出版年:

研究生:

李芝嫻

研究生(英文姓名):

論文名稱:

Ha-ras致癌基因在NIH3T3細胞株中所誘導的細胞計劃性死亡

英文論文名稱:

Study On Ha-ras Induced Apoptosis In NIH3T3 Cell Lines

指導教授:

劉校生

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物及免疫學研究所

學年度:

85

語文別:

中文

論文頁數:

68

關鍵詞:

大腸桿菌 ; Ha-ras

英文關鍵詞:

Escherichia coli

被引用次數:

0

[ 摘要 ]

　　我們利用大腸桿菌（Escherichia, coli）之乳醣調節系統在NIH3T3細胞內建立了一系列帶有可調控之Ha-ras致癌基因之細胞株，其中之7-4細胞當培養於含0.2%牛血清之培養液中並加入誘導子Isopropyl-β-D-thiogalactoside(IPTG)誘導Ha-ras基因過量表達時，發現大部人細胞滯留於細胞週期之S期，並於72小時內死於細胞凋亡。觀察與細胞週期S及G2/M期有關的蛋白質p34cdc2激□之變化，發現其表現量隨培養基中血清量下降而降低，而Ha-ras表現量之上升更促使該激□進一步下降。顯示p34cdc2與7-4細胞週期異常有關。另外蛋白合成抑制劑cycloheximide可抑制7-4細胞的凋亡，顯示Ha-ras所誘導的細胞凋亡是需要蛋白質合成的。若將7-4細胞之培養液換成10%AC-2R（母牛初乳的粹取物，血清之替代品）之培養液，於Ha-ras過量表達下，細胞仍走向凋亡，排除了低血清下之營養不足是造成細胞凋亡因子之一。為了更清楚的證實Ha-ras主導細胞的凋亡，並探討其訊息傳遞之路徑，我們將顯性抑制型突變ras基因和顯性抑制型突變raf基因分別送入7-4細胞內，將ras 之訊息傳遞阻斷在不同的階段，並建立了許多細胞株。於顯性抑制突變ras細胞株Rasl-3中測得ras 活性下降，在軟洋菜膠上形成群落的能力下降，細胞生長速率變慢顯示三株顯性抑制突變ras細胞中ras 的訊息傳遞的確被阻斷，而細胞凋亡能力下降更顯示Ha-ras致癌基因在7-4細胞進行凋亡的過程扮演重要的角色。另外RafE, RafF, RafM等三株顯性抑制型raf細胞株中，raf之活性，軟洋菜膠形成群落的能力及生長速率均下降，顯示raf之訊息傳遞被阻斷，而RafF及RafM細胞株細胞凋亡的現象降低，更顯示此Ha-ras誘發的細胞凋亡是經由Raf之路徑而進行的。總言之本實驗證實Ha-ras的確可主導轉型細胞走向細胞凋亡，ras的確扮演相當主要之角色，而此細胞凋亡是透過raf路徑而進行的。

[ 英文摘要 ]

　　A transformed cell line designated 7-4 cell (derived from NIH3T3) containing an inducible Ha-ras and an E. coli lac I repressor genes has been established in our laboratory. While Ha-ras oncogene was overexpressed by the addition of isopropyl-β-D-thiogalactoside (IPTG) under serum depleted conditron, most of the 7-4 cells were accumulated at S-phase and died of apoptosis. The expression level of protein kinase p34cdc2, which was activated during cell progression from S to G2/M phase, was decreased by serum deprivation as well as by Ha-ras overexpression in 7-4 cells. Cycloheximide, an inhibitor of protein synthesis prevented Ha-ras induced apoptosis indicating de novo protein synthesis is required. The apoptosis of 7-4 cells could not be prevented by substituting 0.2% serum for 10%AC-2R (collistrum, a substitute of serum), indicating that lack of nutrient is not an important factor of 7-4 cell apoptosis. Analysis of dominant negative ras mutant cell lines Rasl-3 (derivatives of 7-4 cells) demonstrate decreased Ap-I activity, colony formation efficiency and cell growth rate, indicating that ras activity was blocked. Furthermore, apoptosis was prevented in these dominant negative ras cell lines, indicating that ras is essential for the apoptosis. Similar to the approach of dominant negative ras gene, three dominant negative raf cell lines RafE, F, and M were esgative ras gene, three dominant negative raf cell lines RafE, F, and M were established. The suppression of raf activity in dominant negative raf mutant cell lines was demonstrated by the falling of Elk activity, colony formation efficiency and cell growth rate. Moreover, apoptosis was prevented in two of the three mutants, indicating that raf pathway was involved. Taken together, we demonstrate the Ha-ras overexpression indeed could induce apoptosis under serum deprived condition, and this apoptosis was through raf pathway.