跳到主要內容

臺灣博碩士論文加值系統

(44.192.92.49) 您好!臺灣時間:2023/06/08 07:44
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:溫世賢
論文名稱:斑馬魚HIF2α 在胚胎發育時期的穩定性
論文名稱(外文):Stability of hypoxia-inducible factor 2 alpha in zebrafish embryo
指導教授:胡清華胡清華引用關係
指導教授(外文):Chin-Hwa Hu
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:2011
畢業學年度:99
語文別:中文
論文頁數:66
中文關鍵詞:低氧誘發蛋白
外文關鍵詞:hypoxia-inducible factor 2 alpha
相關次數:
  • 被引用被引用:1
  • 點閱點閱:159
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
低氧誘發蛋白 (HIFs)是調控細胞內氧恆定生理的主要角色,其本
身為一種轉錄因子,是由對氧濃度相當敏感的alpha次單元和另一個對
氧濃度相當穩定的beta次單元所共同組合成的異偶合體。在面臨缺氧的
環境時低氧誘發蛋白會啟動血球生成、血管新生及無氧代謝等調節適
應的作用。低氧也被認為是調控各種組織型態發育過程的關鍵,像是
血管、血球生成,肺,腦及軟骨的發育。常氧情況下,prolyl hydroxylase
(PHD)具有脯胺酸氫氧化的活性,會在HIFα (HIF1α及HIF2α) ODD
domain上的脯胺酸進行氫氧化作用,氫氧化的HIFα會被Hippel–Lindau
(VHL)E3 ligase complex給辨認,然後協同E2 ligase將氫氧化的HIF加上ubiutin,進而走向蛋白分解路徑。而在缺氧環境下,PHD活性被抑制,
使得HIFα穩定,然後與HIFβ/ARNT結合後,藉由辨識hypoxia response
element (HRE)序列,啟動下游目標基因的表現。
先前實驗中,HIF2α會透過survivin影響腦部中樞神經細胞的存活及
中樞神經細胞分化,顯示HIF2α蛋白在胚胎發育過程中具有相當高的表
現,才會進行下游基因調控的功能,但是對於在常氧環境下胚胎細胞
內如何維持HIF2α的活性則並不清楚。因此本論文主要在探討斑馬魚
HIF2α蛋白在胚胎發育過程中的穩定性,並且探討影響其穩定之因素。
實驗結果發現斑馬魚HIF2α ODD結構連結上綠螢光報導蛋白(EGFP)
在胚胎內非常不安定,但將其ODD結構上的2個脯胺酸點突變或是添加
hydorxylase抑制劑DMOG均可使ODD-EGFP轉為相當安定的蛋白,顯
示HIF2α ODD結構在胚胎內可能受到PHD及VHL調控作用而造成結構
不安定性。phd2, phd3及vhl弱化並不能增加HIF2α ODD的穩定性,但可
以些微提升內生性HIF2α蛋白質量,顯示phd2, phd3及vhl並未直接參與
調節HIF2α ODD的工作。其次將全長的HIF2α連結上綠螢光報導蛋白,
在胚胎細胞內無法表現,但是Western blot卻可偵測到內生性HIF2α蛋白
的存在,顯示這些HIF2α蛋白具有快速的蛋白質更新(protein turnover)
的特性,亦即快速轉譯與快速分解的特性,此可能是細胞快速調適環
境變遷的一種策略所造成的結果。本研究顯示胚胎內應有其他的PHD
與VHL相關蛋白參與了調節HIF2α ODD結構安定性的工作。
目 錄
謝 辭.................................................................................................................................i
摘 要................................................................................................................................ii
Abstract ............................................................................................................................iv
目 錄...............................................................................................................................vi
壹、 前言.............................................................................................................. ‐ 1 ‐
一、低氧誘發蛋白(hypoxia – inducible factor ,HIFs) ...................... ‐ 1 ‐
二、脯胺酸氫氧化酶(Prolyl Hydroxylase,PHD) ........................................ ‐ 4 ‐
三、von Hippel-Lindau tumor suppressor gene product (pVHL) .......... ‐ 6 ‐
四、其他影響HIFα 蛋白穩定因子.................................................................. ‐ 7 ‐
五、研究目的...................................................................................................... ‐ 9 ‐
貳、 實驗方法.................................................................................................... ‐ 10 ‐
一、斑馬魚egln1/phd2、egln3/phd3 及vhl 探針(probe)製作................ ‐ 10 ‐
二、RNA 全覆式原位雜交(RNA wholemount
in situ hybridization) ‐ 15

三、建構重組質體............................................................................................ ‐ 16 ‐
四、藥物Dimethyloxaloylglycine(DMOG)處理........................................... ‐ 18 ‐
