跳到主要內容

臺灣博碩士論文加值系統

(44.211.24.175) 您好!臺灣時間:2024/11/13 05:34
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:簡彤縈
研究生(外文):Tong-Ying Chien
論文名稱:Minocycline對普拿疼造成的肝細胞毒性的影響以及在內質網壓力所扮演的角色
論文名稱(外文):The Effects of Minocycline on Acetaminophen-inducedhepatotoxicity and the roles of ER stress
指導教授:廖經倫朱恆成朱恆成引用關係
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:57
中文關鍵詞:普拿疼內質網壓力抗生素猛爆性肝炎存活率表現量
外文關鍵詞:minocyclineER stress
相關次數:
  • 被引用被引用:0
  • 點閱點閱:489
  • 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
普拿疼(acetaminophen)是臨床上是經常使用的解熱、止痛藥物,在使用上相當方便又有效,但服用過量時,不論是在人體或是實驗動物模式都會造成嚴重的猛爆性肝炎(acute or fulminant hepatitis) ,其造成肝臟毒性有:粒腺體功能的喪失、大量超氧化物的產生肝細胞的凋亡以及壞死。在體外實驗的模式裡,細胞在毒性劑量普拿疼的給予之後會造成細胞凋亡引起細胞死亡,以及粒腺體功能的喪失。另外,有報導指出過量普拿疼會造成腎細胞的ER stress。Minocycline是一種tetracycline類的抗生素,除了廣效抗生素的使用之外另有抗氧化、清除自由基、抗細胞凋亡的效果。由前人的研究結果得知minocycline可以保護由致死劑量普拿疼引起之小鼠猛爆性肝炎,藉由減少過氧化物以及一氧化氮的含量、甚至是降低cytochrome c 的釋放。在本論文裡我們進一步探討在缺乏免疫系統時,minocycline對普拿疼造成之細胞毒性在體外實驗模式中是否也具有同樣保護的效果。藉由觀察minocycline對普拿疼造成之細胞毒性之後細胞的存活率、細胞凋亡和細胞壞死、以及活化IRE1-XBP1路徑的方面來探討minocycline對普拿疼造成之細胞毒性是否有保護的效果。此外,在本論文中欲進一步釐清minocycline保護小鼠由致死劑量普拿疼所引起的猛爆性肝炎是否也與ER Stress有所關聯。我們藉由分析IRE1-XBP1路徑的活化以及CHOP/GADD153蛋白質的表現量來了解minocycline是否具有抑制普拿疼造成小鼠肝臟ER Stress的發生的效果。實驗結果顯示,minocycline的確會降低由普拿疼引起小鼠肝臟CHOP/GADD153蛋白質的表現量而達成降低普拿疼造成之小鼠肝臟ER stress的功能。另外在體外實驗模式中,我們沒有看到minocycline對普拿疼造成之細胞凋亡以及壞死具有明顯保護的效果,這部分還可以再進一步實驗來釐清。藉由本論文的研究結果,藉由抑制普拿疼造成之ER stress可用來當作治療由普拿疼引起之肝毒性的目標。
Acetaminophen (APAP) is a safe and effective analgesic/antipyretic drug at therapeutic levels. However, an acute or cumulative overdose can cause severe liver injury such as acute or fulminant hepatitis. APAP-induced apoptosis and necrosis in both in vivo and in vitro system. It is reported that APAP-induced endoplasmic reticulum (ER) stress, characterized by CHOP/GADD153 upregulation in renal tubular cells. Minocycline, a tetracycline derivative, possesses anti-inflammatory and antioxidative properties, including depletion of oxygen radical, scavenging of peroxinitrite, and inhibition of inductible nitric oxide synthase (iNOS). In the previous study, minocycline can protect APAP-induced fulminant hepatitis in C57BL/6J mice. The protective effect of minocycline on hepatitis in vivo mediated by hepatocyte itself or other immune response is still uncleanly elucidated. Here, we want to study the protection of minocycline on APAP-induced hepatotoxicity on hepatocyte cell lines by analysing the cell viability, apoptosis, necrosis, and the IRE1-XBP1 pathway activation. Further, we study the protection of minocycline on APAP-induced ER stress by using Western Blot and RT-PCR. In this study, pretreatment of minocycline showed a downregulation of CHOP/GADD153 in C57BL/6J mice treated with 800 mg/kg APAP. The results suggest that ER stress as a therapeutic target in APAP-induced hepatotoxicity in our model. But the pretreatment of minocycline did not protect HepG2 and FL83B cells from APAP-induced apoptosis and necrosis. And more experiments should be performed to help us to answer the questions in the future.
