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研究生:吳玉蘭
研究生(外文):Lan-Yu Wu
論文名稱:BNIP3蛋白涉及內質網壓力誘導小鼠神經母細胞死亡
論文名稱(外文):BNIP3 Involves Endoplasmic Reticulum Stress-induced Cell Death in Murine Neuroblastoma Cells
指導教授:唐世杰
指導教授(外文):Shye-Jye Tang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:58
中文關鍵詞:內質網壓力小鼠神經母細胞
外文關鍵詞:BNIP3ER stress
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Bcl-2 nineteen-kilodalton interacting protein (BNIP3)為Bcl-2家族中調控細胞死亡因子的Bcl-2 homology domain (BH3)-only成員之一。BNIP3含有易降解的PEST domain,以及位於C端的高保守性transmembrane (TM) domain,經細胞內壓力,TM domain會促使BNIP3形成穩定的雙聚體複合物(homodimerization complexes)並與粒線體外膜結合。文獻指出,去除BNIP3 TM domain(BNIP3△TM)會降低BNIP3促進細胞死亡能力,可能是TM domain缺陷無法與粒線體外膜結合造成的結果。實驗結果發現BNIP3△TM主要以單體(monomer)形式表現,表示BNIP3的形成雙體為細胞死亡的關鍵因素。研究指出,BNIP3可被磷酸化,而磷酸化BNIP3與BNIP3誘導細胞死亡的作用有關。BNIP3會促細胞凋亡(apoptotic)或非細胞凋亡的細胞死亡路徑,如細胞自噬(autophagy)或細胞壞死(necrosis)。
本實驗利用鈣離子載體A23187處理Neuro-2a細胞導致細胞染色質濃縮(DNA condensation)、細胞自噬(Autophagy)以及粒線體失活的現象之外,同時BNIP3的表現量增加,表示BNIP3參與細胞死亡調控。研究中Neuro-2a細胞轉染 pEGFP-BNIP3處理蛋白酶抑制劑MG132,GFP-BNIP3表現量增加,表示GFP-BNIP3經由proteasome -dependent degradation pathway進行蛋白質降解。
此實驗將BNIP3序列的酪胺酸突變為丙胺酸,製備無法磷酸化的BNIP3(GFP-BNIP3 A33A92A175),或天冬胺酸,製備擬磷酸化的BNIP3(GFP-BNIP3 D33D92D175),結果表示GFP-BNIP3 DDD不形成雙體。進一步將Neuro-2a細胞轉染pEGFP-BNIP3 Y33Y92D175或pEGFP-BNIP3 D33D92Y175,結果發現GFP-BNIP3 D33D92Y175不形成雙體,顯示由Y175對於BNIP3形成雙體有重要的作用,而細胞內BNIP3大量表現會造成粒線體失活。

Bcl-2 nineteen-kilodalton interacting protein (BNIP3), the BH3-only protein, is members of the Bcl-2 family of cell death-regulating factors. The major domains found in BNIP3 contains the PEST domain that targets BNIP3 for degradation, and a conserved transmembrane (TM)domain at its C-terminus ,which targets BNIP3 to form stable homodimerization complexes and localize to the outer membrane of the mitochondria. Several studies have shown that the deletion of the TM domain (△TM) abolished the pro-death activity of the BNIP3. It is possible that this functional defect of the BNIP3△TM mutants might be the result of their inability to localize in the mitochondria. BNIP3△TM major form is monomers, that appears that the unique stable dimerization activity of BNIP3 is not critical for its cell death activity. BNIP3 was found that phosphorylation and elevation of phospho-BNIP3 resulted in apoptotic or nonapoptotic cell death, autophagy or necrosis.
In this study , Neuro-2a cells were treated with Calcium ionophore (A23187), The A23187-induced cell death was showed by DNA condensation, autophagy, mitochondria dysfunction, and the up-regulation of BNIP3 was found in Neuro-2a cells. Proteasome inhibitor, MG132, increased the stability of the protein, indicating that BNIP3 degradation is through proteasome -dependent pathway. To characterize tyrosine phosphorylation involving BNIP3-dependent cell death, tyrosines of BNIP3 were mutated to Alanine (GFP-BNIP3 A33A92A175)to form unphosphorylated BNIP3 or Aspartic acid(GFP-BNIP3 D33D92D175) to mimic tyrosine phosphorylation. After transfection BNIP3 D33D92D175 forms monomers, and the mitochondria dysfunction when BNIP3 overexpression in cells .

目錄

謝辭 I
摘要 II
英文摘要 IV
目錄 VI
圖目錄 VIII
附圖 50
壹、序論 1
1. 氧化壓力(OXIDATIVE STRESS) 1
2. 內質網壓力(ENDOPLASMIC RETICULUM STRESS) 1
3. BCL-2蛋白質家族(BCL-2 FAMILY) 2
4. BNIP3蛋白質 3
貳、研究動機 5
参、實驗材料 6
1. 細胞培養 (CELL CULTURE) 6
2. 酵素 6
3. 試劑 7
4. MAKER 7
5. DNA 8
6. 抗體 8
7. PCR PRIMER 9
8. 緩衝液 (BUFFER SOLUTION) 9
肆、實驗方法 11
1. 冷凍細胞活化 11
2. 細胞繼代 11
3. 計算細胞濃度 12
4. 細胞冷凍保存 12
5. 西方墨點法 ( WESTERN BLOTTING ) 12
6. 反轉錄連鎖聚合反應 (RT-PCR) 16
7. 染色質濃縮(DNA CONDENSATION) 18
8. 細胞自噬(AUTOPHAGY) 19
9. 粒線體活性 19
伍、研究結果 20
陸、討論 25
柒、參考文獻 29



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