五、建構點突變HIF2α ODD domain - EGFP 融合蛋白表現載體.............. ‐ 18 ‐
六、QPCR ............................................................................................................ ‐ 19 ‐
七、斑馬魚胚胎顯微注射 (Microinjection) .............................................. ‐ 19 ‐
八、蛋白質分析................................................................................................ ‐ 22 ‐
九、斑馬魚胚胎浸泡氯化鎘(CdCl2) ............................................................... ‐ 25 ‐
参、實驗結果............................................................................................................ ‐ 26 ‐
1、斑馬魚egln1/phd2、egln3/phd3、hif2α 及vhl 在胚胎表現情形.... ‐ 26 ‐
2、胚胎發育時期斑馬魚HIF2α ODD 區域結構安定性分析........................ ‐ 26 ‐
3、外加性報導質體在胚胎細胞及卵黃囊中的表現差異.............................. ‐ 27 ‐
4、Dimethyloxaloylglycine(DMOG)對HIF2αODD 穩定性的影響.............. ‐ 27 ‐
5、HIF2α ODD domain 脯胺酸對HIF2α ODD 穩定性的影響..................... ‐ 28 ‐
6、胚胎發育時期HIF2α 蛋白穩定分析........................................................ ‐ 28 ‐
7、egln3/phd3 morpholino 效率測試........................................................... ‐ 29 ‐
8、egln1/phd2、egln3/phd3 及vhl 的弱化對於重組質體表現影響......... ‐ 29 ‐
9、egln1/phd2、egln3/phd3 及vhl 的弱化對於胚胎內HIF2α 蛋白表現的影
響........................................................................................................................ ‐ 29 ‐
10、HIF2α 蛋白結構對於自身穩定性的影響............................................... ‐ 30 ‐
11、重金屬鎘對於斑馬魚胚胎HIF2α 蛋白表現影響.................................. ‐ 30 ‐
肆、討論.................................................................................................................... ‐ 32 ‐
伍、圖表.................................................................................................................... ‐ 37 ‐
陸、參考文獻............................................................................................................ ‐ 50 ‐
柒、附錄.................................................................................................................... ‐ 55 ‐
一、實驗材料.................................................................................................... ‐ 55 ‐
二、藥品配製.................................................................................................... ‐ 58 ‐
三、附圖............................................................................................................ ‐ 63 ‐
‐ 50 ‐
陸、參考文獻
李柏翰,2010。HIF2α 在視網膜的發是功能。國立台灣海洋大學生物科技研究
所碩士論文。
黃冠銘,2011。重金屬汞與鎘對斑馬魚胚胎中DNA 錯誤配對(MMR)辨認蛋白MutS
homologag2 及6(MutSα)基因表現之影響與可能機制。國立台灣海洋大
學生物科技研究所碩士論文。
Berra, E., Benizri, E., Ginouves, A., Volmat, V., Roux, D., and Pouyssegur,
J. (2003). HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low
steady-state levels of HIF-1 alpha in normoxia. Embo Journal 22,
4082-4090.
Blouin, C.C., Page, E.L., Soucy, G.M., and Richard, D.E. (2004). Hypoxic
gene activation by lipopolysaccharide in macrophages: Implication of
hypoxia-inducible factor 1 alpha. Blood 103, 1124-1130.
Bruick, R.K. (2003). Oxygen sensing in the hypoxic response pathway:
regulation of the hypoxia-inducible transcription factor. Genes &
Development 17, 2614-2623.
