跳到主要內容

臺灣博碩士論文加值系統

(3.236.23.193) 您好!臺灣時間:2021/07/26 07:40
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:林冠禎
研究生(外文):Kuan-Jen Lin
論文名稱:以RNA干擾探討PICK1在NIH3T3細胞中的功能
論文名稱(外文):Investigation of PICK1 function in NIH3T3 cells by RNA interference
指導教授:林蔚靖
指導教授(外文):Wey-Jinq Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:75
中文關鍵詞:粒線體的形態和功能
外文關鍵詞:mitochondrial morphology and function
相關次數:
  • 被引用被引用:1
  • 點閱點閱:125
  • 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
PICK1是一個含有PDZ domain的支架蛋白 (scaffold protein),在小鼠纖維母細胞NIH3T3中主要位在粒線體。本實驗室之前的研究發現PICK1可以藉由招攬蛋白激酶C�� (PKC��) 至粒線體上而穩定粒線體膜勢 (mitochondrial membrane potential),因此減少藥物etoposide所誘導之細胞凋亡。本論文則利用RNA干擾降低 NIH3T3細胞中PICK1的表現量進一步探討PICK1在細胞內的功能。粒線體為一個動態的胞器,可以不斷融合 (fusion) 和分裂 (fission),這個過程不僅影響粒線體形態同時也在許多細胞功能上扮演重要的角色。我們首次發現在NIH3T3細胞中降低PICK1的表現會明顯增加粒線體斷裂的比例,相反的增加PICK1的表現會降低粒線體斷裂的比例。顯示PICK1在粒線體的形態上扮演重要的角色。若細胞處理PKC�拲M一抑制劑Gӧ6976則會導致粒線體斷裂的比例明顯的上升,推測在親代細胞中需要PKC�悛漪〝囧蚨�持粒線體的正常型態。而降低PICK1 表現的細胞中粒線體型態則對PKC�悇〝坁漕抰鄔囥�顯減低。由這些結果推測也許PICK1可能藉著招攬PKC�悃麮刓u體以維持其型態。粒線體經由有氧呼吸提供大部分細胞所需能量,降低PICK1表現量會降低細胞ATP的含量以及細胞的耗氧量,並且增加細胞中活性氧分子 (ROS),這些結果顯示粒線體氧化磷酸化不能正常運作而造成。此外,這些細胞產生較多的lactate,顯示降低PICK1可能因影響有氧呼吸因而使細胞轉而依賴無氧呼吸提供能量。我的研究顯示PICK1表現量低,細胞的存活和生長均高度依賴葡萄糖,顯示細胞依賴無氧呼吸來維持生存所需的ATP。就以上的結果推測,降低PICK1的表現可能會藉由影響粒線體膜的完整性而使粒線體的電子傳遞鏈以及維持正常粒線體的型態功能受損。
PCK1 is a PDZ-containing scaffold protein. Previous studies showed that PICK1 stabilized mitochondrial membrane potential by recruiting protein kinase C�� (PKC��) and thus reduced etoposide-induced cell death. In this study the functions of PICK1 were further investigated by using RNA interference. Mitochondria are morphologically dynamic organelles that continuously undergo fusion and fission. This process has been shown to play crucial roles in various cellular functions. I found, in this study, that down-regulation of PICK1 by shRNA in NIH3T3 cells significantly increased mitochondrial fragmentation. Furthermore, maintenance of normal mitochondrial network in parental NIH3T3 cells required PKC�� activity as treatment with a PKC��-selective inhibitor resulted in mitochondrial fragmentation. These results suggest that PICK1 may regulate mitochondrial morphology by recruiting PKC�� to mitochondria. Down-regulation of PICK1 also decreased ATP content and oxygen consumption in NIH3T3 cells suggesting a reduced oxidative phosphorylation. PICK1 knock-down cells may thus rely on anaerobic respiration for their ATP supply. This notion is supported by an increased lactate production and dependence on glucose for survival. Taken together, these observations suggest that reduced levels of PICK1 impair the mitochondrial electron transport chain and the maintenance of normal mitochondrial morphology which may be due to a destabilization of mitochondrial membrane integrity.
