本論文主要探討2-(4-aminophenyl)-7-methoxybenzothiazole (7-OMe-APBT)誘導人類急性白血病U937細胞死亡透過TNF-α介導的死亡訊息傳遞路徑。 7-OMe-APBT透過活化caspase-8/mitochondria的死亡路徑引發細胞凋亡，進一步活化p38 MAPK致使TNF-α及TNFR2表現增加； p38 MAPK誘導c-Jun磷酸化導致TNFR2 promoter活性增加，預先處理p38 MAPK抑制劑 (SB202190)能抑制7-OMe-APBT上調TNFR2 promoter冷光活性及mRNA表現的效應；另一方面，7-OMe-APBT透過PP2Acα介導TTP的降解誘導TNF-α mRNA穩定性增加，前處理SB202190可抑制PP2Acα及TNF-α 表現增加，PP2A抑制劑 (Okadaic acid)則可抑制7-OMe-APBT誘導TNF-α表現增加和TTP的降解。 將TTP大量表現能抑制TNF-α 表現增加以及可回復7-OMe-APBT處理之U937細胞的存活率。 我們研究結果顯示7-OMe-APBT誘導TNF-α表現增加透過活化p38 MAPK/PP2Acα 促使TTP表現下降，由此可得知TNF-α引發的死亡訊息傳遞路徑參與7-OMe-APBT誘導U937細胞的死亡。

The aim of this study was to explore the relation of tumor necrosis factor-α(TNF-α)-mediated death pathway to 2-(4-aminophenyl)- 7-methoxybenzothiazole (7-OMe-APBT)-induced death of human leukemia U937 cells. 7-OMe-APBT elicited apoptosis of U937 cells via caspase-8/mitochondria-dependent death pathway. 7-OMe-APBT elicited up-regulation of TNF-α and TNFR2 expression through activation of p38 MAPK. p38 MAPK-induced c-Jun phosphorylation up-regulated TNFR2 promoter activity. SB202190 (p38 MAPK inhibitor) treatment abrogated 7-OMe-APBT effect on promoting TNFR2 promoter activity and mRNA level. On the other hand, 7-OMe-APBT induced increased TNF-α mRNA stability via protein phosphatase 2A catalytic subunit α(PP2Acα)-mediated tristetraprolin (TTP) degradation. Pretreatment with SB202190 abolished 7-OMe-APBT-induced PP2Acα and TNF-α up-regulation. PP2A inhibitor (Okadaic acid) attenuated the ability of 7-OMe-APBT to induce TNF-α up-regulation and TTP degradation. TTP over-expression suppressed 7-OMe-APBT-induced TNF-α up-regulation and restored the viability of 7-OMe-APBT-treated cells. Our data indicate that 7-OMe-APBT elicits TNF-α up-regulation via p38 MAPK/PP2Acα-mediated TTP down-regulation, and suggest that the TNF-α-mediated death pathway is involved in 7-OMe-APBT-induced death of U937 cells.

論文審定書 i
致謝 ii
摘要 iii
Abstract iv
目錄 v
圖次 vii
前言 1
2-(4-aminophenyl) benzothiazole (APBT) 1
細胞凋亡(apoptosis) 1
白血病(Leukemia) 3
實驗目的 4
研究方法與材料 5
實驗藥品 5
細胞培養 6
細胞藥物處理 6
細胞存活率測試 6
測定粒腺體膜電位(ΔΨm) 7
cytochrome c釋放分析 7
測定Bcl-2 family蛋白質表現 8
聚丙烯醯胺膠體電泳法(SDS- PAGE) 8
西方點墨法 8
流式細胞儀分析細胞表面TNF-α及TNFR2表現 8
RT-PCR偵測TNF-α、TNFR2及PP2Acα表現 9
TNF-α、TNFR2及PP2Acα promoter之冷光活性分析 10
TNR-α mRNA穩定性分析 10
DNA細胞轉殖(DNA transfection)與RNA Interference 10
統計分析 11
結果 12
7-OMe-APBT誘導U937細胞凋亡 12
7-OMe-APBT影響U937細胞粒線體膜電位(ΔΨm)與Bcl-2家族蛋白質表現 12
7-OMe-APBT誘導死亡接受器的活化 13
7-OMe-APBT增加TNF-α mRNA穩定性及TNFR2 啟動子的活性 13
7-OMe-APBT誘導活化p-38 MAPK及抑制ERK活性 13
7-OMe-APBT透過活化p-38 MAPK參與調控c-Jun誘導TNFR2表現上升 14
7-OMe-APBT誘導PP2Acα 表現增加導致TTP表現下降 14
7-OMe-APBT誘導TTP表現下降導致TNF-αmRNA穩定性增加 15
討論 16
附圖 30
參考文獻 34
會議發表著作 44

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