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研究生:石志榮
研究生(外文):Shyr, Chih-Rong
論文名稱:乙醯膽鹼對甲狀腺釋放激素促進泌乳激素基因表現增強作用的研究
論文名稱(外文):Studies of the potentiation effect of Acetylcholine on the TRH-stimulated prolactin gene expression
指導教授:陳芬芳陳芬芳引用關係
指導教授(外文):Wang, Fung-Fang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:1993
畢業學年度:81
語文別:中文
中文關鍵詞:甲狀腺乙醯膽鹼
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先前實驗顯示,GH3腦下腺垂體癌細胞株,經Acetyl-choline(ACh)24小時前處理後,Thyrotropin-releasingHormene(TRH)引發prolactin基因表現的作用會受到增強。本論文進一步探討ACh增強作用的可能機轉。TRH可經由促進PI turnover釋放鈣離子,活化PKC。以鈣離子的離子泳動劑Ionomycin(1μm)使胞內鈣離子濃度上升,或以Diaclglyc-erol(DG)的analog phorbol myristate acetate(pMA)(20nm)活化PKC發現ACh前處理,均無法增強ionomycin或是PMA對prolactin基因的刺激作用。顯示單純的鈣離子釋放或PKC活化都無法模擬ACh的增強作用。進一步以TMB-8或H7抑制細胞內鈣離子的釋放或是PKC的活性,結果發現上述抑制劑加入的時間不論是在ACh處理前或是TRH處理前,都無法去除增強作用,排除了鈣離子釋放或是PKC活化牽涉在增強作用內。直接觀察細胞內鈣離子的流動,顯示ACh前處理並不影響TRH對胞內鈣離子的刺激程度。然而,鈣離子通道的激動劑Bay-k8644對prolactin mRNA表現的刺激作用卻可以受ACh的增加,而且vasoactive intestinal peptide(VIP)或者是cAMP的analog 8-b-cAMP其刺激作用皆會被ACh的增強。
TRH或是VIP刺激c-fos表現亦被ACh所增強,由於FOS為一transcription factor是否c-fos的表現與ACh作用有關,尚待進一步實驗証明。Transfection的研究,顯示TRH刺激prolactin promoter所帶動之CAT reporter的表現,而且此作用可被ACh所增強。而在 VIP存在下2.5kb PRL-CAT,2xCRE-CAT活性都上升,但ACh的增強作用只發生在2xCRE-CAT上。這些結果指出ACh的增強作用與鈣離子從細胞外內流所激發的訊息,或是cAMP傳遞的路徑有關。
We have previously shown that when GH3 rat pituitary tumor cells were pretreated with acetylcholine (ACh) for 24h, the thyrotropin releasing hormone (TRH) stimulated-prolactin (PRL) gene expression was potentiated. In the present study, we examined the mechanisms underlying this potentiation effect.
Upon binding to its receptors, TRH stimulates phosphoinositide turnover which, in turn, elevates intracellular calcium levels and activates protein kinase C (PKC). Utilysing calcium ionophore, ionomycin, or PKC activator, phorbol myristic acetate (PMA), we have shown that these agents alone have little stimulatory effect on PRL mRNA expression, and their effects were not potentiated by ACh. In the presence of TMB-8 to inhibit the intracellular calcium mobilization, the augmentation on TRH-stimulated PRL mRNA expression by ACh persisted; moreover, PKC inhibitor H7 was unable to inhibit the potentiation.
Direct measurement of intracellular calcium concentrations by Fura-2 indicated that the TRH stimulated-[Ca+2]i was not affected by ACh. On the other hand, Bay K8644, the L-type calcium channel agonist, increased PRL mRNA expression; this effect was shown to be potentiated by ACh. Both vasoactive intestinal peptide (VIP) and 8-Br- cAMP stimulated-PRL gene expression were potentiated in the ACh conditioned cells.
TRH or VIP induced a rapid and transient increase in c-fos mRNA expression, and this effect was potentiated by ACh. Whether c-fos plays a role in PRL gene expression should await further investigation.
The possibility that the augmentation by ACH was exerted at the transcriptional level was examined with fusion gene constructs composed of chloramphenicolacetyl transferase (CAT) reporter gene driven by PRL promoter (2.5PRL-CAT) or 2 copies of cAMP response element (2xCRE/tk-CAT). Transfection of either 2.5PRL-CAT or 2xCRE/tk-CAT into GH3 cells show that TRH stimulated-CAT activity in both gene constructs was increased by ACh pretreatment. On the other hand, the VIP-stimulated CAT activity was potentiated only with the 2xCRE/tk-CAT transfected cells.
Our results suggest that although the TRH-stimulated intracellular calcium levels was not affected by ACh, the signalling pathway downstream of calcium appears to be responsible for the potentiation effect that eventually leads to accelerated PRL gene transcription in the ACh conditioned GH3 cells.



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