目錄……………………………………………………………………………………..1
縮寫表………………………………………………………………………………….2
圖次目錄………………………………………………………………………………4
中文摘要………………………………………………………………………………7
英文摘要………………………………………………………………………………9
緒論..…………………………………………………………………………………...11
研究目標……………………………………………………………………………...18
實驗材料……………………………………………………………………………...19
實驗方法……………………………………………………………………………...22
實驗結果……………………………………………………………………………...29
討論…………………………………………………..………………………………...38
參考文獻……………………………………………………………………………...43
圖表……………………………………………………………………..……………...52
1. Staudinger, J., Zhou, J., Burgess, R., Elledge. S. J., and Olson, E. N. PICK1: a perinuclear binding Protein and substrate for protein kinase C isolated by the yeast two-hybrid system. J. Cell Biol. 128,263-271 (1995).
2. Xu, J., and Xia, J. Structure and Function of PICK1. Neurosignals. 15, 190-201 (2006).
3. Perez, J. L., Khatri, L., Chang, C., Srivastava, S., Osten, P., and Ziff, E. B. PICK1 targets activated protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2. J. Neurosci. 21, 5417-5428 (2001).
4. Staudinger, J., Zhou, J., Burgess, R., Elledge, S. J., and Olson, E. N. PICK1:a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system. J. Cell Biol. 128,263-271 (1995).
5. Wang, W. L., Yeh, S. F., Chang, Y. I., Hsiao, S. F., Lian, W. N., Lin, C. H., Huang, C. Y., and Lin, W. J. PICK1, an Anchoring Protein That Specifically Targets Protein Kinase Cα to Mitochondria Selectively upon Serum Stimulation in NIH 3T3 Cells. J. Cell Biol. 287, 37705–37712 (2003).
6. Wetsel, W. C., Khan, W. A., Merchenthaler, I., Rivera, H., Halpern, A. E., Phung, H. M., Negro-Vilar, A., and Hannun, Y. A. Tissue and cellular distribution of the extended family of protein kinase C isoenzymes. J. Cell Biol. 117,121-133 (1992).
7. Nishizuka, S. Protein kinase C alpha (PKC alpha):regulation and biological function. J. Biochem. Tokyo 132, 669-675 (2002).
8. Wang, W. L., Yeh, S. F., Huang, E. Y., Lu, Y. L., Wang, C. F., Huang, C. Y., and Lin, W. J. Mitochondrial anchoring of PKCα by PICK1 confers resistance to etoposide-induced apoptosis. Apoptosis. 12, 1857-1871 (2007).
9. Zhang, Y., and Chan, D. C. New insights into mitochondrial fusion. FEBS Lett. 581, 2168-2173 (2007).
10. Manoli, I., Alesci, S., Blackman, M. R., Su, Y. A., Rennert, O. M., and Chrousos, G. P. Mitochondria as key components of the stress response. Trends Endocrinol Metab. 18, 190-198 (2007).
11. Hatefi, Y. The mitochondrial electron transport and oxidative phosphorlyayion system. Annu. Rev. Biochem. 54,1015-1069 (1985).
12. Taanman, J. W. The mitochondrial genome:structure,transcription,translation and replication. Biochim. Biophys. Acta. 1410,103-123 (1999).
13. Matsuno-Yagi, A., and Hatefi, Y. Role of energy in oxidative phosphorylation. J. Bioenerg. Biomembr. 20, 481-502 (1988).
14. Golden, T. R., and Melov, S. Mitochondrial DNA mutations,oxidative stress, and aging. Mech. Ageing. Dev. 122,1577-1589 (2001).
15. Wallace, D. C. Mitochondrial disease in man and mouse. Science 283,1482-1488 (1992).
16. Jeˇzek, P., and Hlavat´a, L. Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism. Int. J. Biochem. Cell. Biol. 75, 1357-2725 (2005).
17. Pfeiffer, S., and Mayer, B. Protein tyrosine nitration and peroxynitrite:reply. FASEB. J. 16.1854 (2002).
18. Sagai, M., and Ichinose, T. Age-related changes in lipid peroxidation as measured by ethane, ethylene, butane and pentane in respired gases of rats. Life Sci. 27, 731-738 (1980).
