中草藥地龍對史旺細胞增生與轉移之分子機轉探討

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題名:	中草藥地龍對史旺細胞增生與轉移之分子機轉探討 Proliferation and migration molecular mechanisms induce by earthworms (Pheretima aspergilum) in Schwann cells.
作者:	張永明;Yung-Ming Chang
貢獻者:	中醫學院中國醫學研究所博士班
關鍵詞:	地龍;存活;增生;移動;史旺神經膠細胞;周邊神經再生;earthworm;survival;proliferation;migration;RSC96 Schwann cells;peripheral nerve regeneration.
日期:	2009
上傳時間:	2010-09-29 12:00:01 (UTC+8)
摘要:	地龍 (Earthworm) 為現今廣泛使用的中草藥，具有移除斑塊 (Stasis) 與促進傷口癒合之功能。Schwann cell則具有支持神經再生之能力，故Schwann cell的移動和增生對受損神經來說相當重要；然而，目前有關地龍是否能誘導Schwann cell移動和增生及其分子機制仍不清楚。根據文獻資料，本實驗推測了兩個可能機制: (1) 移動方面 (Migration)- MAPKs (Mitogen-activated protein kinases) 協調的 PAs 與 MMP2/9 訊息路徑；(2) 存活與增生方面 (Survival and Proliferation)- IGF-I (Insulin- like growth factor-I) 協調的PI3K/Akt訊息路徑及細胞週期之調控。利用MTT與移動實驗，發現125 μg/ml的地龍萃取物作用於細胞24H，可以顯著地刺激Schwann cell增生與移動。此外，western blot數據顯示地龍萃取物會隨著時間誘導ERK1/2 和 p38磷酸化增加 (但不包含JNK)，及提升其下游PAs (Plasminogen activators) 與 MMPs (Matrix metalloproteinases) 蛋白表現。但細胞以化學性抑制劑 (U0126和SB203580) 及ERK1/2 與 p38 之siRNA (Small interfering RNA) 處理後，會顯著地抑制Schwann cell移動及uPA相關訊息路徑。另一方面，地龍萃取物也能誘導IGF-I 協調的 PI3K/Akt 訊息路徑之蛋白磷酸化，並隨時間活化PCNA (proliferating cell nuclear antigen) 及細胞週期調節蛋白 (cyclin D1, cyclin E and cyclin A) 表現；且加速細胞週期G1期的進行，顯著地增加S期進行DNA複製之細胞數目。我們同樣利用PI3K siRNA 進行基因knockdown，可以發現不僅會導致抗凋亡蛋白Bcl2表現量降低，也會阻礙細胞G1至S期的變遷 (transition)。綜合上述結果，可以發現地龍具有顯著促進Schwann cell移動與增生之功能；因此，地龍於未來很有潛力做為臨床治療神經再生之藥物。 The earthworm, which has stasis removal and wound healing functions, is a widely used Chinese herbal medicine in China. Schwann cell migration and proliferation are critical for the regeneration of injured nerves. Schwann cells provide an essentially supportive activity for neuron regeneration. However, the molecular migration and proliferation mechanisms induced by earthworms in Schwann cells remain unclear. Here, we investigate the molecular mechanisms, which includes : (1) migration signaling, MAPKs (Mitogen-activated protein kinases) mediated PAs and MMP2/9 pathway ; (2) survival and proliferative signaling, IGF-I (Insulin-like growth factor-I) mediated PI3K/Akt pathways and cell cycle regulation. Using MTT and migration assay, we confirmed that treatment with 125 μg/ml earthworm for 24 h appears to stimulate cell proliferation and migration. In addition , examined by western blotting, found that earthworm extract induced phosphorylation of ERK1/2 and p38, but not JNK, activate the downstream signaling expression of PAs (Plasminogen activators) and MMPs (Matrix metalloproteinases) in a time-dependent manner. Earthworm stimulated ERK1/2 and p38 phosphorylation was attenuated by pretreatment with chemical inhibitors (U0126 and SB203580), and small interfering ERK1/2 and p38 RNA, resulting in migration and uPA related signal pathway inhibition。 On the other hand, earthworm extract treatment also induces the phosphorylation of IGF-I mediated PI3K/Akt pathway, activate protein expression of PCNA (proliferating cell nuclear antigen) and cell cycle regulatory proteins (cyclin D1, cyclin E and cyclin A) in a time dependent manner. And accelerated G1 phase progression with earlier S phase entry and that significant numbers of cells entered the S phase.siRNA mediated knockdown of PI3K, that significantly reduces PI3K protein expression levels, resulting in Bcl2 survival factor reduction, revealing a marked blockage of G1 to S transition in proliferating cells. Taken together, our data reveals the unknown RSC96 cell migration and proliferation mechanism induced by earthworm extract, which find use as a new medicine for nerve regeneration.
顯示於類別:	[中國醫學研究所] 博碩士論文

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