牛樟芝萃取物維持幹細胞多能性之應用

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题名:	牛樟芝萃取物維持幹細胞多能性之應用 The application of Antrodia camphorata extracts used for maintaining stem cell pluripotency
作者:	李怡萱;Yi-Hsuan Lee
贡献者:	基礎醫學研究所碩士班
关键词:	胚胎幹細胞;牛樟芝;多能性;ES cells;Antrodia camphorata;pluripotency
日期:	2013-08-04
上传时间:	2013-10-02 11:15:26 (UTC+8)
出版者:	中國醫藥大學
摘要:	多能性胚胎幹細胞具有分化成三個胚層的能力，因此在臨床上具有很大的應用價值。然而在胚胎幹細胞的培養上需要添加leukemia inhibitory factor (LIF)，透過LIF來開啟Jak2/Stat3 signaling pathway以維持胚胎幹細胞的自我更新及多能性。然而LIF卻是一種很昂貴的藥劑，因此在本篇研究當中，便想找出傳統中草藥取代LIF並且維持胚胎幹細胞之多能性。根據先前本實驗室初篩的結果顯示酒精萃取牛樟芝 (ethanol extract Antrodia Camphorata, EEAC) 可以增加老鼠胚胎纖維母細胞(Mouse embryonic fibroblast, MEF) Oct4及Sox2的基因表現，因此我們推測EEAC具有潛力取代LIF培養胚胎幹細胞。而我們利用了鹼性磷酸?及免疫螢光染色來檢測處理了EEAC的ES cells是否依舊有胚胎幹細胞的特性，結果顯示處理了EEAC的ES cells仍然具有胚胎幹細胞的特性，像是鹼性磷酸?、Nanog及SSEA1，尤其是在0.8 μg ml-1的濃度最有效果。接下來便用含有0.8 μg ml-1濃度的EEAC取代LIF對ES cells連續培養六代並且使用EB formation來檢測其多能性。而分化的結果直接證明了EEAC所培養的ES cells確實可以分化成三胚層，像是Tuj1、α-SMA及Gata4。最後我們有興趣想要去探討EEAC透過哪一條pathway來維持ES cells的pluripotency。藉由西方墨點法、Q-PCR及ELISA 的結果顯示EEAC 會活化 Jak2/Stat3 pathway並且增加cytokine的基因表現使得胚胎幹細胞得以維持自我更新及多能性。結論是EEAC透過活化Jak2/Stat3 signaling pathway並且維持胚胎幹細胞的多能性。 Embryonic stem (ES) cells are pluripotent stem cells, that have the ability to differentiate into all types of cells. For this reason, ES cells have the potential to clinical application. Cultivation of the ES cells need leukemia inhibitory factor (LIF) to maintain ES cells self-renewal by activating Jak2/Stat3 signaling pathway. However, LIF is an expensive reagent. The goal in this study is to find out a traditional Chinese medicine extract which can replace LIF to maintain the pluripotency of ES cells. In our previous study, we found that Ethanol Extracts Antrodia Camphorata (EEAC) could increase the gene expression leves of Oct4 and Sox2, the genes that could maintain the stemness of stem cells. For this reason, AC has the potential to replace LIF in ES cells cultivation. ES cells were treated with different concentrations of EEAC and identified the stemness of ES cells by Alkaline phosphatease (AP) and immunofluorescent staining. We observed these cells expressed the characteristic of ES cells, including AP, Nanog, and SSEA1 at the concentration of 0.8 μg ml-1. Furthermore, the ES cells were passaged for six generation by the culture medium containing EEAC 0.8 μg ml-1, then used embryoid body formation to identify the pluripotency of ES cells. The results showed that the ES cells still could differentiate to the three germ layer including Tuj1, α-SMA, and Gata4. Finally, we wanted to know why EEAC could maintain the stem cell pluripotency and find the major pathway. By the data of western blot, Q-PCR and ELISA, EEAC activated Jak2/Stat3 pathway and increased the gene expression of related cytokines resulting in maintaining the ES cells self-renewal and pluripotency. In summary, we demonstrated that EEAC could maintain ES pluripotency by activing Jak2/Stat3 signaling pathway.
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