9 pixel2, AMD3100 0.1μg/ml : 1411.01±107.7 pixel2, AMD3100 0.5μg/ml : 906.20±97.8 pixel2, AMD3100 1.0μg/ml : 626.60±66.3 pixel2). We also found that AMD3100 decreased the expression percentage of Ki67-positive cells (Control:52.25±8.45%, AMD3100 1.0μg/ml: 32.67±3.82%). It indicated that CXCR4 signaling blockage inhibited NSC proliferation. Moreover, the results from flow cytometry analysis showed that blocking CXCR4 signaling in NSCs by AMD3100 decreased the percentage of nestin-positive cells (Control : 96.87±0.7%, AMD3100 1.0 μg/ml : 90.6±2.6%); nevertheless, increased the percentage of β III tubulin-positive cells (Control : 52±1.4%, AMD3100 1.0 μg/ml : 67.7±5.1%). It suggested that CXCR4 signaling blockage might promote NSC differentiation and result in the reduction of NSC property. Western blot showed that both REST and Notch expression level were decreased in AMD3100-treated neurosphere, and it represented the character of NSCs were probably changed and prompted to neuronal differentiation. Meanwhile, the pAkt and pS6 activity were significantly elevated, and it’s suggested that the induction of neuronal differentiation by AMD3100 was possibly through Akt-mTOR signaling activation. Besides, after induced neurosphere differentiation, the neurites length and branching level of those differentiated neurons from AMD3100 treatment may significantly increase (Control : 153.21±88.3μm, AMD3100 1.0 μg/ml : 385.93±47.6 μm) (Control : 1.08±0.12 level, AMD3100 1.0 μg/ml : 2.12±0.17 level). It indicated that AMD3100 have a potential ability to promote NSC maturation. On the other hand, loss of SDF-1 function at the proliferation stage of NSC might reduce NSCs proliferation abilities, and ultimately decreased the protein levels of differentiated neurons after induced differentiation. Conclusions : Our results showed that AMD3100 once blocking the SDF-1/CXCR4 signaling pathway, Akt-mTOR signaling pathway might involve in the decrease of NSC property and the increase in NSC differentiation. It indicated that SDF-1/CXCR4 signaling is quite important for NSC maintenance, and it could be a great target for induced NSCs in the future.

並列關鍵字

NSC ； neurogenesis ； SDF-1(Stromal derived factor-1) ； neurosphere ； CXCR4 antagonist ； Akt-mTOR signaling pathway

參考文獻

Identification and Characterization of Neural Progenitor Cells in the Adult Mammalian Brain Series: Advances in Anatomy, Embryology and Cell Biology, Vol. 203

Ahmed, S. (2009). "The culture of neural stem cells." J Cell Biochem 106(1): 1-6.

Bai, Y., M. Cui, et al. (2009). "Ectopic expression of angiopoietin-1 promotes neuronal differentiation in neural progenitor cells through the Akt pathway." Biochem Biophys Res Commun 378(2): 296-301.

Bajetto, A., S. Barbero, et al. (2001). "Stromal cell-derived factor-1alpha induces astrocyte proliferation through the activation of extracellular signal-regulated kinases 1 2 pathway." Journal of Neurochemistry 77: 1226-1236.

Ballas, N., C. Grunseich, et al. (2005). "REST and its corepressors mediate plasticity of neuronal gene chromatin throughout neurogenesis." Cell 121(4): 645-657.

國際替代計量

SDF-1調控神經幹細胞之生長及分化機轉之研究

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