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研究生:朱俐人
研究生(外文):Li-jen Chu
論文名稱:鋰鹽抑制C6神經膠瘤細胞增生之機制
論文名稱(外文):Mechanisms by which Lithium inhibits C6
指導教授:李宏謨李宏謨引用關係
指導教授(外文):Horng-Mo Lee
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:生物醫學技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:63
中文關鍵詞:鋰鹽c6神經膠瘤細胞細胞週期鹼性磷酸脢
外文關鍵詞:LithiumC6 glioma cellsCell cycle arrestAkt/PKB
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鋰鹽(Lithium Chloride)經常被長期用於治療憂鬱症及躁鬱症。近來的研究發現鋰鹽可以抑制細胞週期的運行及誘導細胞計劃性死亡(apoptosis)。鋰鹽還可抑制Glycogen synthase kinase-3活性並保護細胞免於apoptosis。本研究發現鋰鹽(LiCl)可抑制細胞的生長和DNA合成,並且20 mM LiCl可造成 cyclinB、Cyclin E 增加及cdk-2 減少。使用10 mM LiCl隨著處理時間增加,可發現其可暫時的抑制p-Akt,可造成 p21CIP-1及Cyclin E 的增加。LiCl使C6神經膠瘤細胞的細胞週期停滯於S階段,以Flow cytometry分析,發現細胞並無apoptosis的現象。除此之外,並且可抑制DNA合成。這個現象同時伴隨著可誘導p21CIP-1、抑制P27KIP-1蛋白表現和抑制AKT/PKB Thr308 和Ser473的磷酸化,處理24小時的過程中發現10及21小時p-Akt表現及活性均有受到抑制的現象。一旦使細胞內的Akt/PKB過度表現時可影響由鋰鹽(LiCl)所抑制的Akt/PKB磷酸化,也改變了由鋰鹽(LiCl)所刺激的p21CIP-1的現象。但是,Thymidine incorporation 時Akt/PKB過度表現時並有改善鋰鹽所造成的抑制現象,因此認為鋰鹽對細胞可能仍透過其他機制抑制細胞DNA的合成。MTT的實驗中發現給予外源性inositol時並不影響因LiCl所抑制細胞生長的現象,因此,認為鋰鹽的抑制作用並非因抑制PI-Cycle而導致細胞內inositol循環再利用不足所致。綜合以上結果,鋰鹽抑制C6神經膠瘤細胞生長作用中,且其中鋰鹽抑制Akt/PKB的作用扮演了一重要的角色。

Lithium has been widely used to treat manic depression and bipolar disorder behavior. Recently, lithium has been shown to inhibit cell cycle progression and induced programmed cell death. In contrast, lithium inhibits glycogen synthase kinase-3 activity and provides protection against cell apoptosis. Here, we show that lithium inhibits cell cycle progression at S phase without inducing apoptosis in C6 glioma cells. The effects were parallelled with induction of p21cip-1 and suppression of p27KIP-1 protein expressions. The time course of p21cip-1 induction correlates with inhibition of Akt/PKB phosphorylation on Thr308 and Ser473 regulatory sites. Overexpression of Akt/PKB abolished the lithium-mediated inhibtion of Akt/PKB phosphorylation, reversed the lithium-mediated p21cip induction. Overexpression of Akt/PKB did not reverse the lithium inhibition of DNA synthesis. Supplement with exogenous inositol had no effect on the lithium-mediated inhibition of Akt/PKB phosphorylation, indicating that the mechanism of the lithium is not due to inositol depletion. Taken together, these data indicated that inhibition of Akt/PKB signaling is involved in the modulation of cell cycle progression by Lithium in C6 glioma cells.

目錄---------------------------------------------------------I
圖目錄-------------------------------------------------------III
縮寫表--------------------------------------------------------IV
中文摘要-------------------------------------------------------1
英文摘要-------------------------------------------------------3
附圖1.--------------------------------------------------------18
附圖2.--------------------------------------------------------19
附圖3.--------------------------------------------------------20
第一章 前言
一、緒論-------------------------------------------------------4
二、文獻回顧---------------------------------------------------8
第二章 實驗儀器與材料
一、實驗細胞株------------------------------------------------21
二、藥品試劑-------------------------------------------------21
三、常用儀器--------------------------------------------------23
第三章 實驗方法
一、C6神經膠瘤細胞株(C6 glioma cells)的培養------------------25
二、細胞蛋白質的測定-----------------------------------------25
三、Akt 活性測定---------------------------------------------26
四、細胞週期的分析-------------------------------------------28
五、Transfectopn----------------------------------------------28
六、[3H]-thymidine incorporation------------------------------29
七、統計分析--------------------------------------------------30
第四章 實驗結果
一、LiCl抑制細胞增生------------------------------------------27
二、LiCl抑制細胞DNA合成--------------------------------------27
三、以Flow cytometry 來分析LiCl對細胞週期運行的影響-----------28
四、不同劑量對細胞週期調節蛋白表現的影響----------------------28
五、加藥時間不同時對細胞週期調節蛋白表現的影響----------------28
六、p-Akt kinase assay----------------------------------------29
七、藉Akt overexpression方式了解LiCl的作用--------------------29
八、外加Inositol無法改變Lithium對細胞造成的抑制現象-----------30
九、LiCl對Hsp 70的影響----------------------------------------30
第五章討論----------------------------------------------------50
第六章 參考文獻-----------------------------------------------53

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