臺灣博碩士論文加值系統

造血素Erythropoietin(EPO)，可在低氧情況下(例如：CoCl2的加入)，於腎臟及胎兒肝臟中產生。並且，在人類肝癌細胞株Hep G2與Hep 3B中，其EPO蛋白質及mRNA 可被誘發。而報告指出，細胞中氧氣偵測系統會受低氧或金屬鈷及H2O2的作用，而影響EPO的表現。但有趣的是，在Hep G2與Hep 3B細胞中，可以表現EPO基因，但在J5及SK-Hep-I細胞中卻缺乏EPO基因表現的能力。所以，不同分化程度的肝癌細胞，其分化程度在調控EPO基因表現上，是否扮演一個重要的角色？另一方面，可調控內生性麩胱甘肽(glutathione, GSH)合成的酵素(g-glutamylcysteine synthetase, g-GCS)是否能調節EPO基因表現也是一個重要而有趣的問題。
在此實驗中，由有幾個方向證明了內生性GSH在控制及調控EPO基因表現方面扮演重要角色。第一、使用分化程度不同的五株肝癌細胞株當作實驗模式，其內生性GSH含量也不同，依序為Hep G2 >Hep 3B >J5 >Mahlavu >SK-Hep-I細胞，並且我們也發現g-GSH重鏈單位的活性也與此有關。而這些肝癌細胞株中，只有分化程度好的Hep G2及Hep 3B有EPO基因表現，意味著其表現可能依賴於GSH，並且其EPO基因表現可能需跨過一個最低的門檻才能達到最好的表現。第二、進一步證實GSH的角色，我們加入非致死濃度的N-acetylcysteine來增加GSH的產生。我們發現，此舉可誘發EPO基因在J5及SK-Hep-I細胞中表現。後來我們更選取GCS30細胞加入比較，GCS30細胞是由γ-GCSh cDNA 永久轉染入SK-Hep-I細胞而來的，並且也已經被證實，轉染此DNA序列的GCS30細胞株中，GSH含量高過SK-Hep-I細胞；因為NAC屬外源性增加GSH，所以我們利用GCS30細胞內源性增加GSH的特點，單獨比較其與SK-Hep-I細胞之間的EPO表現量是否不同，並且於RT-PCR及EPO蛋白質表現的實驗中證實GCS30細胞株其EPO表現量比SK-Hep-I細胞為高。
綜合上述結果，我們首先證明了，除了厭氧及氯化鈷之外，內生性GSH含量在EPO基因表現方面扮演正面調控的角色，而GSH活性如何來調控EPO基因表現的機制，則尚待進一步的實驗來加以釐清。

Erythropoietin (EPO) is produced in the kidney and in fetal liver in response to hypoxia as well as to CoCl2. The EPO protein and mRNA can be induced in response to both stimuli in the human hepatoma cell (HCC) lines Hep 3B and Hep G2. An oxygen sensing mechanism in which a ligand dependent conformational change in the heme protein produces H2O2 in respone to either hypoxia or Cobalt has been demonstrated. However, an intriguing question can be raised as to why some HCC sublines, such as Hep G2 and Hep 3B are capable of expressing EPO gene, whereas in other HCC sublines, such as J5 and SK-Hep-I are completely devoid of the ability to express EPO gene. Along this line, does “differentiation status” of these HCC cells play a pivotal role in regulating the expression of EPO gene? Next in line, how a differentiation-associated upregulation of g-glutemylcysteine synthetase (g-GCS), which tightly regulating the biosynthesis of endogenous glutathione(GSH) can modulate the expression of EPO. The objective of this research project was designed to address all these questions. Reported herein are several lines of evidence to demonstrate that endogenous GSH contents do play a pivotal role in the control and regulation of the expression of EPO gene. Firstly, using a group of five HCC lines with varying degrees of differentiation as the experimental model, we demonstrated that the endogenous GSH contents of these HCC cells were differentially upregulated depending on the degree of differentiation with an order of abundance being Hep G2> Hep 3B> J5> Mahlavu> SK-Hep-I. Coincidently, we also found that g-GCS heavy subunit activities as well as its mRNA correlated precisely with this order. Among these HCC cell lines tested, only two well-differentiated sublines, Hep G2 and Hep 3B expressed EPO gene implying that the latter process was dependent upon GSH and suggested a notion that a threshold level might be required for its optimal reactivation. Secondly, to further obtain the evidence to substantiate this possible role of GSH, we then supplemented to the cell culture media with an excessive quantity of nonlethal N-acetylcysteine for the purpose of reinforcing the endogenous GSH biosynthesis. Interestingly, we found that this manipulation could revert the reactivation of EPO gene in cell lines, such as J5 and SK-Hep-I, in which their EPO gene expressions were ortherwise shut down under a normal circumstance. Finally, we were able to demonstrated using RT-PCR and western blotting that the expression of EPO gene was reverted in GCS30, a SK-Hep-I subline that was permanently transfected with g-GCSh and is capable of overly expressing endogenous GSH. Taken together, we demonstrated herein for the first time that, besides hypoxia and CoCl2, endogenous GSH contents can also act as a positive regulator for the expression of EPO gene. The underlying mechanism of how GSH exerts its action in the regulation of EPO expression awaits further clarification.

中文摘要.....2
英文摘要.....4
緒論(背景介紹)6
材料與方法16
結果......27
討論......45
總結......49
參考資料....50
附錄......56

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