目 錄 I
圖目錄 III
中文摘要 V
英文摘要 V
前言 1
一、什麼是普拿疼(Acetaminophen) 1
二、普拿疼的代謝與其造成之肝毒性致病機轉 1
1. 普拿疼代謝後所引起之肝毒性 1
2. 粒腺體的失能在普拿疼所引起之肝毒性扮演的角色 1
3. 細胞凋亡與細胞壞死在普拿疼引起之肝毒性所扮演的角色 2
4. 普拿疼造成的肝毒性與免疫系統的關係 3
5. ER Stress在普拿疼所引起之肝毒性在細胞凋亡中所扮演的角色 3
三、Minocycline 的介紹 4
四、Minocycline 可以保護C57BL/6J小鼠因致死劑量普拿疼引起之猛爆性肝炎 5
五、在本論文裡我的實驗目的 5
材料與方法 6
一、細胞培養 6
1、細胞株 6
2、細胞株培養與繼代 6
二、反應試劑以及藥物 6
三、本論文所使用之抗體 7
四、生物反應試劑組 7
五、本論文所用的引子 7
六、RNA的抽取以及反轉錄-聚合酵素連鎖反應 7
七、西方墨點法 8
八、實驗老鼠品系 8
結果 9
一、普拿疼(acetaminophen)於體外引發小鼠肝臟細胞株(FL83B)的細胞毒性模式之建立 9
二、Minocycline於體外小鼠肝臟細胞株(FL83B)劑量之選擇 9
三、Minocycline的前處理在普拿疼於體外引發小鼠肝臟細胞株(FL83B)的細胞毒性之影響 9
1. 前處理minocycline 24小時之後再給予普拿疼24小時其細胞毒性之影響 9
2. 前處理minocycline 24小時之後再給予普拿疼48小時其細胞毒性之影響 10
四、普拿疼於體外造成小鼠肝臟細胞株(FL83B)的細胞凋亡以及細胞壞死於不同時間點之情形 10
五、Minocycline的前處理在普拿疼於體外引發小鼠肝臟細胞株(FL83B)的細胞凋亡以及細胞壞死之影響 10
1. 前處理Minocycline 18小時對之後給予之普拿疼24小時造成小鼠肝臟細胞株(FL83B)其細胞凋亡以及細胞壞死的影響 11
2. 前處理Minocycline 18小時對之後給予之普拿疼48小時造成小鼠肝臟細胞株(FL83B)其細胞凋亡以及細胞壞死的影響 11
3. 前處理Minocycline 24小時對之後給予之普拿疼24小時造成小鼠肝臟細胞株(FL83B)其細胞凋亡以及細胞壞死的影響 11
4. 前處理Minocycline 24小時對之後給予之普拿疼48小時造成小鼠肝臟細胞株(FL83B)其細胞凋亡以及細胞壞死的影響 12
六、Minocycline的前處理在普拿疼於體外引發人類肝臟細胞瘤株(HepG2)的細胞凋亡以及細胞壞死之影響 12
1. 前處理Minocycline 24小時對之後給予之普拿疼24小時造成人類肝臟細胞瘤株(HepG2)其細胞凋亡以及細胞壞死的影響 12
2. 前處理Minocycline 24小時對之後給予之普拿疼48小時造成人類肝臟細胞瘤株(HepG2)其細胞凋亡以及細胞壞死的影響 12
七、Minocycline的前處理在普拿疼於體外引發小鼠肝臟細胞株(FL83B)之ER Stress 13
1. 前處理Minocycline 18小時再給予普拿疼24小時活化小鼠肝臟細胞株(FL83B)其ER Stress中XBP1路徑之影響 13
2. 前處理Minocycline 24小時再給予之普拿疼24小時活化小鼠肝臟細胞株(FL83B)其ER Stress中XBP1路徑之影響 13
八、Minocycline的前處理在致死劑量普拿疼造成小鼠猛爆性肝炎其ER Stress的影響 13
1. 前處理Minocycline 24, 12, 0小時3針之後給予致死劑量普拿疼造成小鼠 猛爆性肝炎中活化ER Stress中XBP1路徑 13
2. 前處理Minocycline 24, 12, 0小時3針之後給予致死劑量普拿疼造成小鼠猛爆性肝炎其CHOP/GADD153蛋白質的表現的影響 14
討論 15
參考文獻 20
1.Amin, A. R., M. G. Attur, G. D. Thakker, P. D. Patel, P. R. Vyas, R. N. Patel, I. R. Patel, and S. B. Abramson. 1996. A novel mechanism of action of tetracyclines: effects on nitric oxide synthases. Proc Natl Acad Sci U S A 93:14014-9.
