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研究生:徐偉倫
研究生(外文):Wei-Lun Hsu
論文名稱:貝它類澱粉蛋白會活化一個嶄新的防禦機制以維護細胞的存活:血清及糖皮質素誘導激酶-訊息傳遞與轉錄活化因子1/訊息傳遞與轉錄活化因子2訊息傳遞的角色
論文名稱(外文):A novel defense mechanism that is activated on amyloid-beat insult to mediate cell survival: role of SGK1-STAT1/STAT2 signaling
指導教授:邱蔡賢邱蔡賢引用關係李小媛李小媛引用關係
指導教授(外文):Tsai-Hsien ChiuHsiao-Yuan Lee
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:189
中文關鍵詞:貝它類澱粉蛋白42細胞外信號調節激酶1/2血清及糖皮質素誘導激酶訊息傳遞與轉錄活化因子1訊息傳遞與轉錄活化因子2抗凋亡蛋白抗凋亡機制
外文關鍵詞:Abeat42ERK1/2SGK1STAT1STAT2Mcl-1Anti-apoptosis
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阿茲海默氏症 (Alzheimer's disease) 是一種神經退化性疾病,大量的老化斑 (senile plaques) 堆積於神經細胞外是此疾病目前病理上最顯著的特徵,老化斑主要的組成物質是貝它類澱粉蛋白 (beta amyloid,Aβ),已知貝它類澱粉蛋白可產生神經毒性造成神經細胞損傷及死亡,而其下游的機制也被廣泛的研究,但對於貝它類澱粉蛋白所引發的細胞自我保護的機制卻不甚清楚。
  本實驗以MTT方法分析細胞的存活程度,及TUNEL分析法測定細胞凋亡程度,結果顯示貝它類澱粉蛋白對PC12細胞具有神經毒性,且其毒性與劑量成正相關,細胞的半致死劑量 (lethal dosage) 約為 0.1 uM。然而細胞在0.1 uM貝它類澱粉蛋白作用下可促進SGK1蛋白質絲胺酸78位置 (SGK1 Ser 78) 的磷酸化,但不會影響到SGK1蛋白質另外二個磷酸化位置,酥胺酸256 (SGK1 Thr 256) 及絲胺酸422 (SGK1 Ser 422) 的活性。同時ERK1/2、STAT1酪胺酸701 (STAT1 Tyr 701)、STAT1絲胺酸727 (STAT1 Ser 727) 及STAT2酪胺酸690 (STAT2 Tyr 690) 蛋白質的磷酸化也有同步增加的現象,此外抗凋亡蛋白Mcl-1的訊息核醣核苷酸及蛋白表現量也會增加。但是當細胞分別轉染ERK1/2、SGK1、STAT1、STAT2干擾核醣核苷酸,則會抑制貝它類澱粉蛋白所誘發的磷酸化變化及Mcl-1蛋白質的表現。而細胞隨著轉染HA-SGK1S78D 質體的濃度增加,可有效減緩貝它類澱粉蛋白所產生的毒性,並促進細胞內Mcl-1表現,同時SGK1蛋白質可直接在STAT1蛋白質的酪胺酸701及絲胺酸727的位置上進行磷酸化,並間接磷酸化 STAT2蛋白質的酪胺酸690位置,而磷酸化的STAT1及STAT2蛋白質會促進Mcl-1蛋白質的表現,進而減緩貝它類澱粉蛋白所造成的細胞凋亡現象,但是細胞轉染Mcl-1干擾核醣核苷酸反而會促進貝它類澱粉蛋白對細胞所造成的毒性。而SGK1蛋白質絲胺酸78突變為丙氨酸 (Ala 78) 後,可抑制貝它類澱粉蛋白所促進的STAT1及STAT2蛋白質的磷酸化及Mcl-1蛋白質的表現。而STAT1蛋白質酪胺酸701突變為苯丙氨酸 (Phe 701)、絲胺酸727突變為丙氨酸及STAT2蛋白質酪胺酸690突變為苯丙氨酸,也會抑制貝它類澱粉蛋白及SGK1 所誘發的Mcl-1蛋白質表現。
  這些結果顯示在低濃度的貝它類澱粉蛋白作用下,可經由活化ERK1/2蛋白質而磷酸化SGK1蛋白質絲胺酸78位置,而活化態的SGK1蛋白質會直接磷酸化 STAT1蛋白質的酪胺酸701、絲胺酸727的位置,並間接磷酸化STAT2酪胺酸690的位置,促使STAT1和STAT2蛋白質結合形成異雙聚體,從細胞質位移至細胞核內,並結合在Mcl-1基因啟動子區域的ISRE特定序列上,啟動了Mcl-1基因的表現。一旦大量增加細胞內Mcl-1基因的表現將減緩貝它類澱粉蛋白對細胞所造成的傷害,增加細胞的存活。所以我們的論文是首先證明當細胞受到貝它類澱粉蛋白毒性時,能夠活化ERK1/2-SGK1-STAT1/STAT2-Mcl-1蛋白質的訊息傳遞,啟動細胞的自我防禦機制以抵抗貝它類澱粉蛋白的傷害,增加細胞的存活能力。
Amyloid-beta (Abeta) is known to induce apoptotic cell death and its underlying mechanism has been studied extensively, but the endogenous protection mechanism that results from Abeta insult is less known. In this study we have found that Abeta 1-42 produced a dose-dependent decrease in cell viability and dose-dependent increase in apoptotic cell death in PC12 cells. Meanwhile, Abeta 1-42 (0.1 uM) increased the phosphorylation of serum- and glucocorticoid-inducible kinase1 (SGK1) at Ser-78 specifically. A parallel increase in ERK1/2, STAT1 and STAT2 phosphorylation and the anti-apoptotic gene Mcl-1 expression was also observed. Transfection of rat siRNAs against ERK1/2, SGK1, STAT1 and STAT2 abolished these effects of Abeta. Transfection of sgkS78D, the constitutively active SGK1, dose-dependently protected against Abeta -induced apoptosis and dose-dependently increased the expression of Mcl-1. SGK1 activation further phosphorylates STAT1 at Tyr-701 and Ser-727 directly, and activates STAT2 at Tyr-690 indirectly. Phosphorylation of STAT1/STAT2 upregulated Mcl-1 expression which in turn protected against Abeta -induced apoptosis. But Mcl-1 siRNA transfection enhanced Abeta -induced apoptosis. Mutation of SGK1 at Ser-78 blocked the effect of Abeta on STAT1/STAT2 phosphorylation and Mcl-1 expression. Further, mutation of STAT1/STAT2 prevented the effect of both Abeta and SGK1 on Mcl-1 expression. These results together showed a novel endogenous protection mechanism that is activated on Abeta insult to mediate cell survival.
