臺灣博碩士論文加值系統

鳥胺酸去羧化酶（ornithine decarboxylase，ODC）為多胺酸起始與決定反應速率的酵素，對細胞週期、細胞生長、分化及細胞凋亡扮演一重要角色；在尋找腫瘤治療及預防藥物研究中，純化植物天然物質是其中來源之一，研究者篩選藥物時，測定對鳥胺酸去羧化酶活性是否具抑制效果是一個重要的試驗。在我們篩選天然化合物實驗中，發現rottlerin抑制鳥胺酸去羧化酶活性最具成效，之前的研究人員已知蛋白激酶Cd調節鳥胺酸去羧化酶的活性，因此在人類白血病HL60細胞處理rottlerin的實驗中發現加入rottlerin二十四小時後，細胞進行計劃性死亡，並可偵測到DNA片段、細胞核濃縮及sub-G1提增；而細胞處理藥物十小時後與控制組細胞比較，觀察到鳥胺酸去羧化酶的蛋白表現與酶活性下降50﹪以上；除此以外，測定鳥胺酸去羧化酶去磷酸化的情況，發現於酥胺酸位置有顯著差異，而絲胺酸和酪胺酸位置並無差異。為了偵測鳥胺酸去羧化酶是否能夠負調節rottlerin所引發的細胞凋亡，我們將具有鳥胺酸去羧化酶cDNA載體穩定轉染至HL60細胞內，此細胞稱為HL60-ODC細胞，另轉染載體的細胞稱為HL60-pcDNA3細胞；實驗發現HL60-ODC細胞中的鳥胺酸去羧化酶蛋白為HL60-pcDNA3細胞與HL60細胞表現之2.5倍，同時HL60-ODC細胞提升鳥胺酸去羧化酶蛋白表現，能夠減少rottlerin造成之細胞凋亡現象，並且此蛋白在HL60-ODC細胞中酥胺酸磷酸化較HL60-pcDNA3細胞表現高。綜合以上結果顯示，rottlerin使得細胞鳥胺酸去羧化酶處於酥胺酸去磷酸化狀況，而使鳥胺酸去羧化酶穩定性下降而導致細胞凋亡；反之，增高鳥胺酸去羧化酶表現，並增加鳥胺酸去羧化酶酥胺酸之磷酸化，這樣的結果，使得細胞凋亡的現象被抑制，因此，本研究推論，高量的鳥胺酸去羧化酶之蛋白質表現並磷酸化的結果，可以對抗rottlerin引發的細胞凋亡。

Ornithine decarboxylase (ODC), a rate-determining enzyme of the polyamine biosynthetic pathway, is involved in the control of cell cycle, differentiation, cell growth and apoptosis. Previous studies described that protein kinase Cd (PKCd) regulates ODC activity. In this thesis, human leukemia cell line, HL60 cell, was used for the treatment with a specific PKCd inhibitor, rottlerin, to measure the effects on cell growth. Our data are showed that apoptosis parameters including DNA fragmentation, nuclear morphological change and sub-G1 fraction, appeared after 24 h by 5 μM rottlerin treatment. Furthermore, rottlerin caused a decrease in 72% of ODC protein expression and an increase in threonine, rather than serine and tyrosine dephosphorylation on ODC enzyme compared with the control after incubation for 10 h. To determine whether ODC could down-regulate rottlerin-induced apoptosis, HL60 cells were stably transfected with ODC cDNA. HL60-ODC cells exhibited a 2.5-fold increase in ODC protein compared with vector only (HL60-pcDNA3) or HL60 cells. Restoration of ODC expression in the HL60-ODC cells resulted in rescue of rottlerin-induced apoptosis of HL60 cells. Simultaneously, phosphothreonine-ODC of rottlerin-treated HL60-ODC cells was highly elevated comparing with HL60-pcDNA3 cells. Taken together, we suggested that rottlerin, a PKCd inhibitor, might decrease phosphothreonine-ODC to reduce both protein stability and enzyme activity of ornithine decarbooxylase and finally resulted in cell apoptosis. Our results also revealed that elevation of ODC protein and phosphotheronine-ODC prevents apoptosis in rottlerin treated cells. This study provided for the first time a new and important linkage between programmed cell death and the regulation of the posttranslational modification of ODC.

目錄 頁次
序言 ------------------------------------------- 1
材料與方法 ------------------------------------- 7
結果 ------------------------------------------- 16
討論 ------------------------------------------- 21
結論 ------------------------------------------- 25
圖與表 ----------------------------------------- 26
參考文獻 --------------------------------------- 41
附錄 ------------------------------------------- 49

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