2.Amin, A. R., R. N. Patel, G. D. Thakker, C. J. Lowenstein, M. G. Attur, and S. B. Abramson. 1997. Post-transcriptional regulation of inducible nitric oxide synthase mRNA in murine macrophages by doxycycline and chemically modified tetracyclines. FEBS Lett 410:259-64.
3.Boulares, A. H., A. J. Zoltoski, B. A. Stoica, O. Cuvillier, and M. E. Smulson. 2002. Acetaminophen induces a caspase-dependent and Bcl-XL sensitive apoptosis in human hepatoma cells and lymphocytes. Pharmacol Toxicol 90:38-50.
4.Chen, M., V. O. Ona, M. Li, R. J. Ferrante, K. B. Fink, S. Zhu, J. Bian, L. Guo, L. A. Farrell, S. M. Hersch, W. Hobbs, J. P. Vonsattel, J. H. Cha, and R. M. Friedlander. 2000. Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nat Med 6:797-801.
5.Chu, H. C., Y. L. Lin, H. K. Sytwu, S. H. Lin, C. L. Liao, and Y. C. Chao. 2005. Effects of minocycline on Fas-mediated fulminant hepatitis in mice. Br J Pharmacol 144:275-82.
6.Cornet, S., B. Spinnewyn, S. Delaflotte, C. Charnet, V. Roubert, C. Favre, H. Hider, P. E. Chabrier, and M. Auguet. 2004. Lack of evidence of direct mitochondrial involvement in the neuroprotective effect of minocycline. Eur J Pharmacol 505:111-9.
7.Dai, Y., and A. I. Cederbaum. 1995. Cytotoxicity of acetaminophen in human cytochrome P4502E1-transfected HepG2 cells. J Pharmacol Exp Ther 273:1497-505.
8.Denicola, A., and R. Radi. 2005. Peroxynitrite and drug-dependent toxicity. Toxicology 208:273-88.
9.Du, Y., Z. Ma, S. Lin, R. C. Dodel, F. Gao, K. R. Bales, L. C. Triarhou, E. Chernet, K. W. Perry, D. L. Nelson, S. Luecke, L. A. Phebus, F. P. Bymaster, and S. M. Paul. 2001. Minocycline prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson's disease. Proc Natl Acad Sci U S A 98:14669-74.
10.Fernandez-Gomez, F. J., M. F. Galindo, M. Gomez-Lazaro, C. Gonzalez-Garcia, V. Cena, N. Aguirre, and J. Jordan. 2005. Involvement of mitochondrial potential and calcium buffering capacity in minocycline cytoprotective actions. Neuroscience 133:959-67.
11.Fiorucci, S., E. Antonelli, A. Mencarelli, B. Palazzetti, L. Alvarez-Miller, M. Muscara, P. del Soldato, L. Sanpaolo, J. L. Wallace, and A. Morelli. 2002. A NO-releasing derivative of acetaminophen spares the liver by acting at several checkpoints in the Fas pathway. Br J Pharmacol 135:589-99.