19. Ray, G., and Husain, S. A. Oxidants, antioxidants and carcinogenesis. Indian. J. Exp. Biol. 40, 1213-1232 (2002).
20. Meister, A. New aspects of glutathione biochemistry and transport:selective alteration of glutathione metabolism. Fed. Proc. 43, 3031-3042 (1984).
21. Sies, H. Strategies of antioxidant defense. Eur. J. Biochem. 215, 213-219 (1993).
22. Blokhina, O., Virolainen, E. & Fagerstedt, K.V. Antioxidant,oxidative damage and oxygen deprivation stress:a review. Ann. Bot. London 91, 179-94 (2003).
23. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479-489 (1997).
24. Susin, S. A., Zamzami, N., Castedo, M., Hirsch, T., Marchetti, P., Macho, A., Daugas, E., Geuskens, M., and Kroemer, G. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184, 1331-1341 (1996).
25. Okamoto, K., and Shaw, J. M. Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes. Annu Rev Genet. 39, 503-536 (2005).
26. Hermann, G. J., and Shaw, J. M. Mitochondrial dynamics in yeast. Annu. Rev. Cell. Dev. Biol. 14, 265-303 (1998).
27. Yaffe, M. P. The machinery of mitochondrial inheritance and behavior. Science 283, 1492-1497 (1999).
28. Jensen, R. E., Hobbs, A. E., Cerveny, K. L., and Sesaki, H. Yeast mitochondrial dynamics: fusion, division, segregation, and shape. Microsc. Res. Tech. 51, 573-583 (2000).
29. Lee, S., Jeong, S. Y., Lim, W. C., Kim, S., Park, Y. Y., Sun, X., Youle, R. J., and Cho, H. Mitochondrial fission and fusion mediators, hFis1 and OPA1, modulate cellular senescence. J. Cell Biol. 281, 22977–22983 (2007).
30. Meinhardt, A., Wilhelm, B., and Seitz, J. Expression of mitochondrial marker proteins during spermatogenesis. Hum. Reprod. Update 5, 108-119 (1999).
31. Motta, P. M., Nottola, S. A., Makabe, S., and Heyn, R. Mitochondrial morphology in human fetal and adult female germ cells. Hum. Reprod. 15, Suppl. 2, 129-147 (2000).
32. Chan, D. C. Mitochondrial fusion and fission in mammals. Annu. Rev. Cell. Dev. Biol. 22, 79-99 (2006).
33. Aleardi, A. M., Benard, G., Augereau, O., Malgat, M., Talbot, J. C., Mazat, J. P., Letellier, T., Dachary-Prigent, J., Solaini, G. C., and Rossignol, R. Gradual alteration of mitochondrial structure and function by beta-amyloids: importance of membrane viscosity changes, energy deprivation, reactive oxygen species production, and cytochrome c release. J. Bioenerg. Biomembr. 37, 207-225 (2005).
34. Heath-Engel, H. M., and Shore, G. C. Mitochondrial membrane dynamics, cristae remodelling and apoptosis. Biochim Biophys Acta 1763, 549-560 (2006).
35. Bereiter-Hahn, J., and Voth, M. Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria. Microsc Res Tech. 27, 198-219 (1994).
36. Griparic, L., van der Wel, N. N., Orozco, I. J., Peters, P. J., and van der Bliek, A. M. Loss of the intermembrane space protein Mgm1/OPA1 induces swelling and localized constrictions along the lengths of mitochondria. J. Biol. Chem. 279, 18792-1898 (2004).
37. Karbowski, M., and Youle, R. J. Dynamics of mitochondrial morphology in healthy cells and during apoptosis. Cell Death. Differ. 10, 870–880 (2003).
38. Knott , A. B., and Bossy-Wetzel, E. Impairing the mitochondrial fission and fusion balance: a new mechanism of neurodegeneration. Ann. N. Y. Acad. Sci. 1147, 283-92 (2008).
39. Cerveny, K. L., Tamura, Y., Zhang, Z., Jensen, R. E., and Sesaki, H. Regulation of mitochondrial fusion and division. Trends Cell Biol. 17, 563-569 (2007).
