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研究生:李如萍
研究生(外文):Ru-Ping Lee
論文名稱:核仁磷酸蛋白在膀胱癌細胞經由UV照射所誘導的程式性細胞死亡所扮演的角色
論文名稱(外文):The Roles of Nucleophosmin/B23 in UV-Induced cell Apoptosis in Bladder cancer cell line MGH-U1
指導教授:翁一鳴 老師
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:54
外文關鍵詞:apoptosisnuclear matrixNucleophosmin(NPM)/B23
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先前研究指出,大量增加B23表現量可以增加細胞受到UV照射後DNA的修復,能抑制apoptosis的現象,然而降低B23表現量可以增加apoptosis的現象。利用siRNA的技術,在MGH-U1的細胞中建立knockdown B23的stable cell lines (siB23-cells,vector-cells)。起先比較這些clones之間的基本功能是否一致,所以觀察doubling time和saturation density及invasion。接著利用Flow Cytometry分析sub-G1的表現,發現降低B23使細胞死亡的情形明顯增加。顯示siB23-cells經UV照射後,apoptosis情形會增加。在之前文獻指出B23和hnRNA是nuclear matrix中的主要蛋白,所以想觀察在nuclear matrix中B23是否能夠調節其他蛋白的表現量,以及是否會影響其他蛋白在Nuclear matrix的localization能力,於是利用二維電泳的方式去比較siB23-cells和Vector-cells之間蛋白的差異性,目前MALDI-TOF分析結果在Vector較多的蛋白有GRP78、PA2G4、PHD finger protein、annexin 2、VDAC 2、 enolase 1;siB23較多的有NADH dehydrogenase、 HCG2039812、 hnRNP D、hnRNP K,較有趣的是GRP78,它能調節AKT和NF-κB的功能,這些蛋白功能都能促進細胞存活,抑制apoptosis,B23和GRP78兩者間是否具有生理上意義或是B23是否能調節GRP78功能的能力仍須進一步求證。
Previously, over expression of nucleophosmin (NPM)/B23 can increase DNA repair activity caused by UV treatment and inhibit apoptosis. But knockdown B23 expression can raise apoptosis. Use technology of siRNA, I produce stable transfected clone in MGH-U1 cells, first our observe basal function; include doubling time, saturation, density and invasion. And then observe sub-G1 by flow cytometry, when after UV irradiation, exhibit knockdown B23 can apparently raise apoptosis.
Previously, the hnRNP and B23 are abunt in nuclear matrix, so we want observe whether B23 of nuclar matrix can regulate expression of nuclear matrix proteins are altered and whether effect localization of nuclear matrix proteins.
GRP78 can regulate function of both AKT and NF-κB that can promote cell survival, inhibit apoptosis. And Both B23 and GRP78 whether have physiological significance or B23 can regulate function of GRP78 still need to prove.
Chapter I Introduction
Nucleophosmin (NPM) /B23……………………………………….......1
Nuclear matrix……………………………………………………….......3
Signaling Pathways of Apoptosis and Apoptosis marker………4
Objective of this study………………………………………………6
Chapter II Materials and Methods
Plasmid……………………………………………………………...7
Stable Transfected Clone Selection…………………………………7
Cell culture………………………………………………………….8
Flow cytometry…………………………………………………...…8
Western blotting…………………………………………………….8
Samples preparation----Nuclear Matrix Protein Isolation…………..9
Gradient gel………………………………………………………..10
Two-dimensional gel electrophoresis……………………………...11
In gel digest………………………………………………………..12
In Vitro Cell Invasion Assay……………………………………....13
Chapter III Results
The efficiency value of transfection and B23-knockdown clones...14
Doubling time and Saturation density of stable lines…………...…14
Invasion of Knockdown of B23/ (NPM)…………………………..15
Knockdown of nucleophosmin/B23-siRNA cells promote apoptosis
to compare with control vector-transfected cells…………………..15
Knockdown of B23/ (NPM) which is promot apoptosis compare with
vector by PARP cleaved……………………………………...16
Silver-stained 2-DE gels of 400 µg nuclear matrix proteins from
stable lines ………………………………………………………...17
Mass spectrometric analysis of GRP78…………………………....19
Effects of B23 knockdown on GRP78 protein expression of nuclear
matrix………………………………………………………………19
Chapter IV Discussion…………………………………………………..21
Chapter V References…………………………………………………...24
Chapter VI Figures……………………………………………………...34
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