12.Gabler, W. L., J. Smith, and N. Tsukuda. 1992. Comparison of doxycycline and a chemically modified tetracycline inhibition of leukocyte functions. Res Commun Chem Pathol Pharmacol 78:151-60.
13.Goldin, R. D., I. D. Ratnayaka, C. S. Breach, I. N. Brown, and S. N. Wickramasinghe. 1996. Role of macrophages in acetaminophen (paracetamol)-induced hepatotoxicity. J Pathol 179:432-5.
14.Gonzalez, F. J. 2005. Role of cytochromes P450 in chemical toxicity and oxidative stress: studies with CYP2E1. Mutat Res 569:101-10.
15.Gotoh, T., and M. Mori. 2006. Nitric Oxide and Endoplasmic Reticulum Stress. Arterioscler Thromb Vasc Biol.
16.Gyamlani, G. G., and C. R. Parikh. 2002. Acetaminophen toxicity: suicidal vs. accidental. Crit Care 6:155-9.
17.He, Y., S. Appel, and W. Le. 2001. Minocycline inhibits microglial activation and protects nigral cells after 6-hydroxydopamine injection into mouse striatum. Brain Res 909:187-93.
18.Hengartner, M. O. 2000. The biochemistry of apoptosis. Nature 407:770-6.
19.Hinson, J. A., S. L. Pike, N. R. Pumford, and P. R. Mayeux. 1998. Nitrotyrosine-protein adducts in hepatic centrilobular areas following toxic doses of acetaminophen in mice. Chem Res Toxicol 11:604-7.
20.Jaeschke, H. 2006. How relevant are neutrophils for acetaminophen hepatotoxicity? Hepatology 43:1191-4.
21.Jaeschke, H., and M. L. Bajt. 2006. Intracellular signaling mechanisms of acetaminophen-induced liver cell death. Toxicol Sci 89:31-41.
22.Jaeschke, H., G. J. Gores, A. I. Cederbaum, J. A. Hinson, D. Pessayre, and J. J. Lemasters. 2002. Mechanisms of hepatotoxicity. Toxicol Sci 65:166-76.
23.Kim, R., M. Emi, K. Tanabe, and S. Murakami. 2006. Role of the unfolded protein response in cell death. Apoptosis 11:5-13.
24.Klein, N. C., and B. A. Cunha. 1995. Tetracyclines. Med Clin North Am 79:789-801.
25.Knight, T. R., and H. Jaeschke. 2002. Acetaminophen-induced inhibition of Fas receptor-mediated liver cell apoptosis: mitochondrial dysfunction versus glutathione depletion. Toxicol Appl Pharmacol 181:133-41.
26.Kon, K., J. S. Kim, H. Jaeschke, and J. J. Lemasters. 2004. Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes. Hepatology 40:1170-9.
27.Laskin, D. L., C. R. Gardner, V. F. Price, and D. J. Jollow. 1995. Modulation of macrophage functioning abrogates the acute hepatotoxicity of acetaminophen. Hepatology 21:1045-50.
28.Laskin, D. L., and K. J. Pendino. 1995. Macrophages and inflammatory mediators in tissue injury. Annu Rev Pharmacol Toxicol 35:655-77.
29.Lawson, J. A., M. A. Fisher, C. A. Simmons, A. Farhood, and H. Jaeschke. 1999. Inhibition of Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice. Toxicol Appl Pharmacol 156:179-86.
30.Li, C. H., S. L. Tzeng, Y. W. Cheng, and J. J. Kang. 2005. Chloramphenicol-induced mitochondrial stress increases p21 expression and prevents cell apoptosis through a p21-dependent pathway. J Biol Chem 280:26193-9.
31.Liu, Z. X., S. Govindarajan, and N. Kaplowitz. 2004. Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity. Gastroenterology 127:1760-74.
32.Liu, Z. X., D. Han, B. Gunawan, and N. Kaplowitz. 2006. Neutrophil depletion protects against murine acetaminophen hepatotoxicity. Hepatology 43:1220-30.
33.Macanas-Pirard, P., N. S. Yaacob, P. C. Lee, J. C. Holder, R. H. Hinton, and G. E. Kass. 2005. Glycogen synthase kinase-3 mediates acetaminophen-induced apoptosis in human hepatoma cells. J Pharmacol Exp Ther 313:780-9.
