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研究生:范文林
研究生(外文):Wen-Lin Fan
論文名稱:探討細胞外組織蛋白促進敗血症嚴重度及其機制與臨床病人的關連性
論文名稱(外文):Investigation of extracellular histones enhancing septic severity and the mechanisms in septic model and the correlation in clinical patients
指導教授:吳淑芬吳淑芬引用關係
指導教授(外文):Shu-Fen Wu
口試委員:李沁戴建國李繼源
口試委員(外文):Chin LiChien-Kuo TaiChi-Yuan Li
口試日期:民國100年7月28日
學位類別:碩士
校院名稱:國立中正大學
系所名稱:分子生物研究所
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:英文
論文頁數:73
中文關鍵詞:敗血症組織蛋白
外文關鍵詞:sepsishistone
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敗血症常伴隨著不可預期的疾病預後,一直以來都是一項嚴重的臨床病症。在重症照護中,敗血症引起的休克及嚴重器官功能失調是引起死亡的主因。有研究指出在全身發炎反應或敗血症生物體血液中的細胞外組織蛋白將會升高,這可能是白血球或其他免疫細胞對發炎組織細胞的破壞所造成。而且當細胞外組織蛋白升高時將會伴隨內皮細胞的毒害甚至造成實驗動物的死亡。
我們推測,細胞外組織蛋白是敗血病症的重要免疫介質之一,因此我們藉以探討組織蛋白造成細胞毒性可能的機轉。在活體外觀察,組織蛋白會造成人類內皮細胞劑量相關的細胞毒性,而血液白蛋白會阻斷此毒性作用。為了進一步了解造成細胞毒性的死亡機轉,我們利用RT-PCR來觀察此細胞毒性所牽涉的基因調控機制,結果發現DR5訊息升高而XIAPF訊息下降,而且白蛋白一樣可以抑制DR5訊息的上升,這些結果更證實細胞外組織蛋白引起的細胞死亡有部分是經由DR5的表現來進行。另外我們利用老鼠盲腸的結紮和穿刺引發敗血症的動物模式來驗證細胞外組織蛋白是否會加重敗血症病程的嚴重度。我們發現靜脈注射組織蛋白會造成敗血症老鼠的病情加重,而且也呈現和劑量正相關的趨勢。最後我們也觀察敗血症病人,隨敗血症病程的進展來監測敗血症病人血中組織蛋白的濃度變化,藉以分析觀察組織蛋白在敗血症病情進展和嚴重度的相關性。
統整我們的結果發現細胞外組織蛋白的確會造成內皮細胞的毒性乃至死亡,而且呈現劑量和時間的正相關趨勢,另外也發現白蛋白會阻斷上述的細胞毒性作用。而組織蛋白造成的細胞毒性,其引發的死亡機轉是經由DR5等細胞凋亡相關的基因所調控。最後我們更證實了細胞外組織蛋白會加重實驗敗血症動物的疾病嚴重度和死亡率,我們也藉此推論敗血症病人若出現較高濃度的細胞外組織蛋白可能會有較差的預後。
Sepsis remains a serious clinical problem accompanied with unpredictable disease outcome. Septic shock and multiorgan dysfunction are the most common causes of death in patients with sepsis under intensive care. As previous reports indicated that the level of extracellular histone in plasma is elevated in systemic inflammation and sepsis, probably due to damages inflicted to the inflamed tissues by neutrophils or other inflammatory cells. Moreover, elevated extracellular histone is associated with cytotoxicity of endothelial cells and leads to death of mice in experimental septic model.
We speculate that extracellular histone is an important mediator of the septic syndrome and set out to investigate the mechanism of histone-dependent cytotoxicity. In vitro, addition of histone mixture in the culture medium caused the death of human endothelial cells in a dosage-dependent manner. In addition, the cytotoxic effect of histone can be blocked by serum albumin, suggesting that serum albumin is able to reduce the adverse effect of histone to endothelial cells. In order to identify the response of endothelial cells to extracellular histone, the expression levels of a cohort of genes functioning in inflammation were determined by RT-PCR, and the result shows that the levels of DR5 and XIAPF were increased and decreased, respectively, upon histone treatment. Increase of DR5 expression is suppressed if albumin is present during histone treatment, confirming regulation of DR5 expression specifically in response to extracellular histone. To support the notion that serum histone plays a role in increasing the severity of sepsis, we discovered that, in a cecal ligation and puncture mouse model, injection of histone mixture leads to drastic increase in mortality rate in a dosage-dependent pattern. We are currently collecting the data in patients suffering from sepsis to correlate serum histone concentration with progression and severity of sepsis.
