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研究生:陳永佳
研究生(外文):Yung-Chia Chen
論文名稱:冬蟲夏草刺激小鼠萊氏腫瘤細胞固醇類生成機制之探討
論文名稱(外文):The Mechanism of Cordyceps sinensis on stimulating steroidogenesis in MA-10 Mouse Leydig Tumor Cells
指導教授:黃步敏
指導教授(外文):Bu-Miin Huang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:細胞生物及解剖學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:82
中文關鍵詞:冬蟲夏草固醇類生合成
外文關鍵詞:Cordyceps sinensissteroidogenesis
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  在先前的研究中我們發現,冬蟲夏草會刺激小鼠萊氏腫瘤MA-10細胞株產生黃體酮,目前為止相關的訊息傳導路徑仍所知有限。本論文將以冬蟲夏草處理MA-10細胞,並同時加入各種不同訊息傳導分子的活化劑或抑制劑,以確定冬蟲夏草刺激MA-10細胞的機制。首先,我們先檢測環腺嘌呤-蛋白激酶這條訊息傳遞路徑。實驗結果顯示,在冬蟲夏草刺激MA-10細胞產生黃體酮過程中可能會有至少一種G蛋白的參與;然而參與其中的G蛋白極有可能是Gsa而非Gia的次單位。至於腺甘酸環化酶及蛋白激酶A的部分,實驗結果顯示冬蟲夏草可能不會影響腺甘酸環化酶活性,但卻透過活化蛋白質激酶A的訊號傳導路徑以促進MA-10細胞產生黃體酮。此外,蛋白質的新合成對冬蟲夏草刺激MA-10細胞產生黃體酮的效用也是必要的,而且西方墨點法的實驗結果也顯示,在以冬蟲夏草處理過的MA-10細胞其類固醇速控蛋白 (StAR protein)會增加表現,而且在冬蟲夏草刺激MA-10細胞使其產生黃體酮過程中,粒腺體內外膜氫離子濃度梯度也是必須的。由上可知,冬蟲夏草會藉由蛋白激酶A路徑來刺激MA-10細胞固醇類生合成,然而其效果僅佔約百分之三十七,這暗示我們會有其他的訊號傳遞路徑也可能參與其中。實驗結果顯示,蛋白激酶C路徑約佔冬蟲夏草刺激MA-10細胞固醇類生合成的百分之五十二,而且磷脂酶C也參與在冬蟲夏草所刺激之效用中。令人驚訝的是,同時處理蛋白質激酶A及蛋白質激酶C抑制劑,冬蟲夏草刺激MA-10細胞的黃體酮生成會被抑制達百分之六十一;由此我們推論,冬蟲夏草刺激MA-10細胞的固醇類生合成會活化蛋白質激酶A及蛋白質激酶C之路徑。另一方面,鈣離子是另一個高度調控固醇類生合成的相關物質。而我們的結果顯示,鈣離子確實參與在冬蟲夏草刺激MA-10細胞固醇類生合成作用中,而且可能是透過T型而非L型鈣離子通道來作用。在固醇類生合成的酵素方面,冬蟲夏草會抑制細胞色素邊鏈切割酵素活性而不改變3 beta-羥基類固醇脫氫酶的活性,使其達到影響MA-10細胞固醇類生合成的效果。至於冬蟲夏草在刺激固醇類生合成作用中卻抑制細胞色素邊鏈切割酵素的活性,必須再被探討。總結來說,冬蟲夏草刺激MA-10細胞固醇類生合成作用中,蛋白質激酶A訊息傳導路徑、蛋白質激酶C訊息傳導路徑、鈣離子以及固醇類生合成中其速率決定步驟的類固醇速控蛋白都扮演著重要的角色。
  The stimulatory effects of crude CS on steroidogenesis in MA-10 mouse Leydig tumor cells have been demonstrated. However, the related signal transduction pathways are still unknown. MA-10 cells were treated with different reagents without or with CS (10 mg/ml) to determine the mechanisms. The cAMP-protein kinase A (PKA) signaling pathway was first examined. Results demonstrated that CS might activate at least one kind of G protein to affect production of progesterone in MA-10 cells, and Gsa subunit, but not Gia, might be involved in CS-stimulated steroidogenesis. With regards to adenylate cyclase and PKA, results indicated that adenylate cyclase might not be involved in CS-stimulated steroidogenesis, whereas PKA was activated by CS to stimulate MA-10 cell steroidogenesis. Moreover, de novo protein synthesis was required for CS-stimulated steroidogenesis. Besides, Western blot analysis illustrated that the expression of steroidogenic acute regulatory (StAR) protein increased in CS-treated cells. Result showed that the existence of mitochondria chemical gradient was also required for CS-stimulated steroidogenesis. PKA pathway involved almost 37% in CS-stimulated MA-10 cell steroidogenesis, which indicated that other pathways might also be involved. Thus, protein kinase C (PKC) pathway was examined. Results showed that activation of PKC was responsible for CS-stimulated steroidogenesis at about 52%, and phospholipase C was also required for CS-stimulated steroidogenesis. Therefore, both PKA and PKC signaling pathways were partially activated by CS in