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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/6312

    Title: 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
    Authors: 吳柏宗;Bo-Tsung Wu
    Contributors: 生命科學研究所
    Keywords: 綠茶唲茶素;EGCG
    Date: 2003-07-08
    Issue Date: 2009-09-22 10:17:24 (UTC+8)
    Publisher: 國立中央大學圖書館
    Abstract: 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。 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.
    Appears in Collections:[生命科學研究所 ] 博碩士論文

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