34.Malhi, H., G. J. Gores, and J. J. Lemasters. 2006. Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology 43:S31-44.
35.Manov, I., M. Hirsh, and T. C. Iancu. 2004. N-acetylcysteine does not protect HepG2 cells against acetaminophen-induced apoptosis. Basic Clin Pharmacol Toxicol 94:213-25.
36.McCullough, K. D., J. L. Martindale, L. O. Klotz, T. Y. Aw, and N. J. Holbrook. 2001. Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state. Mol Cell Biol 21:1249-59.
37.Mori, K. 2003. Frame switch splicing and regulated intramembrane proteolysis: key words to understand the unfolded protein response. Traffic 4:519-28.
38.Nakagawa, T., and J. Yuan. 2000. Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J Cell Biol 150:887-94.
39.Oyadomari, S., K. Takeda, M. Takiguchi, T. Gotoh, M. Matsumoto, I. Wada, S. Akira, E. Araki, and M. Mori. 2001. Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway. Proc Natl Acad Sci U S A 98:10845-50.
40.Ray, S. D., L. M. Kamendulis, M. W. Gurule, R. D. Yorkin, and G. B. Corcoran. 1993. Ca2+ antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen. Faseb J 7:453-63.
41.Ray, S. D., V. R. Mumaw, R. R. Raje, and M. W. Fariss. 1996. Protection of acetaminophen-induced hepatocellular apoptosis and necrosis by cholesteryl hemisuccinate pretreatment. J Pharmacol Exp Ther 279:1470-83.
42.Ray, S. D., C. L. Sorge, J. L. Raucy, and G. B. Corcoran. 1990. Early loss of large genomic DNA in vivo with accumulation of Ca2+ in the nucleus during acetaminophen-induced liver injury. Toxicol Appl Pharmacol 106:346-51.
43.Ryan, M. E., and R. A. Ashley. 1998. How do tetracyclines work? Adv Dent Res 12:149-51.
44.Salas, V. M., and G. B. Corcoran. 1997. Calcium-dependent DNA damage and adenosine 3',5'-cyclic monophosphate-independent glycogen phosphorylase activation in an in vitro model of acetaminophen-induced liver injury. Hepatology 25:1432-8.
45.Wang, J., Q. Wei, C. Y. Wang, W. D. Hill, D. C. Hess, and Z. Dong. 2004. Minocycline up-regulates Bcl-2 and protects against cell death in mitochondria. J Biol Chem 279:19948-54.
46.Wang, X., S. Zhu, M. Drozda, W. Zhang, I. G. Stavrovskaya, E. Cattaneo, R. J. Ferrante, B. S. Kristal, and R. M. Friedlander. 2003. Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington's disease. Proc Natl Acad Sci U S A 100:10483-7.
47.Whiteman, M., and B. Halliwell. 1997. Prevention of peroxynitrite-dependent tyrosine nitration and inactivation of alpha1-antiproteinase by antibiotics. Free Radic Res 26:49-56.
48.Xu, W., L. Liu, I. G. Charles, and S. Moncada. 2004. Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress response. Nat Cell Biol 6:1129-34.
49.Yoshida, H., T. Matsui, A. Yamamoto, T. Okada, and K. Mori. 2001. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107:881-91.
50.Yrjanheikki, J., R. Keinanen, M. Pellikka, T. Hokfelt, and J. Koistinaho. 1998. Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci U S A 95:15769-74.
51.Zhang, H., J. Cook, J. Nickel, R. Yu, K. Stecker, K. Myers, and N. M. Dean. 2000. Reduction of liver Fas expression by an antisense oligonucleotide protects mice from fulminant hepatitis. Nat Biotechnol 18:862-7.
52.Zhu, S., I. G. Stavrovskaya, M. Drozda, B. Y. Kim, V. Ona, M. Li, S. Sarang, A. S. Liu, D. M. Hartley, C. Wu du, S. Gullans, R. J. Ferrante, S. Przedborski, B. S. Kristal, and R. M. Friedlander. 2002. Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature 417:74-8.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top