MA-10 cells, respectively. Interestingly, the stimulatory effect of CS was inhibited 61% with cotreatment of PKA and PKC inhibitors, which confirmed that the effect of CS might simultaneously activate PKA and PKC pathways to stimulate MA-10 cell steroidogenesis. The role of calcium, which profoundly influences steroidogenesis, was also investigated. Results indicated that calcium might be involved in the stimulatory effect of CS on MA-10 steroidogenesis, and T-type calcium channel, but not L-typed, was required for the event. In steroidogenic enzyme activities, CS might inhibit P450 side chain cleavage enzyme activity, but not 3 beta-hydroxysteroid dehydrogenase enzyme, to regulate MA-10 cell steroidogenesis. In conclusion, CS might activate both PKA and PKC signal transduction pathways with calcium ion involved to stimulate steroidogenesis in MA-10 cells, and StAR protein played an important role in this stimulation.
ACKNOWLEDGEMENTS i
INDEX TO FIGURES v

Abstract
  Chinese .viii
  English .x
INTRODUCTION 1
MATERIALS AND METHODS .9
  Chemicals 9
  Cell Culture and Experiments 10
  Radioimmunoassay (RIA) .11
  Protein Assay .12
  Immunoblot Analysis .13
  Statistics .13
RESULTS
  Effect of CS on progesterone production in MA-10 cells .14
  Effects of CS on cholera toxin-treated progesterone production in MA-10 cells 14
  Effects of CS on GDP-b-S-treated progesterone production in MA-10 cells 15
  Effects of CS on pertussis toxin-treated progesterone production in MA-10 cells 16
  Effects of CS on forskolin-induced progesterone production in MA-10 cells 17
  Effects of SQ 22536 on CS-stimulated progesterone production in MA-10 cells .17
  Effects of 2’,5’ dideoxyadenosine on CS-stimulated progesterone production in MA-10 cells .18
  Effects of CS on dbcAMP-induced progesterone production in MA-10 cells 18
  Effects of Rp-cAMP on CS-stimulated progesterone production in MA-10 cells 19
  Effects of H89 and KT 5720 on CS-stimulated progesterone production in MA-10 cells 19
  Effects of PKI on CS-stimulated progesterone production in MA-10 cells 20
  Effect of actinomycin D on CS-stimulated steroidogenesis in MA-10 cells 20
  Effect of cycloheximide on CS-stimulated steroidogenesis in MA-10 cells 20
  Effect of mCCCP on CS-stimulated steroidogenesis in MA-10 cells 21
  Effects of CS on StAR protein expression in MA-10 cells .21
  Effect of CS on the PMA-treated steroidogenesis in MA-10 cells .21
  Effect of CS on the OAG-treated steroidogenesis in MA-10 cells .22
  Effects of staurosporine on CS-stimulated steroidogenesis in MA-10 cells 22
  Effect of calphostin C on CS-stimulated steroidogenesis in MA-10 cells 23
  Effect of neomycin sulfate on CS-stimulated steroidogenesis in MA-10 cells 23
  Effect of CS on the A23187-treated steroidogenesis in MA-10 cells 24
  Effect of W7 on CS-stimulated steroidogenesis in MA-10 cells .24
  Effect of cadmium on CS-stimulated steroidogenesis in MA-10 cells 25
  Effect of nifedipine on CS-stimulated steroidogenesis in MA-10 cells 25
