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系統識別號 U0007-1707200823073300
論文名稱(中文) GSK-3β在人類腦神經膠質瘤細胞株誘導細胞自噬作用所扮演的角色
論文名稱(英文) Roles of GSK-3β in the induction of autophagy in U87-MG human glioma cells
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 96
學期 2
出版年 97
研究生(中文) 曹玫芬
研究生(英文) Mei-Fen Tsao
學號 G160095006
學位類別 碩士
語文別 中文
口試日期 2008-07-02
論文頁數 72頁
口試委員 指導教授-李宏謨
委員-李新城
委員-高淑慧
中文關鍵字 GSK-3β  細胞自噬作用  細胞週期細胞增生 
英文關鍵字 GSK-3β  autophagy  cell cycle  cell proliferation 
學科別分類
中文摘要 GSK-3β(Glycogen Synthase Kinase 3β) 對於細胞週期的進行、細胞凋亡、細胞自噬作用和侵襲性有著深遠的影響。然而,GSK-3β在其中所扮演的角色卻未完全了解。本研究利用各種激酶活性的GSK3β突變種(mock、 wild type、 S9A 和 K85R)轉染U87-MG人類腦神經膠質瘤細胞株,藉此分子探子來研究GSK-3β對於細胞週期的進行、細胞凋亡、細胞自噬作用和侵襲性的影響。本實驗發現wild type (GSK-3β) 或 kinase dead (GSK-3β K85R)增加了 GSK-3β本身在 serine9 的磷酸化。GSK-3β K85R持久表達的U87-MG細胞株細胞有促細胞增生的情形,然而constitutive active (GSK-3β S9A)卻沒有影響。細胞週期進行需cyclins, Cdks and Cdk inhibitors的調控,由實驗得知GSK-3β S9A持久表達的U87-MG細胞株p27蛋白表現增加。本研究並探討GSK-3β活性對於U87-MG細胞株是否會產生抗凋亡的特性,研究結果顯示Bcl-xL, Bcl and Mcl-1等抗凋亡蛋白及Bax and Bad 等促凋亡蛋白的表現,並沒有顯著差異,推測GSK-3β活化可能不會導致或抑制引發細胞凋亡的產生。此外,近來的研究證實了在MEF 和 COS-7 兩種細胞株中,當活化GSK-3β時,會誘導細胞自噬作用(macroautophagy)。本研究中,接著探討當活化GSK-3β時,在U87-MG人類腦神經膠質瘤細胞株中是否會刺激細胞自體吞噬作用的產生。利用持續保持激酶活性的GSK3β?n?n-S9A轉染U87-MG細胞株時,LC3-II的蛋白表現量增加;若利用失去激酶活性的GSK3β-K85R,轉染U87-MG細胞株時,LC3-II的蛋白表現量減少。所以,我們推測GSK3β?n?n的活化和自體吞噬小體(autophagosome)的形成有相關。進一步利用帶有綠色螢光蛋白的LC3的質體(GFP-LC3),再次轉染這些持續表現不同GSK3β突變株的U87-MG人類腦神經膠質瘤細胞株,觀察到GSK3β被抑制時,自噬小體形成也跟著減少。由這些研究結果,我們可以得知GSK3β在誘導細胞自體吞噬作用的過程,扮演著很重要的角色,並藉此探討GSK3β下游的基因是否有受到影響。而這樣的結果也可能是引起Doxorubicin這個化療用藥,常在乳癌的病人身上產生抗藥性的原因。先前研究已知GSK3β在Wnt訊息傳遞路徑扮演重要的角色,而本研究由轉染GSK3β?n?n-S9A U87-MG細胞株的侵襲性比wild type降低,推測GSK3β的活性亦直接影響了癌細胞的侵襲力。綜合以上數據本研究證實GSK3β的活性可調控U87-MG人類腦神經膠質瘤細胞株細胞週期的進行、細胞自噬作用和侵襲性,但對細胞凋亡沒有影響。
英文摘要 GSK-3β exerts profound effects on cell cycle progression, apoptosis, autophagy and invasion. However, the mechanism by which GSK-3β exerts these functions are not completely understood. In the present study, we constructs GSK-3β mutants and transfected into U87-MG human glioma cells. U87-MG cells transfected with mock, wild type, S9A and K85R express various kinase activities were used to examine the effects of GSK-3β on cell cycle progression, apoptosis, autophagy and invasion. Ectopic expressing