臺灣博碩士論文加值系統

鳥胺酸去羧化酶 (Ornithine Decarboxylase; ODC) 在許多生物功能，包括細胞增生、分化及細胞凋亡上扮演著重要的角色。在本研究中，過度表現鳥胺酸去羧化酶於人類乳癌及子宮頸上皮癌細胞中發現，當細胞受到低劑量 UVB (Ultraviolet B) 照射後，會使細胞內氧化壓力 (Reactive oxygen species；ROS) 堆積下降，最後使自噬作用 (Autophagy) 被抑制。當利用 shRNA 下調鳥胺酸去羧化酶後發現，自噬作用會被活化。另外，當過度表現鳥胺酸去羧化酶之人類乳癌及子宮頸上皮癌細胞受到高劑量 UVB 照射後，發現細胞中腺苷單磷酸活化蛋白激酶表現量會上升，進而誘發自噬作用的發生，同時並抑制細胞走向細胞凋亡 (Apoptosis)。當利用 shRNA 及氯奎寧 (Chloroquine; CQ) 將過度表現鳥胺酸去羧化酶之人類乳癌及子宮頸上皮癌細胞中自噬作用下調後 (shAtg5、shBECN1、shAtg12) 也發現，細胞走向凋亡的比率會增加。而利用 shRNA 將鳥胺酸去羧化酶下調後也可發現當細胞受到高劑量 UVB 處理後，自噬作用表現會跟著受到抑制，最後導致細胞發生凋亡。由於 Atg12 這個蛋白扮演著可調控細胞發生細胞凋亡及自噬作用的能力，因此利用定位突變 (site directed mutagenesis) 的方式分別在過度表現鳥胺酸去羧化酶之人類乳癌及子宮頸上皮癌細胞上，將調控細胞凋亡及自噬作用的胺基酸位點突變後發現，當 Atg12 調控細胞凋亡的位點被突變後，發現細胞較不容易凋亡；相對地，當 Atg12 調控自噬作用的位點被突變後可發現，當自噬作用被抑制後，細胞發生凋亡的比例會增加。因此，在未來治療人類乳癌及子宮頸上皮癌細胞上，可藉由同時抑制鳥胺酸去羧化酶及自噬作用並搭配抗癌用藥來當作治療的方針。

Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. In this study, we revealed that overexpression of ODC in HeLa and MCF-7 cells decreased cellular ROS (Reactive oxygen species) after low dose of UVB radiation, leading autophagy inhibited, and it was restored by knocking down ODC (shODC) in ODC overexpressing HeLa and MCF-7 cells. Furthermore, the results demonstrated that AMPK was increased after high dose of UVB radiation in ODC ovexpressing HeLa and MCF-7 cells, leading to autophagy induced and apoptosis inhibited. We demonstrated that knocked down of autophagy by shRNA (shAtg5, shBECN1, and shAtg12) and chloroquine (CQ) could enhance high dose of UVB induced cell death in ODC overexpressing HeLa and MCF-7 cells. Here, we also observed that knocked down of ODC in ODC overexpressing HeLa and MCF-7 cells inhibited autophagy and enhanced high dose of UVB radiation. Because of Atg12 can regulate cell apoptosis and autophagy. Site directed mutagenesis was used to mutate the amino acid which can regulate cell apoptosis and autophagy on Atg12, respectively in these two ODC overexpressing cells. According to the results, mutated the amino acid which can regulate apoptosis on Atg12 leading the cells more survival. Relatively, mutated the amino acid which can regulate autophagy on Atg12 leading the cells died. Therefore, inhibition of ODC and autophagy could be a promising strategy for adjuvant chemotherapy in human breast and cervical cancers.

目錄
誌謝 I
縮寫表 II
中文摘要 III
Abstract IV
圖次 VII
前言 (Introduction) 1
實驗結果 (Results) 5
Low intensity of UVB induces autophagy, and high intensity of UVB provokes apoptosis in parental HeLa and MCF-7 cells 5
Diverse intensity of UVB radiation interferes with the protein expression of ODC 6
Autophagy was induced after UVB irradiation and overexpression of ODC resists low dose UVB-induced autophagy 7
Overexpression of ODC prevents UVB-induced cell death and induces autophagy in HeLa and MCF-7 cells 8
Knockdown of ODC attenuates autophagy and enhances UVB-induced cell death 9
Inhibition of autophagy enhances UVB-induced cell death 9
Atg12 plays an important role in ODC-overexpressing HeLa and MCF-7 cells to regulate cell autophagy and apoptosis 10
Overexpression of ODC declines UVB-induced intracellular ROS and DNA damage in HeLa and MCF-7 cells 11
The AMPK/mTOR axis mediates the effect of ODC on autophagy and cell survival during UVB radiation 12
討論 (Discussion) 14
材料方法 (Materials and Methods) 18
Materials 18
Cell Culture and UV Irradiation 18
Cloning of human ornithine decarboxylase (odc) and atg12 genes 18
Site-directed mutagenesis of atg12 gene 19
Cell transfection 19
Cell viability and acridine orange (AO) staining assay 20
Total RNA isolation and reverse-transcription polymerase chain reaction (RT-PCR) 20
DNA fragmentation analysis 21
Protein expression knockdown by shRNA technology 21
GFP-mcherry-LC3B fluorescence microscopy 22
Immunoblotting 22
Detection of intracellular ROS 22
Statistical Analysis 23
參考文獻 (References) 24
Figures and Figure legends 31
附錄 93
Vector Map 93
RNAi資料庫 96
PCR Primers 100
實驗器材 100
藥品配置 103

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