臺灣博碩士論文加值系統

Notch1訊息傳遞路徑參與許多生理及病理的調控，包括細胞增生、凋亡、維持幹細胞特性以及參與調控紅血球和巨核細胞的分化。本論文發現Transient receptor potential ankyrin 1 (TRPA1)是Notch1活化態受體(Notch1 intracellular domain, N1IC)活化的基因之一。TRPA1是一個非選擇性鈣離子通道蛋白，當TRPA1活化後會促使神經傳導物質的釋放，且發炎相關的細胞激素會促使其表現。發炎會抑制紅血球分化及導致貧血，而TRPA1是否和血球分化有關目前尚未明瞭。實驗結果顯示，N1IC透過CBF1非依賴性路徑調控TRPA1啟動子，且N1IC會透過Ets-1調控TRPA1啟動子上並促進其表現。N1IC活化TRPA1亦透過啟動子甲基化的調控，且N1IC和Ets-1協同抑制DNA methyltransferase 3B (DNMT3B)表現。以hemin誘導K562及HEL細胞往紅血球分化的情況會受到TRPA1的抑制。TRPA1活化劑AITC可抑制K652及HEL細胞往紅血球分化，且ERK磷酸化程度亦增加，此現象在前處理TRPA1拮抗劑或是EGTA則被回復。N1IC和Ets-1亦透過TRPA1抑制細胞往紅血球分化。由PMA誘導細胞往巨核細胞分化的現象會被TRPA1所促進，且巨核細胞標誌表現量皆隨之上升。而抑制Notch1或Ets-1表現後會降低巨核細胞分化，此現象在大量表現TRPA1後有所回復。抑制DNMT3B後TRPA1之表現量上升，且減少K562細胞往紅血球分化，並促進往巨核細胞分化。此外，抑制TRPA1後會促進K562細胞之移行、侵襲及細胞聚落形成能力。綜合以上結果顯示，N1IC誘導TRPA1表現，並且是調控紅血球及巨核細胞分化的重要基因。

Notch1 signaling involves in several physiological and pathological cellular processes, including proliferation, apoptosis, stem cell maintenance and regulation of erythroid and megakaryocyte differentiation. Notch1 intracellular domain (N1IC), the activated form of Notch1, induced TRPA1 expression. TRPA1 is a non-selective calcium channel. Inflammatory cytokines enhance TRPA1 expression, and TRPA1 activation induces neurotransmitter release. Inflammatory cytokines suppress erythroid differentiation and result in anemia. The roles of TRPA1 in erythroid/megakaryocyte differentiation are poorly understood. Herein, the data indicated that N1IC activated TRPA1 promoter in a CBF1-independent manner. N1IC enhanced TRPA1 promoter activity via Ets-1, and both of them bound to TRPA1 promoter. N1IC modulated TRPA1 promoter depend on promoter methylation, and N1IC and Ets-1 inhibited DNA methyltransferase 3B (DNMT3B) expression synergistically. TRPA1 decreased hemin-induced erythroid differentiation of K562 and HEL cells. TRPA1 agonist AITC suppressed erythroid differentiation and increased phosphorylation of ERK in K562 and HEL cells, which were reversed by TRPA1 antagonist or EGTA pretreatment. TRPA1 mediated N1IC- or Ets-1- restrained erythroid differentiation. TRPA1 improved PMA-induced megakaryocyte differentiation, and the levels of megakaryocytic markers were increased. Notch1 receptor or Ets-1 knockdown reduced me megakaryocyte differentiation, which could be restored by TRPA1 expression. Knockdown of DNMT3B increased TRPA1 level, inhibited erythroid differentiation as well as promoted megakaryocyte differentiation. Moreover, TRPA1 inhibition enhanced migration, invasion and colony forming abilities of K562 cells. These results demonstrate that N1IC-induced TRPA1 play a critical role in the regulation of erythroid and megakaryocyte differentiation.