40. Benard, G., Bellance, N., James, D., Parrone, P., Fernandez, H., Letellier, T., and Rossignol, R. Mitochondrial bioenergetics and structural network organization. Science 120, 838-848 (2007).
41. Chan, D. C. Mitochondrial fusion and fission in mammals. Annu. Rev. Cell. Dev. Biol. 22, 79-99 (2006).
42. Koshiba, T., Detmer, S. A., Kaiser, J. T., Chen. H., McCaffery, J. M., and Chan, D. C. Structural basis of mitochondrial tethering by mitofusin complexes. Science 305, 858-862 (2004).
43. Mozdy, A. D., McCaffery, J. M., and Shaw, J. M. Dnm1p GTPase-mediated mitochondrial fission is a multi-step process requiring the novel integral membrane component Fis1p. J. Cell Biol. 151, 367-380 (2000).
44. Chen, H., Chomyn, A., and Chan, D. C. Disruption of fusion results in mitochondrial heterogeneity and dysfunction. J. Cell Biol. 280, 26185-26192 (2005).
45. Tieu, Q., Okreglak, V., Naylor, K., and Nunnari, J. The WD repeat protein, Mdv1p, functions as a molecular adaptor by interacting with Dnm1p and Fis1p during mitochondrial fission. J. Cell Biol. 158, 445-452 (2002).
46. Shaw, J. M., and Nunnari, J. Mitochondrial dynamics and division in budding yeast. Trends Cell Biol. 12, 178-184 (2002).
47. Benard, G., Bellance, N., James, D., Parrone, P., Fernandez, H., Letellier, T., and Rossignol, R. Mitochondrial bioenergetics and structural network organization. Science 120, 838-848 (2007).
48. Carlucci, A., Lignitto, L., and Feliciello, A. Control of mitochondria dynamics and oxidative metabolism by cAMP, AKAPs and the proteasome. Trends Cell Biol. 18, 604-613 (2008).
49. Suen, D. F., Norris, K. L., and Youle, R. J. Mitochondrial dynamics and apoptosis. Genes Dev. 22, 1577–1590 (2008).
50. Frezza, C., Cipolat, S., Martins de Brito, O., Micaroni, M., Beznoussenko, G. V., Rudka, T., Bartoli, D., Polishuck, R.S., Danial, N. N., and De Strooper, B. OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 126, 177–189 (2006).
51. Karbowski, M., Norris, K. L., Cleland, M. M., Jeong, S. Y., and Youle, R. J. Role of Bax and Bak in mitochondrial morphogenesis. Nature 443, 658–662 (2006).
52. Ulle, E., Djikeng, A., Shi, H., and Tschudi, C. RNA interference: advances and questions. Philos Trans R Soc Lond B Biol Sci. 357, 65-70 (2002).
53. Heggeness, M. H., Simon, V., and Singer, S. J. Association of mitochondria with microtubules in cultured cells. Proc. Natl Acad. Sci. USA 75, 3863-3866 (1978).
54. Juel, C., and Halestrap, A. P. Lactate transport in skeletal muscle - role and regulation of the monocarboxylate transporter. J Physiol. 517.3, 633-642 (1999).
55. Kuznetsov, A. V., and Margreiter, R. Heterogeneity of Mitochondria and Mitochondrial Function within Cells as Another Level of Mitochondrial Complexity. Int J Mol Sci. 10, 1911-1929 (2009).
56. Mootha, V. K., Wei, M. C., Buttle, K. F., Scorranol, L., Panoutsakopoulou, V., Mannella, C. A., and Korsmeyer, S. J. A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c. EMBO J. 20, 661-671 (2001).
57. Dolinsky, V. W., and Dyck, J. R. Role of AMP-activated protein kinase in healthy and diseased hearts. Am J Physiol Heart Circ Physiol. 291, H2557-H2569 (2006).
58. Young, L. H., Li, J., Baron, S. J., and Russell, R. R. AMP-activated protein kinase: a key stress signaling pathway in the heart. Trends Cardiovasc Med. 15, 110-118 (2005).
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top