Brunelle, J.K., Bell, E.L., Quesada, N.M., Vercauteren, K., Tiranti, V.,
Zeviani, M., Scarpulla, R.C., and Chandel, N.S. (2005). Oxygen sensing
requires mitochondrial ROS but not oxidative phosphorylation. Cell Metab
1, 409-414.
Chandel, N.S., McClintock, D.S., Feliciano, C.E., Wood, T.M., Melendez,
J.A., Rodriguez, A.M., and Schumacker, P.T. (2000). Reactive oxygen
species generated at mitochondrial complex III stabilize
hypoxia-inducible factor-1 alpha during hypoxia - A mechanism of O-2
sensing. Journal of Biological Chemistry 275, 25130-25138.
Compernolle, V., Brusselmans, K., Acker, T., Hoet, P., Tjwa, M., Beck,
H., Plaisance, S., Dor, Y., Keshet, E., Lupu, F., et al. (2002). Loss of
HIF-2 and inhibition of VEGF impair fetal lung maturation, whereas
treatment with VEGF prevents fatal respiratory distress in premature mice.
Nat Med 8, 702-710.
Counts, D.F., Cardinale, G.J., and Udenfriend, S. (1978). Prolyl
hydroxylase half reaction: peptidyl prolyl-independent decarboxylation
of alpha-ketoglutarate. Proc Natl Acad Sci U S A 75, 2145-2149.
Crews, S.T. (1998). Control of cell lineage-specific development and
transcription by bHLH-PAS proteins. Genes & Development 12, 607-620.
Ema, M., Hirota, K., Mimura, J., Abe, H., Yodoi, J., Sogawa, K., Poellinger,
L., and Fujii-Kuriyama, Y. (1999). Molecular mechanisms of transcription
‐ 51 ‐
activation by HLF and HIF1 alpha in response to hypoxia: their
stabilization and redox signal-induced interaction with CBP/p300. Embo
Journal 18, 1905-1914.
Ema, M., Taya, S., Yokotani, N., Sogawa, K., Matsuda, Y., and
FujiiKuriyama, Y. (1997). A novel bHLH-PAS factor with close sequence
similarity to hypoxia-inducible factor 1 alpha regulates the VEGF
expression and is potentially involved in lung and vascular development.
Proceedings of the National Academy of Sciences of the United States of
America 94, 4273-4278.
Eng, C., Kiuru, M., Fernandez, M.J., and Aaltonen, L.A. (2003). A role
for mitochondrial enzymes in inherited neoplasia and beyond. Nat Rev
Cancer 3, 193-202.
Epstein, A.C.R., Gleadle, J.M., McNeill, L.A., Hewitson, K.S., O'Rourke,
J., Mole, D.R., Mukherji, M., Metzen, E., Wilson, M.I., Dhanda, A., et
al. (2001). C-elegans EGL-9 and mammalian homologs define a family of
dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107, 43-54.
Gerald, D., Berra, E., Frapart, Y.M., Chan, D.A., Giaccia, A.J., Mansuy,
D., Pouyssegur, J., Yaniv, M., and Mechta-Grigoriou, F. (2004). JunD
reduces tumor angiogenesis by protecting cells from oxidative stress.
Cell 118, 781-794.
Gordeuk, V.R., Stockton, D.W., and Prchal, J.T. (2005). Congenital
polycythemias/erythrocytoses. Haematologica 90, 109-116.
Haase, V.H. (2005). The VHL tumor suppressor in development and disease:
Functional studies in mice by conditional gene targeting. Seminars in Cell
& Developmental Biology 16, 564-574.
Hirsila, M., Koivunen, P., Gunzler, V., Kivirikko, K.I., and Myllyharju,
J. (2003). Characterization of the human prolyl 4-hydroxylases that
modify the hypoxia-inducible factor. Journal of Biological Chemistry 278,
30772-30780.
Huang, L.E., Arany, Z., Livingston, D.M., and Bunn, H.F. (1996).
