博碩士論文 90224011 詳細資訊




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姓名 吳柏宗(Bo-Tsung Wu)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 Cdk2在綠茶唲茶素調節3T3-L1前脂肪細胞的生長和細胞凋亡扮演著必要性的角色
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摘要(中) 在我們之前的研究中發現綠茶中所含有的唲茶素,尤其是
EGCG,能夠抑制3T3-L1 前脂肪細胞的生長或是誘發其凋亡,然而
這些現象可能導因於EGCG 改變了細胞週期的動態變化。因此,我
們利用pTargeTTM 載體建構出Cdk2 過度表達和顯性抑制(將Cdk2 基
因序列上Asp145 突變為Asn145)的穩定表達的鼠類3T3-L1 前脂肪細胞
株作為研究素材,且更進一步地實驗確認Cdk2 是否在EGCG 所調控
的3T3-L1 前脂肪細胞的生長或凋亡上,扮演一必要性之角色。在實
驗中,我們發現在沒有EGCG 處理的環境下,Cdk2 過度表達的細胞
數較Cdk2 顯性抑制型的細胞數約增加了50%,且降低了細胞週期
G1 期約20%,增加了磷酸化Histone H1 的活性;另外,Cdk2 過度表
達的細胞的生長速度也較Cdk2 顯性抑制型、Lac Z 轉殖型的細胞來
得快。在不同轉殖的細胞中處理20~100μM 的四種綠茶唲茶素後,
只有EGCG 可以降低野生型和Lac Z 轉殖型的細胞數,而對Cdk2 過
度表達型的細胞影響不大;但所有的綠茶唲茶素皆對Cdk2 顯性抑制
型的細胞沒有產生任何作用。同樣地,EGCG 會使野生型的細胞週期
延滯約10%,對於Cdk2 過度表達的細胞週期影響不大,但卻不會改
變Cdk2 顯性抑制的細胞週期動態。Cdk2 顯性抑制型的細胞不論在
EGCG 有無的環境下,皆會有細胞凋亡而產生DNA 梯度、Sub-G1 期
的現象。利用免疫沉澱將有無處理EGCG 之不同轉殖細胞的Cdk2 沉
澱下來,再用西方點墨法觀察細胞週期抑制蛋白p18、p21、p27 與
Cdk2 的結合量,結果並沒有很大的差異。因此,我們斷定EGCG 在
抑制3T3-L1 前脂肪細胞的生長及誘發其凋亡上,是透過一Cdk2 途
徑,且有別於其他的綠茶唲茶素;其作用機制則細胞週期抑制蛋白與
Cdk2 的結合無關。
摘要(英) Previously we have found that green tea catechins, particularly EGCG, inhibit
growth and induce apoptosis of 3T3-L1 preadipocytes and such effects result from its
altering different phases of cell cycle. Using 3T3-L1 preadipose cell lines
transfected with pTargeTTM plasmid containing either wild type of cyclin-dependent
kinase 2 (CDK2) or CDK2-dominant negative (CDK2-dn) gene mutated from Asp145
of CDK2 to Asn145, we further examined whether CDK2 was essential in the EGCG
modulation of preadipocyte growth and apoptosis. As indicated by 50% increases in
cell number, 20% lower in G1 phase of cell cycle, and higher activity in
phosphorylating Histone H1, CDK2-overexpressed cells in the absence of EGCG
treatment grew faster than vector-transfected cells or CDK2-dn cells. At 20-100 µM,
EGCG, but not structurally-related EC, ECG, or EGC, reduced cell number by 30-50%
in wild type of cells and vector-transfected cells, and, to a lesser extent, in
CDK2-overexpressed cells; however, all catechins had no effect on CDK2-dn cells.
Also, EGCG arrested normal cells by 10% and, to a much lesser extent,
CDK2-overexpressed cells in G1 phase, but did not alter the G1 percentage of
CDK2-dn cell cycle. CDK2-dn cells displayed the similar extent of apoptosis, as
indicated by the appearance of DNA ladder and sub-G1 phase of cell cycle, when they
were treated with or without EGCG. CDK2 immunoprecipitates from normal,
vector-transfected, CDK2-transfected, or CDK2-dn cells treated with or without
EGCG contained the corresponding amounts of CDK2 inhibitors such as p18, p21, or
p27. We conclude that EGCG mediate growth and apoptosis of preadipocytes
through CDK2-dependent pathway and such pathway of EGCG action seems different
from other green tea catechins and may not rely on the binding between CDK2 and
CDK2 inhibitors.
關鍵字(中) ★ 綠茶唲茶素 關鍵字(英) ★ EGCG
論文目次 中文摘要1
英文摘要2
第一章:緒論3
一、Cdk2 的簡介3
二、綠茶唲茶素6
三、綠茶與Cdk2 9
四、研究動機與目的10
第二章:材料與方法12
一、實驗材料12
二、實驗方法13
1. 細胞培養13
2. 細胞處理13
3. 細胞計數方法14
4. Cdk2-overexpress 與Cdk2-dominant negative 表現載體
(expressing vector )之構築14
4.1 定點突變14
4.2 Total RNA 萃取15
4.3 cDNA 的合成16
4.3.1 primer 的設計16
4.3.2 反轉錄酶反應(Reverse Transcriptase, RT) 16
4.3.3 聚合酶鏈反應(Polymerase chain reaction) 17
4.4 PCR 產物的製備18
4.4.1 PCR 產物的分析18
4.4.2 PCR 產物的純化18
4.5 接和反應(Ligation) 19
4.6 大腸桿菌勝任細胞之製備(Preparation of E. coli
competent cells) ,, 19
4.7 大腸桿菌的轉型作用(Transformation) ,20
4.8 篩選(Screening) 20
4.8.1 藍白篩選21
4.8.2 質體DNA 的少量製備21
4.8.3 核酸限制酶剪切22
4.9 核酸定序(DNA sequencing) 22
5. Cdk2-overexpressed 與Cdk2-dominant negative 基因於
哺乳類細胞中之表現23
5.1 質體pTargeT-Cdk2-overexpress 及pTargeT- Cdk2-
dominant negative 的轉染(Transfection) 23
5.1.1 細胞的培養23
5.1.2 轉染作用23
6. 細胞流式分析法24
7. 蛋白質濃度定量25
8. 西方點墨法25
9. 蛋白質免疫沉澱法26
10. Cdk2 活性測定27
11. DNA 梯度分析法27
第三章:結果29
一、Cdk2 過度表達穩定表現細胞株之建立29
二、Cdk2 顯性抑制穩定表現細胞株之建立29
三、比較野生型、過度表達型、顯性抑制型三種細胞株
的生長曲線30
四、比較野生型、過度表達型、顯性抑制型三種細胞株
的細胞週期31
五、比較野生型、過度表達型、顯性抑制型三種細胞株
的Cdk2 表達量及活性變化32
六、綠茶EGCG 影響野生型、過度表達型、顯性抑制型三種
細胞株的生長32
七、不同綠茶唲茶素對於野生型、過度表達型、顯性抑
制型三種細胞株的生長影響之比較34
第四章:討論35
第五章:結論與展望39
第六章:參考文獻40
第七章:附錄60
表圖目錄V
縮寫與全名對照表Ⅵ
參考文獻 第六章參考文獻
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指導教授 高永旭(Yung-Hsi Kao) 審核日期 2003-7-25
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