臺灣博碩士論文加值系統

我們利用大腸桿菌乳糖調節系統所建立之7-4細胞株其內含有可調控之
Ha-ras基因，已證實於低血清狀態下，若Ha-ras過量表達會造成細胞
apoptosis，此過程中訊息之傳遞主要是經由RasRRafR MAPK途徑。
PI (3)-kinase在細胞內具多重功能，主要與細胞之增殖、骨架重組及
抑制apoptosis有關。為了研究PI (3)-kinase在Ha-ras轉型之纖維母
細胞扮演的角色，我們從短暫性基因轉殖之7-4細胞及持續活化
PI (3)-kinase之永久細胞株兩方面分別證實PI (3)-kinase之活化確實
可以阻斷Ha-ras過度表達所產生之細胞凋亡。另外轉錄因子NF-kB活性之
分析結果顯示PI (3)-kinase活性增加與NF-kB活化成正相關，推論
PI (3)-kinase可能透過NF-kB傳遞細胞之生死訊息。此外活化態
PI (3)-kinase能促進細胞之轉型，如：群落形成能力。當Ha-ras及
PI (3)-kinase同時過度活化則細胞轉型更遽烈，因此推論PI (3)-kinase
及Ha-ras在細胞轉型上扮演重要角色。建立之永久細胞株bcl2-3A中含有
Tet/Lac雙重調控系統，可同時調控Ha-ras及bcl-2兩個基因，結果發現
在飢餓刺激下Ha-ras過度表達時會抑制Bcl-2的表現而使得細胞無法免於
apoptosis，顯示Bcl-2之量扮演重要角色。此外bcl2-3A細胞在一般培養
之狀態下生長速率較7-4-2慢，而且單獨表現Bcl-2不能促使細胞進一步轉型。於飢餓刺激下將blc2-3A細胞內之Ha-ras與bcl-2同時過度表達，結果發覺細胞進行apoptosis，且此二蛋白質會被coimmunoprecipitate下來，顯示彼此間可能會相互作用，其機制有待進一步之研究證實。

A transformed cell line designated 7-4 cell (derived from
NIH/3T3) containing an inducible Ha-ras and an E. coli lacI
repressor genes has been established in our laboratory. While
Ha-ras oncogene was overexpressed by the addition of
isopropyl-b-D-thiogalactosidase (IPTG) under serum depleted
condition, most of the 7-4 cells underwent apoptosis.
The signaling of 7-4 apoptosis was through RasRRaf-1RMAPK
pathway. PI (3)-kinase is a multi-functional protein. Its
effects include cell proliferation, cytoskeletal organization,
and apoptosis. The apoptosis of 7-4 cells could be prevented by
overexpression of PI (3)-kinase using either transient
transfection or stable cell line analysis. We also demonstrated
that PI (3)-kinase activity positively correlates with NF-kB
activity, indicating that NF-kB may be a downstream effector of
PI (3)-kinase. Overexpression of activated PI (3)-kinase in
7-4 cells increased colony formation efficiency, demonstrating
that PI (3)-kinase and Ha-ras play important roles in cell transformation. In the dual-inducible cell line, bcl2-3A, we found that under serum deprived condition, Ha-ras ovexpression inhibited the expression of bcl-2, and induced apoptosis. This result suggested that the levels of Bcl-2 may be critical for cells to prevent apoptosis in our system. Moreover, overexpression of bcl-2 gene alone in 7-4 cells was insufficient for colony formation in soft agar, whereas combining Ha-ras gene ovexpression increased the efficiency of colony formation, indicating that ovexpression of bcl-2 and Ha-ras potentiates cell transformation. Finally, we demonstrated that in bcl2-3A cells, overexpressed Ha-ras and Bcl-2 could be co-immunoprecipated under serum depleted condition. However, the mechanism behind this phenomenon remains to be explored.

目錄
頁次
一．摘要
I中文摘要 i
II英文摘要 ii
二. 誌謝 iii
三. 目錄 iv
四. 圖目錄 v
五. 緒論 1
六. 材料與方法
I. 菌種與細胞株 8
II. 細菌與細胞之培養. 8
III. 質體及其製備 10
IV. 篩選與建立持續表現外源DNA之細胞株 12
V. 蛋白質電泳及西方墨點 13
VI. 細胞計劃性死亡的定量分析 16
VII. DNA斷裂分析 17
VIII. 細胞軟洋菜膠生長之分析 19
IX. 細胞生長之微量分析 20
X. Chloramphenicol acetyltransferase (CAT) 分析 21
XI PI (3)-kinase的活性分析 22
XII. 活化態PI (3)-kinase對7-4細胞凋亡的影響 24
XIII. Ha-ras及Bcl-2的coimmunoprecipitation 24
七. 結果
I. 過量表達外源性PI (3)-kinase時，對7-4細胞之影響
1. 7-4細胞內短暫性轉殖的活化態PI (3)-kinase DNA量愈多，PI (3)-kinase活性愈高
25
2. 以短暫性基因轉殖方法觀察PI (3)-kinase對細胞命運之影響
25
II. 進一步確認PI (3)-kinase的過量活化與7-4細胞凋亡之關係
1. 持續活化PI (3)-kinase細胞株的建立 26
2. 持續活化PI (3)-kinase之細胞株在軟洋菜膠之生長能力比7-4細胞強 26
3. 持續活化PI (3)-kinase之細胞株其生長速率與7-4並無差異
27
4. 於0.2%低血清刺激下持續活化PI (3)-kinase之細胞株其PI (3)-kinase的活性仍明顯高於7-4細胞 27
5. 持續活化PI (3)-kinase之細胞株DNA斷裂現象被阻
斷 28
6. 持續活化PI (3)-kinase之細胞株凋亡之細胞減少 28
7. 持續活化PI (3)-kinase之細胞株其NF-kB活性較7-4細胞明顯增加 28
III.用短暫性基因轉殖的方式證明雙重可誘導系統是可時調控兩個基因
IV.利用Tet/Lac雙重可誘導系統建立永久細胞株研究Bcl-2與Ha-ras之相互關係
1. 建立Tet/Lac雙重可誘導系統之永久細胞株 30
2. Tet/Lac雙重可誘導系統之細胞株在軟洋菜膠上之生長情形 30
3. Tet/Lac雙重可誘導系統之細胞株的生長速率較7-4
細胞慢 31
4. Tet/Lac雙重可誘導系統之細胞株DNA斷裂情形 31
5. Tet/Lac雙重可誘導系統之細胞株細胞凋亡之情形 32
6. Ha-ras與Bcl-2的相互作用 32
八. 討論 33
九. 參考文獻 38
十. 自述
十一. 著作聲明

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