Activation of hypoxia-inducible transcription factor depends primarily
upon redox-sensitive stabilization of its alpha subunit. Journal of
Biological Chemistry 271, 32253-32259.
Huang, L.E., Gu, J., Schau, M., and Bunn, H.F. (1998). Regulation of
hypoxia-inducible factor 1 alpha is mediated by an O-2-dependent
degradation domain via the ubiquitin-proteasome pathway. Proceedings of
the National Academy of Sciences of the United States of America 95,
7987-7992.
‐ 52 ‐
Hyvarinen, J., Parikka, M., Sormunen, R., Ramet, M., Tryggvason, K.,
Kivirikko, K.I., Myllyharju, J., and Koivunen, P. (2010). Deficiency of
a Transmembrane Prolyl 4-Hydroxylase in the Zebrafish Leads to Basement
Membrane Defects and Compromised Kidney Function. J Biol Chem 285,
42023-42032.
Isaacs, J.S., Jung, Y.J., Mole, D.R., Lee, S., Torres-Cabala, C., Chung,
Y.L., Merino, M., Trepel, J., Zbar, B., Toro, J., et al. (2005). HIF
overexpression correlates with biallelic loss of fumarate hydratase in
renal cancer: Novel role of fumarate in regulation of HIF stability.
Cancer Cell 8, 143-153.
Kaelin, W.G. (2005). The von Hippel-Lindau protein, HIF hydroxylation,
and oxygen sensing. Biochemical and Biophysical Research Communications
338, 627-638.
Kallio, P.J., Okamoto, K., O'Brien, S., Carrero, P., Makino, Y., Tanaka,
H., and Poellinger, L. (1998). Signal transduction in hypoxic cells:
inducible nuclear translocation and recruitment of the CBP/p300
coactivator by the hypoxia-inducible factor-1 alpha. Embo Journal 17,
6573-6586.
Kallio, P.J., Pongratz, I., Gradin, K., McGuire, J., and Poellinger, L.
(1997). Activation of hypoxia-inducible factor 1 alpha:
Posttranscriptional regulation and conformational change by recruitment
of the Arnt transcription factor. Proceedings of the National Academy of
Sciences of the United States of America 94, 5667-5672.
Kallio, P.J., Wilson, W.J., O'Brien, S., Makino, Y., and Poellinger, L.
(1999). Regulation of the hypoxia-inducible transcription factor 1 alpha
by the ubiquitin-proteasome pathway. Journal of Biological Chemistry 274,
6519-6525.
Ko, C.Y., Tsai, M.Y., Tseng, W.F., Cheng, C.H., Huang, C.R., Wu, J.S.,
Chung, H.Y., Hsieh, C.S., Sun, C.K., Hwang, S.P., et al. (2011).
Integration of CNS survival and differentiation by HIF2alpha. Cell Death
Differ.
Kotch, L.E., Iyer, N.V., Laughner, E., and Semenza, G.L. (1999). Defective
vascularization of HIF-1 alpha-null embryos is not associated with VEGF
deficiency but with mesenchymal cell death. Dev Biol 209, 254-267.
Lu, H.S., Dalgard, C.L., Mohyeldin, A., McFate, T., Tait, A.S., and Verma,
A. (2005). Reversible inactivation of HIF-1 prolyl hydroxylases allows
cell metabolism to control basal HIF-1. Journal of Biological Chemistry
280, 41928-41939.
‐ 53 ‐
Madan, A., Varma, S., and Cohen, H.J. (2002). Developmental
stage-specific expression of the alpha and beta subunits of the HIF-1
protein in the mouse and human fetus. Mol Genet Metab 75, 244-249.
Moeller, B.J., Cao, Y.T., Li, C.Y., and Dewhirst, M.W. (2004). Radiation
activates HIF-1 to regulate vascular radiosensitivity in tumors: Role of
reoxygenation, free radicals, and stress granules. Cancer Cell 5,
429-441.
Page, E.L., Robitaille, G.A., Pouyssegur, J., and Richard, D.E. (2002).
Induction of hypoxia-inducible factor-1 alpha by transcriptional and
translational mechanisms. Journal of Biological Chemistry 277,
48403-48409.