中文摘要 i
Abstract ii
縮寫表 iii
目次 iv
圖表目次 vi
緒論 1
Notch受體 (Notch receptor) 1
ETS proto-oncogene 1 (Ets-1) 3
Transient receptor potential cation channel subfamily A member 1 (TRPA1) 3
造血作用 5
研究動機 6
研究材料與實驗方法 8
研究材料 8
細胞株 8
質體 8
RNAi及引子序列 10
抗體 11
實驗材料 12
實驗方法 17
製備勝任細胞及轉型作用 17
萃取質體DNA 17
試管內甲基化 (in vitro methylation) 18
細胞培養 18
細胞轉染 19
冷光報導分析 20
核糖核酸萃取 20
即時聚合酶鏈反應 21
西方點墨法 21
染色質免疫沉澱分析法 23
紅血球分化及benzidine染色 25
巨核细胞分化及Giemsa染色 25
流式細胞儀分析 26
細胞生長分析 26
細胞移行及侵襲能力試驗 27
細胞聚落培養分析 27
統計分析 28
結果 29
N1IC透過非CBF1路徑調控TRPA1表現 29
N1IC透過Ets-1調控TRPA1表現 30
N1IC可藉由DNMT3B調控TRPA1 31
TRPA1抑制K562及HEL細胞往紅血球分化 32
N1IC及Ets-1透過TRPA1抑制K562及HEL細胞往紅血球分化 34
TRPA1促進K562及HEL細胞往巨核細胞分化 34
Notch1及Ets-1透過TRPA1促進K562及HEL細胞往巨核細胞分化 36
抑制DNMT3B減少K562細胞往紅血球分化並促使往巨核細胞分化 36
抑制TRPA1促進K562細胞移行、侵襲及聚落形成之能力 37
討論 38
參考文獻 43
圖表 57

圖表目次
圖一、Notch1受體調控TRPA1之表現。 57
圖二、N1IC透過CBF1非依賴性路徑活化TRPA1啟動子。 59
圖三、Ets-1促進N1IC活化TRPA1啟動子 60
圖四、N1IC透過Ets-1活化TRPA1啟動子。 62
圖五、Ets-1可增加N1IC所促進的TRPA1之mRNA及蛋白質表現量。 63
圖六、N1IC及Ets-1可結合於TRPA1啟動子。 64
圖七、N1IC活化TRPA1啟動子受甲基化抑制且N1IC和Ets-1協同抑制DNMT3B蛋白質表現。。 65
圖八、Notch1透過DNMT3B促進TRPA1之mRNA表現。 67
圖九、TRPA1抑制K562及HEL細胞往紅血球分化。 68
圖十、抑制TRPA1促進K562及HEL細胞往紅血球分化。 69
圖十一、TRPA1透過影響鈣離子流入細胞抑制K562及HEL細胞往紅血球分化。 70
圖十二、N1IC透過TRPA1抑制K562及HEL細胞往紅血球分化。 72
圖十三、Ets-1透過TRPA1抑制K562及HEL細胞往紅血球分化。 74
圖十四、Notch1、Ets-1、TRPA1及DNMT3B巨核細胞分化有關。 76
圖十五、TRPA1促進K562細胞往巨核細胞分化。 77
圖十六、TRPA1增加K562細胞之巨核細胞分化的標誌。 79
圖十七、TRPA1抑制K562細胞生長。 80
圖十八、抑制TRPA1抑制K562細胞往巨核細胞分化。 81
圖十九、TRPA1可回復抑制Notch1所減少巨核細胞分化的情形。 84
圖二十、N1IC透過TRPA1抑制K562及HEL細胞往巨核細胞分化。 85
圖二十一、TRPA1可回復抑制Ets-1所減少巨核細胞分化的情形。 87
圖二十二、Ets-1透過TRPA1抑制K562及HEL細胞往巨核細胞分化。 88
圖二十三、抑制DNMT3B抑制紅血球分化而促進巨核細胞分化 89
圖二十四、抑制TRPA1促進K562細胞移行、侵襲及聚落形成之能力 91
圖二十五、N1IC、Ets-1及DNMT3B調控TRPA1影響細胞分化示意圖 92
表一、RNAi列表 93
表二、PCR引子 94
表三、Real-time PCR引子 95
附圖一、Notch受體結構及訊息傳遞路徑 96
附圖二、造血作用 97

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