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系統識別號 U0026-0812200911542752
論文名稱(中文) 蟲草素對MA-10小鼠萊氏腫瘤細胞之細胞凋亡的研究
論文名稱(英文) The apoptotic effect of cordycepin on MA-10 mouse Leydig tumor cell line
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 94
學期 2
出版年 95
研究生(中文) 林純玉
研究生(英文) Chun-Yu Lin
電子信箱 amanda661003@hotmail.com
學號 t9693107
學位類別 碩士
語文別 英文
論文頁數 61頁
口試委員 口試委員-劉明毅
口試委員-江美治
指導教授-黃歩敏
中文關鍵字 硫胱氨酸蛋白脢  細胞凋亡  蟲草素  腺嘌呤接受器 
英文關鍵字 adenosine receptors  MA-10 cells  caspase  cordycepin  apoptosis 
學科別分類
中文摘要 在先前的研究中就有學者指出,冬蟲夏草對於人體的生理功能具有多方面的療效。包括促進免疫系統的免疫能力,降低血壓,增加肝臟代謝能力,抗氧化,降低低密度脂蛋白的合成,增加生殖系統功能以及抗癌效果。研究冬蟲夏草的成分發現其中的蟲草素(3'去氧腺嘌呤)是切確可以增加能量代謝具有生物活性的物質。蟲草素最早是萃取自冬蟲夏草的菌絲體,它是一種腺嘌呤的類似物;在我們實驗室之前的研究中證實蟲草素可以藉由調控腺嘌呤接受器的表現進而進一步刺激小鼠萊氏細胞製造睪固酮。然而當我們欲利用MA-10這株小鼠萊氏腫瘤細胞株來對蟲草素刺激賀爾蒙生成做更進一步的探討時,卻發現蟲草素非但無法刺激MA-10細胞賀爾蒙生成反而對MA-10細胞造成毒殺作用,因此引發了我們對於蟲草素在MA-10細胞株的毒殺作用探討的興趣。在實驗室之前的實驗中也曾發現冬蟲夏草的萃取物可以毒殺MA-10細胞,然而在其他的報導中也曾經指出蟲草素可以抑制癌細胞的生長。但是對於蟲草素是否對腫瘤細胞有造成細胞凋亡的效果,這方面的相關研究還很少。在我們的實驗中的確發現蟲草素不但可以使MA-10細胞死亡,而且是以細胞凋亡的型態使MA-10細胞死亡。此外,MA-10細胞不但具有四種不同的腺嘌呤接受器,而且在蟲草素作用下更可以改變腺嘌呤接受器傳訊者核醣核酸的表現。當我們利用腺嘌呤接受器拮抗劑去分別抑制四種腺嘌呤接受器的表現時,發現可以大量的回復蟲草素帶來的死亡效果,也就是細胞的死亡被抑制了。從這樣的結果可以得知蟲草素可以經由作用在腺嘌呤接受器後再進一步的去引發MA-10細胞的死亡。然而在早期眾多對於研究細胞凋亡機制的報導中就指出了硫胱氨酸蛋白脢參與在細胞凋亡的機制中。因此,我們也進一步去探討會有哪些硫胱氨酸蛋白脢被活化。結果顯示出蟲草素可以誘發硫胱氨酸蛋白脢-9,-3以及-7的活化,但無法去活化硫胱氨酸蛋白脢-8。綜合本篇實驗結果,我們推測蟲草素可以經由與腺嘌呤接受器作用後再去引發硫胱氨酸蛋白脢-9,-3以及-7的活化,最後使MA-10小鼠萊氏腫瘤細胞走向細胞凋亡的路徑。



英文摘要 Cordyceps sinensis (CS), a fungal parasite on the larvae of Lepidoptera, has been used as herbal tonic in traditional Chinese medicine for a long time. It is well known that CS has multiple pharmacological activities, including tonic supplement for sexual dysfunction, stimulating on Leydig cells and granulose-lutein cells to produce sex steroid, tumor growth inhibitor in human- and monkey-derived tumor cells, and an enhancer in immune system. On the other hand, CS extracts can promote apoptosis of human mesangial cells or U937 leukemia cells. Indeed, our lab has also found that CS induced MA-10 cell apoptosis by activating caspase-8 pathway and suppressing the NF-KB pathway. Cordycepin (3’deoxyadenosine), the analogue of nucleoside, is a constituent isolated from the mycelia of CS. It has been suggested that cordycepin can improve lung function, and increase energy levels and sex drive. Besides, cordycepin also exerts an anti-metastatic action by inhibiting the invasiveness of mouse melanoma cells, and exerts polyadenylate polymerase (PAP) modulations in human epithelial cancer cell lines. Recent study shows that cordycepin could inhibit growth of melanoma cells and lung carcinoma cells via stimulating adenosine A3 receptor. In this study, we wanted to investigate the effect of cordycepin on cell death in MA-10 mouse Leydig tumor cell line. Firstly, in methylthiazolecterazolium (MTT) assay, cordycepin decreased MA-10 cell viability in a dose-dependent manner (10 µM-1 mM). We also identified apoptosis by DNA ladder assay and flow cytometry analysis. Secondly, in Western blotting analysis, protein expressions of caspase-9, caspase-3, and caspase-7 could be induced under cordycepin (100 µM and 1 mM) treatments for 6-36 hr. Furthermore, the expressions of adenosine receptor (AR) subtype mRNA in MA-10 cells could also be detected with cordycepin treatment by RT-PCR. In addition, selective adenosine receptor subtype antagonists could significantly rescue 100 μM cordycepin-induced apoptosis in MA-10 cells. In conclusion, our results suggest that cordycepin might act via the activation of adenosine receptors and then activate caspase-9, -3, and -7 expressions to cause DNA fragmentation and apoptosis in MA-10 mouse Leydig tumor cell line.



論文目次 Abstract in Chinese-----------------------------------I
Abstract----------------------------------------------II
Acknowlegements---------------------------------------IV
Table of Contents--------------------------------------V
List of Table-----------------------------------------VII
List of Figures---------------------------------------VIII
Introduction-------------------------------------------1
Materials and Methods----------------------------------7
Chemicals--------------------------------------------7
Cell culture-----------------------------------------7
MTT cytotoxicity assay-------------------------------8
DNA fragmentation assay--------------------------------8
Flow cytometry analysis--------------------------------9
Immunobloting analysis---------------------------------9
Reverse transcription-polymerase chain reaction (RT-PCR) --------------------------------10
Statistical analysis-------------------------11
Results-------------------------------13
The effect of cordycepin on morphological change in MA-10 cells--------------------13
The death effect of cordycepin on MA-10 cells-----------------------------------------13
The death effect of cordycepin on MA-10 cells is the result of apoptosis-------------14
Adenosine also induced cell death in MA-10 mouse Leydig tumor cells---------------15
RT-PCR analysis for detection of A1, A2a, A2b and A3 adenosine receptor mRNA transcripts in MA-10 cell--------------------------------------15
The mRNA expression of adenosine receptor subtypes in different time scales under cordycepin treatment-------------------------------16
The effect of adenosine receptor antagonists on morphological change in cordycepin- treated MA-10 cells-------------------------------------------16
The effects of adenosine receptor subtype antagonists on cordycepin-induced cell death--------------------------------------------------------16
Effect of cordycepin on caspase proteins expression in MA-10 cells---------------------17
Discussion-------------------------------------------------------------------------------19
References-------------------------------------------------------------------------------52
About the author-------------------------------------------------------------------------61
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