Pugh, C.W., and Ratcliffe, P.J. (2003). Regulation of angiogenesis by
hypoxia: role of the HIF system. Nat Med 9, 677-684.
Reyes, A.E., Rojas, D.A., Perez-Munizaga, D.A., Centanin, L., Antonelli,
M., Wappner, P., and Allende, M.L. (2007). Cloning of hif-1 alpha and hif-2
alpha and mRNA expression pattern during development in zebrafish. Gene
Expression Patterns 7, 339-345.
Richard, S., David, P., Marsot-Dupuch, K., Giraud, S., Beroud, C., and
Resche, F. (2000). Central nervous system hemangioblastomas,
endolymphatic sac tumors, and von Hippel-Lindau disease. Neurosurg Rev
23, 1-22.
Roitbak, T., Surviladze, Z., and Cunningham, L.A. (2011). Continuous
Expression of HIF-1 alpha in Neural Stem/Progenitor Cells. Cell Mol
Neurobiol 31, 119-133.
Rojas, D.A., Perez-Munizaga, D.A., Centanin, L., Antonelli, M., Wappner,
P., Allende, M.L., and Reyes, A.E. (2007). Cloning of hif-1 alpha and hif-2
alpha and mRNA expression pattern during development in zebrafish. Gene
Expr Patterns 7, 339-345.
Semenza, G.L. (1999). Regulation of mammalian O-2 homeostasis by hypoxiainducible
factor 1. Annual Review of Cell and Developmental Biology 15,
551-578.
Stohs, S.J., and Bagchi, D. (1995). Oxidative Mechanisms in the Toxicity
of Metal-Ions. Free Radical Bio Med 18, 321-336.
Tian, H., McKnight, S.L., and Russell, D.W. (1997). Endothelial PAS domain
protein 1 (EPAS1), a transcription factor selectively expressed in
endothelial cells. Genes & Development 11, 72-82.
van Eeden, F.J., van Rooijen, E., Voest, E.E., Logister, I., Korving, J.,
Schwerte, T., Schulte-Merker, S., and Giles, R.H. (2009). Zebrafish
‐ 54 ‐
mutants in the von Hippel-Lindau tumor suppressor display a hypoxic
response and recapitulate key aspects of Chuvash polycythemia. Blood 113,
6449-6460.
van Rooijen, E., Voest, E.E., Logister, I., Korving, J., Schwerte, T.,
Schulte-Merker, S., Giles, R.H., and van Eeden, F.J. (2009). Zebrafish
mutants in the von Hippel-Lindau tumor suppressor display a hypoxic
response and recapitulate key aspects of Chuvash polycythemia. Blood 113,
6449-6460.
Wang, G.L., Jiang, B.H., Rue, E.A., and Semenza, G.L. (1995).
Hypoxia-Inducible Factor-1 Is a Basic-Helix-Loop-Helix-Pas Heterodimer
Regulated by Cellular O-2 Tension. Proceedings of the National Academy
of Sciences of the United States of America 92, 5510-5514.
Wang, G.L., and Semenza, G.L. (1995). Purification and Characterization
of Hypoxia-Inducible Factor-1. Journal of Biological Chemistry 270,
1230-1237.
Wang, M., Kirk, J.S., Venkataraman, S., Domann, F.E., Zhang, H.J., Schafer,
F.Q., Flanagan, S.W., Weydert, C.J., Spitz, D.R., Buettner, G.R., et al.
(2005). Manganese superoxide dismutase suppresses hypoxic induction of
hypoxia-inducible factor-1 alpha and vascular endothelial growth factor.
Oncogene 24, 8154-8166.
Wiesener, M.S., Turley, H., Allen, W.E., Willam, C., Eckardt, K.U., Talks,
K.L., Wood, S.M., Gatter, K.C., Harris, A.L., Pugh, C.W., et al. (1998).
Induction of endothelial PAS domain protein-1 by hypoxia:
Characterization and comparison with hypoxia-inducible factor-1 alpha.
Blood 92, 2260-2268.
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top