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研究生:包秉潔
研究生(外文):Ping-Chieh Pao
論文名稱:腦指蛋白(BrainFingerProtein),ZNF179,在神經細胞分化功能之探討
論文名稱(外文):The functional role of the brain finger protein, ZNF179, in neural differentiation
指導教授:張文昌張文昌引用關係李宜釗
指導教授(外文):Wen-Chang ChangYi-Chao Lee
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:78
中文關鍵詞:腦指蛋白神經分化
外文關鍵詞:neural differentiationbrain finger proteinZNF179
相關次數:
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ZNF179又名腦指蛋白,屬於RING finger家族中的一員。藉由分析從Gene Expression Omnibus (GEO) 所獲得的基因表達資料,我們發現,在亨丁頓疾病 (Huntington’s disease) 之基因轉殖鼠內,ZNF179的表現明顯的下降。此外,ZNF179的表現在肌萎縮性脊髓側索硬化症(Amyotrophic Lateral Sclerosis ;ALS)的動物模式中也是明顯減少的。參考過去文獻已知,於老鼠胚胎發育的過程中,ZNF179基因的表現隨著胚胎發育的天數而逐漸增加,並且於成鼠腦中維持相當高度的表現。顯示著ZNF179在神經系統內似乎扮演著極為重要的角色。因此我們便想探討在神經細胞內,ZNF179的功能性角色究竟為何。
為了研究ZNF179於神經細胞內的功能,我們選用P19細胞來作為實驗模式。已知P19細胞是一株老鼠胚胎瘤細胞,具有分化成多種細胞型態的能力。若是將 P19細胞培養於養菌盤內,P19細胞會慢慢的聚集成一個團塊並持續的進行細胞分裂,若同時伴隨著維他命 A 酸 (Retinoic acid;RA) 的處理,P19細胞便會分化為神經細胞、神經膠細胞以及纖維母細胞。於是我們便利用P19細胞來進行神經細胞的分化,並在此過程中探討ZNF179的功能。我們發現,當利用RA誘導P19細胞進行神經細胞分化,ZNF179基因的表現也隨著神經細胞分化的天數而逐漸增加,並且在已分化的P19細胞內,維持大量的表現。此外,我們也利用EGFP-ZNF179的融合蛋白來研究在神經細胞分化前後,ZNF179於細胞內的分布情況。結果我們發現,在未分化的P19細胞內,EGFP-ZNF179主要是分布於細胞質內,但在分化後的P19細胞中,則可在細胞質以及細胞核內觀察到EGFP-ZNF179的存在。暗示著在神經細胞中,ZNF179可能藉由進入細胞核而執行某些重要的功能。為了更加了解ZNF179於神經細胞內所扮演的功能,於是我們建立了ZNF179 knock-down stable cell line,結果我們發現,knock-down ZNF179明顯抑制了P19細胞進行神經分化。於是我們進一步去探討可能的作用機制。目前我們的研究顯示,ZNF179 knock-down stable cell line處理RA四天的團塊,其AKT Ser473的磷酸化明顯高於P19細胞,因此我們認為knock-down ZNF179可能會透過增加AKT Ser473的磷酸化,而抑制神經分化,但其中詳細的分子機制仍有待進一步的探討。
ZNF179, also known as brain finger protein, is a member of RING finger family. After analyzed the gene expression profile obtained from Gene Expression Omnibus (GEO), we found that ZNF179 was down-regulated in R6/2 Huntington’s disease transgenic mice. In addition to Huntington’s disease, the expression of ZNF179 was also reported to be decreased in other neurodegererated diseases, Amyotrophic Lateral Sclerosis (ALS). The expression of ZNF179 was gradually increased during mouse embryogenesis and maintained a high expression level in the adult brain. These all indicate that the ZNF179 is important to the functions of the brain. Therefore, the functional role of ZNF179 in neural cells is worthwhile for further investigation.
P19 cell line is mouse pluripotent embryonic carcinoma cells. It could be induced to differentiate into neurons, glial cells and fibroblasts when aggregated in the presence of retinoic acid, and is a well-established cell model to investigate the important cellular mechanism during neural differentiation. Using P19 cell line as a neural differentiation cell model, we found that the expression of ZNF179 was noticeably increased in the RA-induced neural differentiated P19 cells. To evaluate the cellular distribution of ZNF179, we created EGFP-ZNF179 fusion protein expression construct and found that the EGFP-ZNF179 fusion protein was distributed in the cytosol of undifferentiated P19 cells. Interestingly, in neural differentiated P19 cells the EGFP-ZNF179 fusion protein translocated from the cytosol into the nucleus. The different cellular distribution patterns imply that the ZNF179 may play its role in the nucleus. Then the ZNF179 knock-down stable cell lines were established to further dissect the exact role and function of ZNF179. Our data showed that knock-down of ZNF179 significantly attenuated the P19 cell neural differentiation. We further investigated the cellular mechanism of ZNF179 in neural differentiation and found that the phosphorylation of PKB/AKT Ser473 but not Thr308 was dramatically increased in the aggregated ZNF179 knock-down stable cell lines after treating with RA 4 days. These results indicate that abnormal phosphorylation of PKB/AKT Ser473 could be involved in the cellular mechanism of ZNF179 in neural differentiation.
目錄

中文摘要 ------------------------------------------------------------------------ Ⅰ
英文摘要------------------------------------------------------------------------- Ⅲ
誌謝 ----------------------------------------------------------------------------- Ⅴ
目錄 ----------------------------------------------------------------------------Ⅶ
圖目錄 ------------------------------------------------------------------------ Ⅷ
附錄目錄 ----------------------------------------------------------------- Ⅸ
縮寫檢索 -----------------------------------------------------------------------Ⅹ
第一章 緒論 -------------------------------------------------------------------- 1
第二章 實驗材料與方法 ----------------------------------------------------- 9
第三章 實驗結果 ------------------------------------------------------------- 33
第四章 討論 ------------------------------------------------------------------- 43
第五章 參考文獻 ------------------------------------------------------------- 49
附圖 ----------------------------------------------------------------------------- 54
附錄 ----------------------------------------------------------------------------- 64
自述 ----------------------------------------------------------------------------- 66
參考文獻

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