(18.204.227.34) 您好!臺灣時間:2021/05/17 06:36
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:魏荷君
研究生(外文):Wei, Ho-Chun
論文名稱:乙型腫瘤生長因子在人類肝癌細胞中訊息傳遞之研究
論文名稱(外文):Studies on the signal transduction pathway of TGF-β in human hepatoma cells
指導教授:周成功
指導教授(外文):Chou, Chen-Kung
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物暨免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1994
畢業學年度:82
語文別:中文
中文關鍵詞:微生物學免疫學乙型腫瘤肝癌細胞
外文關鍵詞:MICROBIOLOGYIMMUNOLOGY
相關次數:
  • 被引用被引用:0
  • 點閱點閱:96
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
乙型腫瘤生長因子(TGF-β)是一種具有多重功能的多胜太。它廣泛地分佈於各種動物細胞中,並且具有重要的調節功能。在我們先前的實驗中發現,以低濃度TGF-β處理經血清匱乏培養之人類肝癌細胞株Hep 3B,即可使之死亡。為了解TGF-β是透過何種機制來造成細胞死亡,以及TGF-β如何將訊息傳入細胞而造成細胞的死亡反應,我們主要從兩方面來進行研究。
首先,TGF-β會造成肝癌細胞的死亡,然而胰島素卻會促進肝癌細胞的分裂與生長。當細胞同時處理胰島素及TGF-β時,我們可以觀察到細胞死亡的現象完全被抑制,換句話說,胰島素刺激生長的訊息強過TGF-β的死亡訊息。
過去已知胰島素刺激細胞生長的作用乃是透過蛋白質磷酸激酉每C(PKC)及ras蛋白質來傳遞訊息,因此我們欲知直接活化上述兩種蛋白質之活性是否可以抑制TGF-β的作用。將Hep 3B細胞同時處理PKC活化劑(TPA)及TGF-β俊發現:細胞死亡現象大為降低。假如細胞先行以TPA處理 24小時以上,廢除PKC的功能後,再處理TGF-β與TPA時,細胞仍然大量死亡。顯示了TPA是透過PKc的活化來抑制TGF-β的致死作用。接著,藉由傳染技術將帶v-H-ras基因的表現質體送入Hep 3B細胞,選殖出可大量表現v-H-ras蛋白質的細胞株,測試這些細胞株對TGF-β的反應後發現,細胞死亡的現象亦大為下降,顯示了ras蛋白質之活性在對抗TGF-β的死亡訊息上,亦扮演重要角色。
許多生長因子會藉由調節致癌基因(oncogene)的表現進而形容細胞的生長與分化。藉由北方點墨分析我們觀察到胰島素及TGF-β均會刺激c-jun的表現,然而對於 c-myc基因而言,胰島素有刺激作用,而TGF-β卻是抑制作用,且結果顯示TGF-β會拮抗胰島素的刺激作用。這意味著TGF-β與胰島素在控制細胞生死及基因表現上乃循不同之途徑。
其次,最近對於TGF-β受體 (receptor)的發現及功能的解析,提供了了解TGF-β如何傳遞其訊息的模式。因此,我們想了解第二型的受體在TGF-β致死作用中,訊息傅導過程所扮演之角色。將Hep 3B細胞迷入不帶打細胞內結構的第二型受體,使之大量表現,以多取勝而使細胞內完整的第二型受體無法作用。我們選殖出表現多量及中等量的穩定細胞株(stable clones),繼之以TGF-β處理後發現,當不完整受性表現越多時,細胞死亡越少。表示第二型受體確實是TGF-β傳遞死亡訊息所必需。另外,我們發現這種大量表現不完整受體的穩定細胞株,其TGF-β調控c-myc基因表現的程度大受影響,亦即當TGF-β處理細胞後,抑制c-myc基因表現的程度大為降低,表示TGF-β對於c-myc之調控也須經由第二型受體來傳達訊息。
在本論文裡,我們証明了TGF-β對肝癌細胞的致死作用及c-myc之調控須透過第二型受體。而胰島素抑制TGF-β致死作用則可能是透過PKC及ras蛋白質的作用途徑未完成。
Transforming growth factor β (TGF-β) is a multifunctional protein that controls an array of functions in animal cells from virtually every lineage. In our previous studies, we have shown that human hepatoma cell line (Hep 3B) undergoes programmed cell death when serum starved Hep 3B cells were treated with very low concentration of TGF-β1. The mechanism by which TGF-β initiates the cell death program is not known.
To dissolve this problem, we adopted two approaches. First, I treated Hep 3B cells with TGF-β plus insulin which stimulated cell proliferation of Hep 3B cells and found that insulin completely block the cell death induced by TGF-β. In other words, the stimulating signal of insulin is dominant to the apoptotic signal of TGF-β in human hepatoma cells. In previous studies, we knew that insulin could stimulate cell proliferation through protein kinase C( PKC) and ras protein. Therefore, we tested whether insulin blocks the apoptotic effect of TGF-β which goes through the action of PKC and/or ras protein. When the cells were simultaneously treated with TPA (150 ng/ml) and TGF-β (3 ng/ml), Hep 3B cells became resistant to die, but when the activity of PKC was down-regulated by TPA (150 ng/ml) long-term pretreatment, TPA no longer protected Hep 3B cells from TGF-β-induced apoptosis. These results indicate that activation of PKC do block TGF-β-induced cell death. Additionally, I transfected Hep 3B cells with v-H-ras gene. Several clones which express high level of v-H-ras protein exhibit strong resistance to TGF-β. This result suggests the activity of ras protein also antagonizes TGF-β-induced cell death.
Most of the growth factors control cell proliferation through regulating the expression of oncogenes. I tested the proto-oncogene expression regulated by insulin and TGF-β. By Northern blot analysis, we found that insulin could stimulate c-myc and c-jun expression, and TGF-β could enhance c-jun expression but suppress c-myc expression. Surprisingly, TGF-β antagonized the stimulating effect of insulin on c-myc expression. These results suggest that TGF-β may regulate gene expression and induce cell death through two distinct pathways.
Recently, molecular cloning of TGF-β receptors provides a molecular basis to address how TGF-β transduce its signals. The expression of three types of TGF-β receptors had been identified by cross-linking experiment in Hep 3B cells. In order to determine the specific roles of type II receptor in the signaling pathway, the negative dominant truncated TGF-β type II receptor was transfected into Hep 3B cells. Then, we evaluated the responses of the transfectants toward TGF-β. The resistant potential to TGF-β of these transfectants depends on the expression level of the truncated receptor. The results indicate that cytoplasmic domain of type II receptor is essential for TGF-β-induced apoptosis. Additionally, we found that the regulatory ability of TGF-β on the expression of c- myc could be partially abolished in the clones which overexpress the truncated receptors. The result indicates that the intracellular domain of TGF-β type II receptor somehow could participate in the signaling pathway.
In my thesis, we demonstrated that the intracellular domain of TGF-β type II receptor is essential for apoptotic action of TGF-β in Hep 3B cells, but TGF-β regulates gene expression by different signaling pathways. Besides this, insulin protects TGF-β-induced apoptosis might be through the actions of PKC and/or ras protein.



QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