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研究生:李昆霖
研究生(外文):Kun-Lin Li
論文名稱:探討各種變異型ING4在人類乳癌細胞行為上的角色
論文名稱(外文):A study of the roles of ING4 variants on the cell behavior in human breast adenocarcinoma
指導教授:梁有志梁有志引用關係
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗生物技術學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:76
中文關鍵詞:變異型
外文關鍵詞:ING4
相關次數:
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目前,已有五種屬於生長抑制蛋白家族(inhibitor of growth)的成員被
確定出來,它們在蛋白結構的C 端都各自擁有高保留性的plant
homeodomain (PHD) finger motif,此外家族本身也被人發現可以接合和
活化一些造成histone 乙醯基化或是去乙醯基化的蛋白質複合體
(HAT/HDAC complexes),以調節基因染色質的結構進而調控基因的轉錄
活性。ING4 是家族的一員,並具有一些腫瘤抑制基因的特性,其特性
包括藉由與p53、NF?羠、HIF-1?捄氐鉯?因子的結合去控制血管新生、接
觸抑制等癌症惡化的現象。最近的研究發現ING4 擁有四種序列差異很
小的變異型可能是由於轉錄初產物的剪接失誤造成,不同的變異型對細
胞的生長速度、型態有著不同的影響,本文對這四種ING4 是依照胺基
酸在序列中減少的數目命名為ING4-d0、-d1、-d3、-d4。
首先,本研究利用RT-PCR 的方式從BALB卅C 小鼠的睪丸組織中,
獲得兩種ING4 變異型的cDNA(ING4-d1 及ING4-d4),我們以定點突變
的方式獲得剩下兩種ING4 分別是ING4-d1 及ING4-d3。之後,將四種
ING4 接在可經tetracycline 誘導表現的載體(Tet on system),再轉殖到
MCF-7 細胞中,經抗生素的篩選後,挑出可穩定表現單一變異型的細胞
株。接著分析其對腫瘤細胞的影響,我們發現各種ING4 的表現,並不
會影響人類乳腺癌生長與細胞週期的分布。以西方墨點法及螢光顯微鏡
分析,發現大部分的ING4- d0、ING4- d1 只侷限於細胞核內,而ING4-
d4 有部份存在於核外。另外,將純化後具有磷酸化活性的Cdk2/4 與純
化的ING4- d0 與ING4- d4 反應,進行放射線激酶活性分析,發現Cdk2/4
可磷酸化ING4- d0 及ING4- d4,但磷酸化的程度,沒有差異。最後,我們偵測雌性素所誘導的cyclin D1 表現,是否會因加入各種不同的
ING4 而有所影響,實驗發現cyclin D1 的表現量會因為ING4-d0 在細胞
內的增加而有些許下降的趨勢,但有趣的是ING4-d1 及ING4-d4 不會影
響cyclin D1 的表現。總結,我們認為無論是野生型的ING4 或是剪接的
變異型,對人類乳腺癌細胞的增生、細胞週期及cyclin D1 的表現都沒
有明顯的影響。
To date, five members of the ING (inhibitor of growth) family have
been identified that all contain a highly conserved plant homeodomain (PHD)
finger motif in the C-terminal end of the proteins. ING proteins are
identified to associate with and modulate the activity of histone acetyl
transferase (HAT) and histone deacetylase (HDAC) complexes in chromatin
remodeling. ING4 is a member of ING family and characterized as a
candidate tumor suppressor gene. Several biological functions of ING4 have
been identified, including regulation of chromatin remodeling, contact
inhibition, and angiogenesis through interaction with transcription factors
such as p53, NF-?羠, and HIF-??. Recent reports demonstrated that ING4 has
four splice variants produced by transcripts wobble splicing with differing
activities in controlling cell proliferation, cell spreading, and cell migration.
In this study, we obtained two splice variants of ING4 cDNA (ING4-d1
and ING4-d4) by RT-PCR from mouse testis. Due to lack of wild type and
one splice variant of ING4 cDNA in mouse testis, we used point mutant
primers and megaprimers to perform PCR and obtained wild type (ING4-d0)
and one splice variant of ING4 (ING4-d3) cDNA.The three splice variants
of ING4 cDNA have different deletions in the exon 4/exon 5
splicing-junction sites. In translation, deletion of one amino acid is named
ING4-d1; deletion of three amino acids is named ING4-d3; delection of four
amino acids is named ING4-d4. Three splice variants and the wild type
ING4 cDNA were inserted to pcDNA4/TO/myc-His vector that is a
tetracycline inducible expression vector (tetracycline on system). Second,
wild type and three ING4 variants expression vector were then introduced
into human MCF7 breast cancer cells, selected with antibiotics, and generated stable cell lines that express wild type or individual ING4 splice
variants. Third, we analyzed the roles of various ING4 variants on the tumor
cell behavior. We found ING4 did not affect cell proliferation and cell cycle
distribution in human breast adenocarcinoma. ING4-d0 and ING4-d1
limitedly expressed in nucleus but ING4-d4 was part expression in cytosol
by Western blot and Immunofluorescence stain. ING4-d0 and –d4 proteins
could be phosphorylated by Cdk2/4 using in vitro kinase assay, however, no
difference was found in the phosphorylation levels between ING4-d0 and
ING4-d4. Finally, we detected the cyclin D1 expression induced by estrogen
in MCF-7 cells with various ING4 variants. The results show that ING4-d1
and ING4-d4 did not affect the cyclin D1 expreesion induced by estrogen.