  Effect of flunarizine, dihydrochloride on CS-stimulated steroidogenesis in MA-10 cells 26
  Effect of cotreatment of PKI and calphostin C on CS-stimulated steroidogenesis in MA-10 cells 26
  Effect of CS on the 22R-hydroxycholesterol-treated steroidogenesis in MA-10 cells 27
  Effect of P450scc enzyme inhibitor, aminoglutethimide on CS-stimulated steroidogenesis in MA-10 cells .27
  Effect of CS on the pregnenolone-treated steroidogenesis in MA-10 cells 28
  Effects of 3b-HSD enzyme inhibitor, trilostane on CS-stimulated steroidogenesis in MA-10 cells 28

DISSCUSSION 60
REFERENCES .67
RESUME 82



Figure Page
The effect of CS on progesterone production in MA-10 cells 29
The effects of CS on cholera toxin-treated steroidogenesis in MA-10 cells .30
The effect of GDP-b-S on CS-stimulated steroidogenesis in MA-10 cells .31
The effects of pertussis toxin (PTX) on CS-stimulated steroidogenesis in MA-10 cells 32
The effects of CS on forskolin-induced steroidogenesis in MA-10 cells .33
The effects of SQ 22536 on CS-stimulated steroidogenesis in MA-10 cells .34
The effects of 2’,5’ dideoxyadenosine on CS-stimulated steroidogenesis in MA-10 cells 35
The effects of CS on dbcAMP-induced steroidogenesis in MA-10 cells .36
The effect of Rp-cAMPS on CS-stimulated steroidogenesis in MA-10 cells 37
The effect of H89 on CS-stimulated steroidogenesis in MA-10 cells .38
The effect of KT 5720 on CS-stimulated steroidogenesis in MA-10 cells 39
The effect of PKI on CS-stimulated steroidogenesis in MA-10 cells 40
The effect of actinomycin D on CS-stimulated steroidogenesis in MA-10 cells 41
The effect of cycloheximide on CS-stimulated steroidogenesis in MA-10 cells .42
The effect of mCCCP on CS-stimulated steroidogenesis in MA-10 cells 43
The effects of CS on StAR protein expression in MA-10 cells .44
The effect of CS on PMA-treated progesterone production in MA-10 cells .45
The effects of CS on 1-Oleoyl-2-acetyl-sn-glycerol- treated progesterone production in MA-10 cells .46
The effects of staurosporine on CS-stimulated steroidogenesis in MA-10 cells 47
The effect of calphostin C on CS-stimulated steroidogenesis in MA-10 cells 48
The effects of neomycin sulfate on CS-stimulated steroidogenesis in MA-10 cells 49
The effects of CS on A23187-treated steroidogenesis in MA-10 cells 50
The effect of W7 on CS-stimulated steroidogenesis in MA-10 cells .51
The effects of cadmium on CS-stimulated steroidogenesis in MA-10 cells 52
The effects of nifedipine on CS-stimulated steroidogenesis in MA-10 cells 53
The effects of flunarizine dihydrochloride on CS-stimulated steroidogenesis in MA-10 cells 54
The effect of cotreatment of PKI and calphostin C on CS-stimulated steroidogenesis in MA-10 cells 55
The effect of CS on 22R-hydroxycholesterol- treated steroidogenesis in MA-10 cells 56
The effect of aminoglutethimide on CS-stimulated steroidogenesis in MA-10 cells 57
The effect of CS on pregnenolone-treated steroidogenesis in MA-10 cells 58
The effect of trilostane on CS-stimulated steroidogenesis in MA-10 cells 59
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