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系統識別號 U0026-0812200911043807
論文名稱(中文) 黃金銀耳對小鼠萊氏腫瘤細胞固醇類生成作用機制之探討
論文名稱(英文) The Effects of Tremella aurantia on Steroidogenesis in MA-10 Mouse Leydig Tumor Cells
校院名稱 成功大學
系所名稱(中) 細胞生物及解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 92
學期 2
出版年 93
研究生(中文) 陳彥文
研究生(英文) Yan-Wen Chen
電子信箱 b8516002@pchome.com.tw
學號 t9690104
學位類別 碩士
語文別 中文
論文頁數 61頁
口試委員 召集委員-楊西苑
口試委員-劉明毅
口試委員-簡基憲
指導教授-黃步敏
中文關鍵字 固醇類生成作用  黃金銀耳  小鼠萊氏腫瘤細胞 
英文關鍵字 MA-10 Mouse Leydig Tumor Cells  Steroidogenesis  Tremella aurantia 
學科別分類
中文摘要 黃金銀耳,是一種金黃色膠狀樣的蘑菇,長久以來在中國傳統社會中被用來當作食品及天然的藥品來改善許多的症狀。最近的研究發現黃金銀耳子實體的萃取物對於患有糖尿病的老鼠具有明顯降血糖的功效,而糖尿病在臨床上常會面臨到性功能的障礙。在先前的實驗中,我們發現黃金銀耳對於正常的老鼠會降低血清中睪固酮,且對糖尿病的老鼠沒有正向的作用。藉此引申出的問題是黃金銀耳是否會直接調控萊氏細胞固醇類生合成,並經由何種傳導路徑來作用。在本論文中將小鼠萊氏腫瘤MA-10細胞株處理人類胎盤絨毛膜激素 (hCG),以黃金銀耳及各種不同製劑來闡明作用的機制,其中也包括固醇類速控蛋白(StAR protein)及固醇類生成酵素。實驗結果顯示,在三小時處理後,10 mg/ml的黃金銀耳會抑制MA-10細胞由人類胎盤絨毛膜激素刺激下生成的黃體酮 (progesterone)。黃金銀耳也會明顯抑制分別由錦紫蘇 (forskolin, 10及100 uM)及二丁酰環腺嘌呤單核苷酸(dbcAMP, 0.5 及1 mM)所誘發的黃體酮生成量。此外,黃金銀耳亦會顯著地抑制由22R-羥基膽固醇處理下的黃體酮產量及部份地減少由孕烯酮處理下的黃體酮生成。此結果顯示黃金銀耳可能抑制P450支鏈裂解酵素及3 beta-羥基固醇類脫氫酵素的活性。然而,在有或沒有人類胎盤絨毛膜激素處理的實驗中,黃金銀耳可以活化固醇類速控蛋白的表現,但對於P450支鏈裂解酵素及3 beta-羥基固醇類脫氫酵素的蛋白質表現則沒有顯著的影嚮。此外,移除了含黃金銀耳培養液後再繼續用人類胎盤絨毛膜激素來處理,結果顯示黃體酮的產量可以在四小時後復原。另外,MTT測試法顯示黃金銀耳對於MA-10細胞不具有毒殺作用。在H-89(蛋白激酶A的抑制劑,50 uM), calphostin-c (蛋白激酶C的抑制劑,50 uM),OAG (蛋白激酶C的活化劑,10 及100 uM),W7 (攜鈣素的拮抗劑,10 uM) 的處理下,黃金銀耳對 MA-10細胞黃體酮生成也沒有任何影嚮。總結來說,黃金銀耳在人類胎盤絨毛膜激素處理下可能主要是透過了蛋白激酶A 的訊息傳導路徑來抑制固醇類生成酵素的活性及固醇類生合成。
英文摘要 Tremella aurantia (TA), a yellow jelly mushroom, has been traditionally used as food and crude medicine to improve several kinds of symptoms in Chinese society for a long time. Recent studies have illustrated that the fractions of fruiting bodies of TA exhibit a significant hypoglycemic activity in diabetic mouse models, which usually suffer from the sexual dysfunction. In previous study, we showed that TA reduced plasma testosterone production in normal rats without any positive effect in diabetic rats. It evolved a question that if TA directly regulated Leydig cell steroidogenesis and by which signaling pathway. In this study, MA-10 Leydig tumor cells were treated with vehicle, human chorionic gonadotropin (hCG, 50 ng/ml), or different reagents without or with TA to clarify the effects. Steroidogenic enzyme activities and steroidogenic acute regulatory (StAR) protein expression, which are essential for steroidogenesis, were also determined. The results showed that 10 mg/ml of TA suppressed hCG-treated progesterone production at 3 hr in MA-10 cells. TA significantly suppressed progesterone production with the presences of forskolin (10 and 100 uM) or dbcAMP (0.5 and 1 mM), respectively. In addition, TA obviously decreased progesterone production with the treatment of 22R-hydroxylcholesterol (50 uM), and partially decreased progesterone production with the presence of pregnenolone (50 uM), which indicated that TA might inhibit P450 side-chain cleavage (P450scc) and/or 3b-hydroxysteroid dehydrogenase (3b-HSD) enzyme activities. However, TA could activate the expression of StAR protein without or with hCG treatments, and there were no influences on the expression of P450scc and 3b-HSD. MTT assay indicated that TA had no any effect on cell proliferation. In addition, removal of TA and then the addition of hCG (2 and 4 hr), progesterone levels were restored in 4 hr. Furthermore, TA had no effect with the treatment of H89 (PKA inhibitor, 50 uM), calphostin-c (PKC inhibitor, 500 nM), OAG (PKC activator, 10, 100 uM), W7 (calmodulin antagonist, 10 uM), and cardiolipin (P450scc activatior, 1, 10, 100 mg/ml). Taken together, these data suggest that TA suppressed hCG-treated steroidogenesis in MA-10 cells by inhibiting PKA signal pathway and steroidogenic enzyme activities.
論文目次 Table of Contents
Page
Acknowlegements…………………………………………………………...……….ii