On the other hands, ING4-d0 slightly inhibited cyclin D1 expression. These
results suggest that ING4 wild type and various splice variants seem have no
significant effects on cell proliferation, cell cycle distribution and cyclin D1
expression in human breast adenocarcinoma.
Abstract .................................................................................................................................... I
摘要.........................................................................................................................................III
目錄..........................................................................................................................................V
第一章序論(Introduction) .................................................................................................... 1
1. ING 分子的基本介紹................................................................................................... 1
2. ING4 分子結構上的相關研究...................................................................................... 2
3. ING4 一些分子機制的探討.......................................................................................... 5
4. ING4 與腫瘤................................................................................................................. 7
5. MCF-7 細胞株.............................................................................................................. 8
第二章實驗材料與方法(Materials and Methods) ...............................................................10
1.小鼠基因體DNA 分析.................................................................................................10
1.1 基因體DNA 萃取(Genome DNA extraction) ..................................................10
1.2 聚合酵素鏈鎖反應(PCR) .................................................................................11
1.3 瓊酯膠體電泳(Agarose electrophoresis) ..........................................................12
1.4 純化瓊酯膠體中DNA 片段(Gel DNA extraction)...........................................12
2.構築質體(plasmids construct)......................................................................................13
2.1 RNA 萃取(RNA extraction) ..............................................................................13
2.2 RNA 定量分析(RNA quantification) ................................................................14
2.3 RT-PCR.............................................................................................................14
2.4 限制酶切割(Restriction enzyme cutting).........................................................15
2.5 接合作用(Ligation)...........................................................................................16
2.6 細菌基因轉殖(transformation).........................................................................16
2.7 細菌培養(E coli culture)...................................................................................17
2.8 質體萃取(Plasmids DNA extraction) ...............................................................17
2.9 DNA 定序(DNA sequence) ................................................................................19
2.10 Megaprimer PCR............................................................................................19
3.細胞實驗.......................................................................................................................20
3.1 細胞株(Cell line)................................................................................................20
3.2 細胞繼代培養(Cell culture).............................................................................21
4.細胞基因轉殖(Cell trransfection) ..............................................................................22
5. 細胞存活率試驗(Cell viability assay).......................................................................22
6. 細胞蛋白質萃取(Protein extraction)........................................................................23
7. 細胞核蛋白質萃取(Nuclear protein extraction) ......................................................24
8. 蛋白質定量分析(Protein quantification ) ................................................................25
9. 西方墨點法(Western blotting)..................................................................................25
SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel)之配製:....................25
9.1 電泳(Electrophoresis): ....................................................................................26
9.2 轉印(Transfer):................................................................................................26
9.3 免疫染色(Immunoblot):....................................................................................27
10. 鋅離子膠體染色(Zinc stain) .....................................................................................27
11. 免疫沉澱法(Immunoprecipitation) ........................................................................28
12. 激酶測定(Kinase assay)............................................................................................29
13. 流式細胞分析儀測定(Flow cytometry assay) ........................................................29
14. 免疫螢光染色(Immunofluorescence) .....................................................................30
15. 軟體分析與預測........................................................................................................31
第三章結果(Results).............................................................................................................32
1. 從正常細胞挑選出ING4 基因與其變異型d1、d4 的發現。..................................32
2. 野生型ING4 及d3 的取得。....................................................................................33
3. 穩定表現ING4s 細胞株的建立。..............................................................................34
4. 各種ING4s 的表現對MCF-7 細胞型態上的影響與一些基礎的分析。..................39
5. 尋找ING4 的轉譯後修飾與蛋白的交互作用。........................................................40
6. 嘗試探討MCF-7 與ING4 之間的關連性。..............................................................41
第四章討論(Discussion)......................................................................................................43
各種ING4 變異型發現的探討........................................................................................43
各種ING4 變異型功能的差異........................................................................................44
MCF-7 是否適合ING4 的實驗.......................................................................................44
Mouse ING4 融合蛋白於此實驗的適用性的探討..........................................................45
未來展望..........................................................................................................................46
第五章參考文獻(Reference) ................................................................................................47
第六章圖表(Figure)……………………………………………………….……………………53
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