In conclusion, we have demonstrated that extracellular histone exhibits cytotoxic effect toward endothelial cells in dosage and time dependent manner in vitro and that additional albumin protects endothelial cells from histone-induced cellular damage. It is likely that DR5 and XIAPF, members of tumor necrosis factor family, are involved in activation of histone-induced cytotoxicity. Finally, extracellular histone exacerbates progression and severity of sepsis and increase the mortality rate in a sepsis mouse model, strongly supporting our hypothesis that histone is a negative indicator for the outcome of sepsis in patients.
致謝 ………………………………………………………………………………….i
摘要 …………………………………………………………………………………ii
Abstract ......................................................................................................................... iii
List of Content ............................................................................................................... v
1. Introduction ........................................................................................................ 1
1.1. Definition of sepsis ............................................................................................ 1
1.2. Pathogenesis of sepsis ........................................................................................ 2
1.3. Therapy of sepsis ............................................................................................... 7
2. Objectives of Study ............................................................................................ 9
3. Materials and Methods ..................................................................................... 10
3.1. Cell culture ....................................................................................................... 10
3.2. Histone cytotoxicity assay ............................................................................... 10
3.3. Sub-G1 assay .................................................................................................... 10
3.4. Flow cytometry assay ....................................................................................... 10
3.5. Time-coursed microscopy assay ...................................................................... 11
3.6. Septic animal model (Cecal ligation and puncture, CLP) ................................ 11
3.7. RNA extraction ................................................................................................ 11
3.8. cDNA synthesis ................................................................................................ 12
3.9. Reverse transcription-PCR (RT-PCR) .............................................................. 12
3.10. Plasma isolation from mice .............................................................................. 12
3.11. Septic blood specimens .................................................................................... 12
3.12. Coomassie blue analysis .................................................................................. 13
3.13. Histological examination ................................................................................. 13
3.14. Mass analysis ................................................................................................... 13
3.15. Statistical analysis ............................................................................................ 14
3.15.1. Clinical data ..................................................................................................... 14
3.15.2. Septic animal model (CLP model) ................................................................... 14
3.15.3. Flow data .......................................................................................................... 14
4. Result ............................................................................................................... 15
5. Discussion ........................................................................................................ 32
6. Figure ............................................................................................................... 36
7. Supplementary Data ......................................................................................... 59
7.1. Supplementary Figure ...................................................................................... 59
7.2. Supplementary Table ........................................................................................ 60
8. Reference ......................................................................................................... 63
1. Karimova A, Pinsky DJ: The endothelial response to oxygen deprivation: biology and clinical implications. Intensive Care Med 2001, 27:19-31.
2. Rivers EP, Kruse JA, Jacobsen G, Shah K, Loomba M, Otero R, Childs EW: The influence of early hemodynamic optimization on biomarker patterns of severe sepsis and septic shock. Crit Care Med 2007, 35:2016-24..
3. Beal AL, Cerra FB. Multiple organ failure syndrome in the 1990s. Systemic inflammatory response and organ dysfunction. J Am Med Assoc 1994;271:226–233.
4. William C Aird: The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood 2003, 101: 3765-3777
5. Dofferhoff A.S.,Bom V.J., de Vries-Hospers H.G., et al. Patterns of cytokines, plasma endotoxin, plasminogen activator inhibitor, and acute-phase proteins during the treatment of severe sepsis in humans. Crit Care Med. 1992,20:185–192.
6. Gutierrez-Ramos J.C., Bluethmann H. Molecules and mechanisms operating in septic shock: lessons from knockout mice. Immunol Today.1997 18:329–334.