wild type (GSK-3β) or kinase dead (GSK-3β K85R) increased GSK-3β serine 9 phosphorylation. Expression of GSK-3β K85R increased cell proliferation, whereas constitutive active (GSK-3β S9A) has not effect on cell proliferation. Cell cycle progression is regulated by cyclins, Cdks and Cdk inhibitors. In U87-MG cells, expression of GSK-3β S9A increased the protein level of p27Kip-1. We next examined whether GSK-3β affect cell apoptosis in U87-MG cells. Permanent expression of various GSK-3β mutants did not alter apoptotic cell death. In agreement, transfection of GSK-3β did not alter the expression of pro-apoptotic proteins including Bax and Bad or anti-apoptotic proteins such as Bcl-xL, Bcl and Mcl-1. We next investigated whether activation of GSK-3β stimulates autophagy while permanent expression of GSK-3β S9A increased LC3-II level, GSK-3β K85R decreased LC3-II. Suggesting that GSK-3β activation is linked to autophagosome formation. Taken together, these data suggest that GSK-3β may play a role in the induction of autophagy, which is important in Doxorubicin drug resistance. In conclusion, these results suggest that GSK-3β may regulate cell cycle progression, autophagy and invasion but not apoptosis.
論文目次 中文摘要 VII
研究特定目標 XII
縮寫表 XIII
壹、 緒論 1
一、 乳癌(Breast cancer) 的特性 1
二、 腦瘤(Brain cancer)的特性 2
三、 Doxorubicin 3
四、 TDZD-8 4
五、 肝醣合成酶激酶3β (Glycogen synthase kinase 3β) 4
六、 Wnt 訊息傳遞路徑(Wnt Signaling Pathway) 6
七、 細胞週期調控 8
八、 細胞自噬機制(autophagy) 8
貳、 研究方法與材料 13
一、 實驗材料 13
1. 藥品試劑 13
2. 儀器設備 16
3. 常用溶液 17
二、 實驗方法 20
1. 細胞的培養 20
2. 質體的轉染 21
3. 細胞蛋白質的測定 22
4. 蛋白質電泳(SDS-聚丙醯胺膠體電泳) 23
5. 螢光顯微鏡觀察 24
6. 細胞侵襲力試驗 (Invasion assay) 24
7. 細胞存活率試驗 (cell viability) 25
8. DNA fragmentation assay 27
9. 電泳 (electrophoresis) 27
10. 統計分析 28
參、 實驗結果 29
一、 MCF-7細胞中內源性及送入的GSK3β蛋白表現情形 29
二、 U87-MG細胞中內源性及送入的GSK3β蛋白表現情形 30
三、 GSK3β活性抑制時有促細胞增生的情形 30
四、 GSK3β持續活化時會使細胞週期停在G0/G1 phase 31
五、 GSK3β持續活化時會使p27蛋白表現量增加 32
六、 GSK3β被活化或抑制時與細胞凋亡的相關性 33
七、 GSK3β被活化或抑制時之抗凋亡蛋白的表現情形 34
八、 GSK3β被活化或抑制時之促凋亡蛋白的表現情形 34
九、 GSK3β活性抑制時會使LC3-II表現量減少 35
十、 GSK3β活性抑制時會使細胞自噬體減少 35
十一、 GSK3β活性改變時β-catenin的表現情形 36
十二、 GSK3β活性改變時上皮間質過度蛋白的表現情形 37
十三、 GSK3β活性改變時與細胞侵襲力的相關性 37
肆、 討論 39
伍、 参考文獻 44
陸、 實驗結果圖表 49

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