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系統識別號 U0026-0812200911330343
論文名稱(中文) 蟲草素對雄性小鼠萊氏細胞固醇類生成之影響
論文名稱(英文) The effect of cordycepin on steroidogenesis in normal mouse Leydig 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 93
學期 2
出版年 94
研究生(中文) 方宋齡
研究生(英文) Song-Ling Poon
電子信箱 songlp@yahoo.com
學號 T9692401
學位類別 碩士
語文別 英文
論文頁數 61頁
口試委員 指導教授-黃步敏
口試委員-楊西苑
口試委員-江美治
中文關鍵字 萊氏細胞  固醇類生成  蟲草素 
英文關鍵字 Leydig cell  steroidogenesis  cordycepin 
學科別分類
中文摘要 先前的研究指出,冬蟲夏草可以影響許多不同生理功能。其中一項功能是能夠增強生殖系統的功能。我們先前的探討也證明,冬蟲夏草具有刺激雄性小鼠萊氏細胞睪固酮的生成及萊氏腫瘤MA-10細胞株黃體酮的生成。然而,此作用是由冬蟲夏草中何種純物質所引發,至今仍然未有定論。蟲草素(3’ 去氧腺嘌呤),是一種腺嘌呤的類似物;亦是從冬蟲夏草菌絲體所萃取出來的純物質。研究指出,腺嘌呤及其拮抗物能夠影響男性生殖系統。在動物實驗中也發現,大鼠睪丸中具有腺嘌呤受器傳訊者核醣核酸的表現。因此,本篇論文的目的在探討蟲草素對小鼠萊氏細胞固醇類生成之影響及調節機制。結果顯示,隨著濃度及時間的增加,蟲草素具有刺激小鼠萊氏細胞睪固酮的生成。另外,我們也在小鼠的萊氏細胞上偵測到A1,A2a,A2b及A3這四種腺嘌呤受器傳訊者核醣核酸的表現。以具有專一性的腺嘌呤拮抗劑與蟲草素一起處理萊氏細胞,我們發現A1,A2a及A3的拮抗劑能個別的抑制由蟲草素所刺激的睪固酮生成。另外,在探討蟲草素對小鼠萊氏細胞固醇類生成之訊息傳導調節部份,我們發先,Rp-cAMPs,H89或是PKI這三種PKA抑制劑皆能個別抑制由蟲草素所誘導的睪固酮生成。MAPK的抑制劑,PD98059,及PKC的抑制劑,staurosporine,對蟲草素所誘導的睪固酮生成則無任何影響。若以cycloheximide這種蛋白質合成抑制劑與蟲草素一起處理萊氏細胞,睪固酮生成能夠有效的被抑制。西方點墨法及反轉錄酶聚合酶鏈聚反應的實驗結果顯示,蟲草素能有效的刺激固醇類速控蛋白(StAR)在蛋白質上及傳訊者核醣核酸的表現。而若以PKA抑制劑,H89,去處理萊氏細胞,蟲草素所刺激的固醇類速控蛋白的表現能夠被抑制。總結,蟲草素可藉由與腺嘌呤受器結合,活化PKA傳遞路徑進而調控固醇類速控蛋白的表現以刺激小鼠萊氏細胞睪固酮之生成。


英文摘要 Studies have shown the multiple pharmacological activities of Cordycep sinensis (CS), one of which is that it can enhance reproductive activity and restore impaired reproductive functions. We have previously demonstrated that CS alone could stimulate steroid production in both normal and tumor mouse Leydig cells. However, the precise substance in CS that plays the role in modulating the mechanism of steroidogenesis remains elusive. Cordycepin (3’deoxyadenosine), the analogue of adenosine, is a constituent isolated from the mycelia of CS. It has long been postulated that adenosine and its antagonists could influence the male reproductive system. Moreover, the mRNA expression of adenosine receptors has been detected in rat testis. Thus, the aim of this study is to investigate the effect and the regulatory mechanism of cordycepin on steroidogenesis in normal mouse Leydig cells. The results showed that cordycepin could stimulate testosterone production with dose- and time-dependent manners in normal mouse Leydig cells. The expression of A1, A2a, A2b and A3 mRNA was detected in normal mouse Leydig cells. By using specific receptor antagonist, cordycepin-stimulated testosterone production could be suppressed by A1, A2a and A3 adenosine receptor antagonists, respectively. PKA inhibitors, H89, PKI & Rp-cAMPs, could suppress the cordycepin-stimulated testosterone production. Both MAPK inhibitor, PD98059, and PKC inhibitor, staurosporine, could not affect the cordycepin-stimulated testosterone production. Cycloheximide, a protein synthesis inhibitor, inhibited the cordycepin-stimulated testosterone production. Also, cordycepin did induce the expression of Steroidogenic acute regulatory protein (StAR) protein and StAR mRNA in a dose-dependent manner. Lastly, using H89 to cotreat with cordycepin, there was a reduction in the cordycepin-stimulated StAR protein expression. Taken together, cordycepin might act through adenosine receptor and the PKA signaling pathway to induce the expression of StAR protein, and thus stimulate the testosterone production in normal mouse Leydig cells.


