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  • 學位論文

研究 dbcAMP 在神經幹細胞的分化效果

The effect of dbcAMP on neural stem cell differentiation

指導教授 : 金亭佑
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摘要


具有自我更新和能分化出多種細胞的老鼠神經幹細胞 (rNSCs)被認為是讓損壞的神經組織細胞替換的好方式。我們利用在體外實驗可將這些幹細胞能分化神經細胞的型態。在這份論文中,我們證明dbcAMP能讓神經幹細胞分化成神經細胞,透過細胞形態以及利用免疫染色方法測量神經標定蛋白 MAP2 和 neurofilement 來鑑定神經細胞特性,我們發現被誘導的神經幹細胞才會有染到 MAP2 和neurofilement 。此外為了要確定誘導的神經幹細胞具有神經細胞的功能,我們添加高鉀溶液讓細胞膜去極化,發現經 dbcAMP 誘導的神經幹細胞有細胞內鈣離子濃度增加以證明其具有神經特性並且會有鈣離子震盪的現象。 dbcAMP 除了誘導胞內鈣離子濃度增加以活化 ERK 途徑,我們利用西方點墨法發現隨著dbcAMP 處理時間會跟 ERK 磷酸化成正比。前處理 BAPTA-AM (一種鈣離子螯合劑) 發現對於神經分化有抑制作用,為了要觀察鈣離子對於神經分化在神經幹細胞中的訊息傳導途徑,我們添加與鈣離子相關的抑制劑如 H89 、 PD98095 、 OA (okadaic acid) 、 KN93 和 U0126 ,在神經幹細胞前處理上述抑制劑發現會抑制 dbcAMP所誘發的神經幹細胞分化作用。這些結果指出細胞內鈣訊號對神經幹細胞分化是重要的。值得一提是 MAPK 另一家族成員 JNK 在神經分化過程中會發生去磷酸化現象,若單獨添加 JNK 抑制劑 SP600125 把 JNK 抑制住,則發現會讓神經幹細胞分化且神經軸突不會生長,因此,我們認為抑制JNK 磷酸化在早期神經分化過程中具有作用證明。總結上述,這些結果支持 dbcAMP 誘導神經分化過程中分別在不同時期的分化過程可能有不同的途徑。

關鍵字

幹細胞 神經分化 鈣離子

並列摘要


Rat neural stem cells (rNSCs) with self-renewal and multi-lineage potential are considered good candidates for cell replacement of damaged nervous tissue. In vitro experimental conditions can differentiate these cells into neuronal phenotypes. The purpose of the present study is to characterize the effect of dibutyryl-adenosine 3', 5'-cyclic monophosphate (dbcAMP) -mediated on rNSC differentiation by which molecular mechanism regulated. Immunostaining labeling to neurons with MAP2 and neurofilement were analyzed for neural characteristics morphology. Functional characterization of these cells was conducted using Ca2+ imaging analysis ststem. A calcium influx and oscillation also showed response to KCl depolarization. Our results indicated that dbcAMP induced rNSCs into a TUJ1 and MAP2 positive neuronal-like cells. In addition, dbcAMP induces activation of extracellular - regulated kinase (ERK) pathway and dbcAMP induced the level of phosphor-ERK1/2 in a time - dependent pattern. Pretreatment of rNSCs with BAPTA/AM, a permeable intracellular calcium chelator, these data suggest that extracellular calcium entry is a critical part of the signaling pathway. We used some pharmacological inhibitor, U0126 and PD98059 (to inhibit MEK), H89 (to inhibit PKA), KN93 (to inhibit CaMKII) and okadaic acid (to inhibit PP2A/2B). These results showed that blockade of each of these pathways could inhibit dbcAMP-induced neural differentiation. A blockade of the JNK pathway by the JNK inhibitor, SP600125, was the most effective in the activation of differentiation but no change of neurite ourgrowth. We consider that dephosphorylated JNK works at an early stage of neural differentiation in rNSCs. Taken together, these results suggest that dbcAMP might lead to the neural differentiation of rNSCs via differential distimct downstream signaling pathways at differential stages of differentiation.

並列關鍵字

fura-2/AM dbcAMP neurogenesis MAPK

參考文獻


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被引用紀錄


王貞琪(2009)。研究鈣離子在神經幹細胞分化過程中的角色〔碩士論文,中原大學〕。華藝線上圖書館。https://doi.org/10.6840%2fcycu200900209

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