Index to Figures…………………………………………………………………..…vi

Abstract …………………………………………………………….…………..……1
中文摘要 …………………………………………………………………….....……3

Introduction ………………………………..……….………………………....…….5
Tremella aurantia ……………………………….………………………...….…….5
Diabetes mellitus (DM)…………………….…………………………….…...……6
Steroidogenesis …………………………………………….………….……...……7
Steroidogenic acute regulatory (StAR) protein ……………….…….………..……8
Protein kinase A (PKA) ……………………………………….…….………....…..9
Protein kinase C (PKC) …………………………………………………………..10
Calcium and steroidogenesis ……………………………………………...….…..10
Aim of this study ………………………………………………………...……….11

Materials and Methods……………………………….….………………...………12
Chemicals ................................................................................……………....…...12
Cell culture ………………………………..………………………………...……13
Experiment design …………………..………………………………….…..…….14
Radioimmunoassay (RIA) ………………...………………………….………..…14
Protein assay …………………………………………………….…………....…..15
Immunoblot analysis ……………………..…….………………………………....16
MTT …………………………………………….………..………………...……..16
Statistics ……………………………………….………….………………...…….17

Results ………………………………………………………....…………….…….18
Dose effects of TA on progesterone production in MA-10 cells………..….……..18
Time course effects of TA on progesterone production in MA-10 cells…………..18
Effects of TA on forskolin-treated progesterone production in MA-10 cells …….19
Effects of TA on dbcAMP-treated progesterone production in MA-10 cells…......19
The expression of StAR protein with TA treatment in MA-10 cells……………...20
The expression of P450 side-chain cleavage (P450scc) enzymes
with TA treatment in MA-10 cells ………………………………...…….…..…21
The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) enzymes
with TA treatment in MA-10 cells ……………………………………………..21
Effects of TA on 22R-hydroxycholesterol-treated steroidogenesis
in MA-10 cells ……………………………...………………………………….21
Effects of TA on pregnenolone-treated steroidogenesis
in MA-10 cells ……………………………...………………..…………….…..22
Toxicity examination of TA on MA-10 cells………………………...……………22
Recovery of progesterone production after TA treatment …….…………….……23
Effects of TA on cardiolipin-treated steroidogenesis
in MA-10 cells………………………………………………………..………..23
Effects of TA on H89-treated steroidogenesis in MA-10 cells ………………..…24
Effects of TA on W7-treated steroidogenesis in MA-10 cells ………………...….24
Effects of TA on OAG-treated steroidogenesis in MA-10 cells ……………..…..25
Effects of TA on calphostin-c-treated steroidogenesis in MA-10 cells .....…….....25


Discussion …………………………………………………….……………...……..43

References………………………………………………………………...…………51

Appendix ……………………………………………………………………………60

Resume ………………………………………………………………..……………61
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