目 錄
圖目錄 1
表目錄 4
英文縮寫 5
中文摘要 11
Abstract 14
緒論 16
第一節、阿茲海默氏症 (Alzheimer’s disease) 16
第二節、貝它類澱粉蛋白前驅蛋白 (amyloid precursor protein,APP) 18
第三節、貝它類澱粉蛋白 (β amyloid protein,A��) 20
第四節、貝它類澱粉蛋白導致神經細胞死亡的毒性機制 21
第五節、血清及糖皮質素誘導激酶 (serum and glucocorticoid- inducible kinase,sgk) 24
第六節、血清及糖皮質素誘導激酶的異構型 25
第七節、血清及糖皮質素誘導激酶參與學習與記憶形成的過程 29
第八節、血清及糖皮質素誘導激酶調控細胞存活 31
第九節、訊息傳遞與轉錄活化因子 (signal transducer and activator of transcription,STAT) 33
第十節、STAT蛋白質在細胞生理功能上所扮演的角色 36
第十一節 STAT1及STAT2蛋白質的細胞生理功能 37
第十二節、抗凋亡基因Mcl-1 (myeloid cell leukemia-1,Mcl-1) 的調控 40
第十三節、細胞外信號調節激酶 (extracellular signal-regulated protein kinase,ERK) 45
第十四節、大鼠腎上線之嗜鉻細胞瘤細胞 (PC12 cell) 47
材料與方法 49
一、細胞實驗 49
1. 細胞培養皿之塗抹 49
2. 活化冷凍細胞 49
3. 細胞培養 50
4. 冷凍保存細胞 51
二、實驗動物與飼養 51
三、藥物的製備 52
四、轉形作用 (Transformation) 53
五、小量質體DNA之製備 (Minipreparation of plasmid DNA) 54
六、藥物處理及細胞轉染 (Transfection) 55
七、MTT細胞存活率分析 (MTT cell viability assay) 57
八、TUNEL試驗 (transferase-mediated dUTP nick end labeling staining,TUNEL assay) 58
九、反轉錄聚合連鎖反應 (Reverse Transcription- Polymerase Chain Reaction,RT-PCR) 59
1. RNA之萃取 59
2. 反轉錄酵素 (Reverse Transcriptase) 反應 60
3. 聚合酵素連鎖反應 (Polymerase Chain Reaction,PCR) 60
十、即時定量聚合酶連鎖反應 (Real-Time Polymerase Chain Reaction,real time-PCR) 61
十一、西方墨點法 (Western blot) 62
1. 蛋白質萃取 62
2. 蛋白質濃度測定 62
3. 西方墨點法 63
十二、免疫沈澱反應 (Immunoprecipitation) 65
十三、定點突變 (Site-directed mutagenesis) 66
十四、His融合蛋白之製備 67
十五、離體激酶分析 (In vitro kinase assay) 68
十六、STATs報告基因表現分析 (Reporter Gene Analysis) 69
十七、海馬迴內質體基因轉染及藥物注射 70
十八、灌流 (Perfusion) 71
十九、免疫組織化學染色法 (Immunohistochemistry) 72
結果 74
一、貝它類澱粉蛋白會造成PC12細胞的凋亡現象,同時促進Mcl-1的表現,進而啟動細胞防禦機制 74
二、貝它類澱粉蛋白活化SGK1 Ser 78,調控細胞存活的機制 79
三、SGK會直接活化STAT1蛋白質的磷酸化並間接調控STAT2蛋白質的磷酸化 86
四、貝它類澱粉蛋白透過活化SGK1 Ser 78,而促進STAT1及STAT2的磷酸化 91
五、SGK1是透過STAT1/STAT2的活化,以促進Mcl-1啟動子的活性,使基因和蛋白質表現表現 95
六、SGK1透過活化STAT1及STAT2可以減緩貝它類澱粉蛋白對細胞產生的神經毒性 100
七、利用干擾核醣核苷酸減弱細胞中內生性基因之表現,證明貝它類澱粉蛋白促進Mcl-1的表現,是受到ERK1/2、SGK1及STAT1/STAT2蛋白質所調控的 102
八、貝它類澱粉蛋白在大白鼠海馬迴的區域,同樣透過活化SGK1訊息傳遞以啟動細胞的防禦機制 105
討論 109
結論 125
參考文獻 167
附圖 183
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