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研究生:劉宗豪
研究生(外文):Tsung-Hao Liu
論文名稱:探討β-lapachone在高血清濃度及低血清濃度培養下引致人類血管內皮細胞凋亡的機制
論文名稱(外文):Investigating the Apoptotic Mechanism Induced by beta-Lapachone on Human Endothelial Cells under
指導教授:周逸鵬
指導教授(外文):Yat-Pang Chau
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:解剖暨細胞生物學研究所
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:2004
畢業學年度:92
語文別:中文
論文頁數:65
中文關鍵詞:內皮細胞血清凋亡
外文關鍵詞:beta-lapachoneendothelial cellserumapoptosis
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血管新生對腫瘤生長和轉移是必需的因素之一,因此,利用抗血管新生在治療癌症的想法由此而生。□-lapachone (3,4,dihydro-2,2- dimethyl-2H-naphtho- [1,2-b] pyran-5, 6- dione) 是一種由lapacho tree 所萃取出的ortho-naphthoquinone,目前已被證實具有抗腫瘤、抗病毒的功效。有文獻指出NAD(P)H:quinone oxidoreductase 1 (NQO1) 會和□-lapachone形成無效的循環 (Futile cycle),造成細胞毒性。而且在我們實驗室之前的研究指出 □-lapachone 可以藉由引發人類血管內皮細胞 (EA.hy926細胞株、人類臍靜脈內皮細胞) 凋亡,達到抗血管增生的作用,然而□-lapachone誘發什麼樣的訊息導致細胞凋亡,以及□-lapachone為何在低血清濃度下培養之下會引發較輕微的細胞毒性,目前尚不了解。因此在我們實驗中,利用MTT assay偵測細胞存活、用Acridine orange 觀察細胞形態,以及西方點墨法偵測 □-lapachone處理過後所引發蛋白質表現上的變化,並利用事先處理MAPK 抑制劑、細胞內鈣離子螯合劑BAPTA-AM和細胞外鈣離子螯合劑EDTA或EGTA來探討在不同血清濃度培養下, □-lapachone毒殺人類血管內皮細胞所引發的訊息有何不同。實驗發現EAhy926細胞培養在2 % 胎牛血清培養下處理2 □M □-lapachone可引發MAPK當中的磷酸化ERK表現下降,JNK表現 量上升,p38變化不是很明顯,EA.hy926細胞在10 % 胎牛血清培養下則造成磷酸化ERK、JNK表現上升,磷酸化的p38則變化一樣不是很明顯;HUVEC在2 % 胎牛血清培養下經10 □M □-lapachone處理後,細胞內磷酸化ERK、JNK、p38表現 量上升;經□-lapachone處理在10 % 胎牛血清培養下的細胞,細胞中的磷酸化ERK蛋白表現反而下降,但磷酸化的JNK、p38表現 量仍會增加。利用ERK、JNK 和p38抑制劑作細胞前處理之實驗顯示,抑制JNK磷酸化可有效保護細胞,減低□-lapachone對內皮細胞的殺害;此外加入細胞內鈣離子螯合劑BAPTA-AM或NQO1抑制劑, dicoumarol, 也可有效抑制□-lapachone對EAhy926內皮細胞的毒殺,使細胞死亡率降低。
綜合以上的結果,我們認為□-lapachone促使人類血管內皮細胞凋亡之訊號主要是經由JNK、鈣離子和NQO1等有關途徑來傳送。
Since angiogenesis is an very important event for tumor growth and metastasis, therefore, anti-neovascular formation has been considered an effective strategy for cancer therapy.beta-Lapachone (3,4,dihydro-2,2-dimethyl
-2H-naphtho-[1,2-b] pyran-5,6-dione), an ortho-naphthoquinone, originally isolated from lapacho tree (Tabebuia avellanedae) has been shown to have anti-cancer and anti-viral effect. In our preliminary studies, we demonstrated that beta-lapachone can induce the human endothelial cells undergoing apoptosis and exert anti-vascularization. However the signaling mechanism underlying the beta-lapachone induced apoptosis of human vascular endothelial cell and its inhibitory effect on human endothelial cells under different serum conditions are still unclear. In this study, we report that beta-lapachone induces the apoptosis of human endothelial cells including EAhy926 endothelial cell line and human umbilical vascular endothelial cells under cultured with 2 % or 10 % serum condition. It was found that 2 uM or 10 uM of beta-lapachone was able to decrease the EAhy926 cells or HUVEC viability. Results from western blot showed that the treatment of human endothelial cells with beta-lapachone caused primarily activation of JNK phosphorylation. Treatment of JNK kinase inhibitors, SP600125, actually protected the beta-lapachone-treated cells. More, addition of intracellular calcium chelator BAPTA-AM, or NQO1 inhibitor decreased the endothelial cell death induced by beta-lapachone treatment. Taken together, we concluded the JNK, intracellular calcium and the activity of NQO1 involving in the signaling pathway of beta-lapachone-induced endothelial cell apoptosis.
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