論文目次 Table of contents

Abstract in Chinese---------------------------------------------------------------------------------I
Abstract----------------------------------------------------------------------------------------------II
Acknowledgements---------------------------------------------------------------------------------IV
Table of Contents------------------------------------------------------------------------------------V
List of Table-----------------------------------------------------------------------------------------VII
List of Figures-------------------------------------------------------------------------------------VIII
Introduction------------------------------------------------------------------------------------------1
Materials and Methods-----------------------------------------------------------------------------7
Chemicals-----------------------------------------------------------------------------------------7
Animals-------------------------------------------------------------------------------------------8
Leydig cell isolation-----------------------------------------------------------------------------9
Cell culture---------------------------------------------------------------------------------------10
Radioimmunoassay (RIA)---------------------------------------------------------------------10
Immunoblot Analysis--------------------------------------------------------------------------11
Isolation of total RNA-------------------------------------------------------------------------12
Reverse transcription-polymerase chain reaction (RT-PCR)-----------------------------13
Statistical analysis------------------------------------------------------------------------------14
Results----------------------------------------------------------------------------------------------16
Cordycepin-induced steroidogenesis with dose-dependent manner in normal
mouse Leydig cells--------------------------------------------------------------------------16
Cordycepin-induced steroidogenesis with time-dependent manner in normal
mouse Leydig cells--------------------------------------------------------------------------16
Expression of specific adenosine receptor subtypes in normal mouse Leydig cells-- 16
Effects of adenosine receptor antagonists on cordycepin-induced
steroidogenesis in normal mouse Leydig cells-------------------------------------------17
Cordycepin-induced steroidogenesis requires the activation of cAMP-
dependent signal pathway in normal mouse Leydig cells------------------------------18
Cordycepin-induced steroidogenesis requires the activation of PKA
signaling pathway in normal Leydig cells----------------------------------------------18
Cordycepin-induced steroidogenesis is MAPK and PKC independent
in normal mouse Leydig cells------------------------------------------------------------19
Cordycepin-induced StAR protein expression in a dose-dependent
manner in normal mouse Leydig cells--------------------------------------------------19
Cordycepin induced StAR mRNA expression in a dose-dependent
manner in normal mouse Leydig cells--------------------------------------------------20
Cordycepin induced StAR protein expression requires the activation of
PKA signal transduction pathway in normal mouse Leydig cells-------------------20
Discussion------------------------------------------------------------------------------------------43
References------------------------------------------------------------------------------------------48
About the author-----------------------------------------------------------------------------------61


List of Table

Table 1 Sequence of primers used for reverse transcription-polymerase chain
reaction (RT-PCR) -------------------------------------------------------------------18


List of Figures

Fig. 1. Dose effect of cordycepin on testosterone production in normal mouse
Leydig cells. ---------------------------------------------------------------------------- 22

Fig. 2. Time course effects of cordycepin on testosterone production in normal
mouse Leydig cells.---------------------------------------------------------------------23

Fig. 3. Characterization of A1 adenosine receptor mRNA expression in normal
mouse Leydig cells.---------------------------------------------------------------------24

Fig. 4. Characterization of A2a adenosine receptor mRNA expression in normal
mouse Leydig cells.---------------------------------------------------------------------25

Fig. 5. Characterization of A2b adenosine receptor mRNA expression in normal
mouse Leydig cells.---------------------------------------------------------------------26

Fig. 6. Characterization of A3 adenosine receptor mRNA expression in normal
mouse Leydig cells.---------------------------------------------------------------------27

Fig. 7. Effects of DPCPX, A1-AR antagonist, on cordycepin-stimulated testosterone production in normal mouse Leydig cells.------------------------------------------28

Fig. 8. Effects of CSC, A2a-AR antagonist, on cordycepin-stimulated testosterone production in normal mouse Leydig cells.-----------------------------------------29

Fig. 9. Effects of MRS 1754, A2b-AR antagonist, on cordycepin-stimulated testosterone production in normal mouse Leydig cells.-----------------------------------------30

Fig. 10. Effects of MRS 1191, A3-AR antagnonist, on cordycepin-stimulated testosterone production in normal mouse Leydig cells.------------------------------------------31

Fig. 11. Effects of cordycepin on forskolin-stimulated testosterone production
in normal mouse Leydig cells.--------------------------------------------------------32

Fig. 12. Effects of cordycepin on dbcAMP-stimulated testosterone production
in normal mouse Leydig cells.-------------------------------------------------------33

Fig. 13. Effects of Rp-cAMPs on cordycepin-stimulated testosterone production
in normal mouse Leydig cells.-------------------------------------------------------34

Fig. 14. Effects of H89 on cordycepin-stimulated testosterone production
in normal mouse Leydig cells.-------------------------------------------------------35

Fig. 15. Effects of PKI on cordycepin-stimulated testosterone production
in normal mouse Leydig cells.-------------------------------------------------------36

Fig. 16. Effects of PD98059 on cordycepin-stimulated testosterone production
in normal mouse Leydig cells.--------------------------------------------------------37

Fig. 17. Effects of staurosporine on cordycepin-stimulated testosterone production
in normal mouse Leydig cells.-------------------------------------------------------38

Fig. 18. Effects of cycloheximide (CHX) on cordycepin-stimulated testosterone production in normal mouse Leydig cells.------------------------------------------------------ 39

Fig. 19. Effects of cordycepin on StAR protein expression in normal mouse
Leydig cells.----------------------------------------------------------------------------40

Fig. 20. Effects of cordycepin on StAR mRNA expression in normal mouse
Leydig cells.----------------------------------------------------------------------------41

Fig. 21. Effects of H89 on cordycepin-induced StAR protein expression in
normal mouse Leydig cells.------------------------------------------------------------42
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