7. Klosterhalfen B.,Hauptmann S., Tietze L., et al. The influence of heat shock protein 70 induction on hemodynamic variables in a porcine model of recurrent endotoxemia. Shock 1997,7:358–363.
8. Muller A.M.,Cronen C., Muller K.M., Kirkpatrick C.J. Heterogeneous expression of cell adhesion molecules by endothelial cells in ARDS. J Pathol 2002,198:270–275.
9. Pinsky MR. Sepsis: a pro- and anti-inflammatory disequilibrium syndrome. Contrib Nephrol 2001;354–366.
10. Morrison D.C., Ulevitch R.J.(1978) The effects of bacterial endotoxins on host mediation systems; a review. Am J Pathol 93:526–617.
11. Henneke P., Golenbock D.T.(2002) Innate immune recognition of lipopolysaccharide by endothelial cells. Crit Care Med 30:S207–213.
12. Angus D.C., Wax R.S.(2001) Epidemiology of sepsis: an update. Crit Care Med 29:S109–116.
13. Stefanec T. Endothelial apoptosis: could it have a role in the pathogenesis and treatment of disease? Chest. 2000;117:841-854
14. Stefanec T. Endothelial apoptosis: could it have a role in the pathogenesis and treatment of disease? Chest. 2000;117:841-854.
15. Hotchkiss RS, Tinsley KW, Swanson PE, Karl IE. Endothelial cell apoptosis in sepsis. Crit Care Med. 2002;30:S225-S228.
16. Haimovitz-Friedman A, Cordon-Cardo C, Bayoumy S, et al. Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation. J Exp Med. 1997;186:1831-1841.
17. Fujita M, Kuwano K, Kunitake R, et al. Endothelial cell apoptosis in lipopolysaccharide-induced lung injury in mice. Int Arch Allergy Immunol. 1998; 117:202-208.
18. Kawasaki M, Kuwano K, Hagimoto N, et al. Protection from lethal apoptosis in lipopolysaccharide- induced acute lung injury in mice by a caspase inhibitor. Am J Pathol. 2000;157:597- 603.
19. Shigeaki Inoue et al. IL-15 prevents apoptosis, reverses innate and adaptive immune dysfunction, and improves survival in sepsis. J Immunol 2010;184:1401-1409
20. Mario Perl et al. Contribution of anti-inflammatory/immune suppressive process to the pathology of sepsis. Frontiers in Bioscience 2006;11:272-299
21. Savill J et al. A blast from the past:clearance of apoptotic cells regulate immune responses. Nature Reviews Immunology 2002:2:965-975
22. Rittirsch D, Flierl MA, Ward PA. Harmful molecular mechanisms in sepsis. Nat Rev Immunol. 2008; 8:776–787. [PubMed: 18802444]
23. Ye X, Ding J, Zhou X, Chen G, Liu SF. Divergent roles of endothelial NF-kappaB in multiple organ injury and bacterial clearance in mouse models of sepsis. J Exp Med 2008;205:1303-15. [Erratum, J Exp Med 2008;205:1509.]
24. Gröger M, Pasteiner W, Ignatyev G, et al. Peptide Bbeta( 15-42) preserves endothelial barrier function in shock. PLoS One 2009;4(4):e5391
25. Brinkmann et al. Neutrophil extracelular traps (NETs) kill bacteria. Science, 303:1532-1535, 2004.
26. Clark et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat. Med. 13, 463-469, 2007.
27. Brinkmann V, Zychlinsky A: Beneficial suicide: why neutrophils die to make NETs.Nat Rev Microbiol 2007;5:577–582.
28. Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, Weinrauch Y, Brinkmann V, Zychlinsky A : Novel cell death program leads to neutrophil extracellular traps.J Cell Biol 2007; 176: 231–241.
29. Jun Xu et al. Extracellular histones are major mediators of death in sepsis. Nature Medicine 2009;15:1318-132
30. Catherine C et al. Sepsis: the dark side of histones. Nature Medcine 2009;15:1245-1246
31. David F. Gaieski et al. Impact of time to antibiotics on survival in patients with severe sepsis or septic shock in whom early goal-directed therapy was initiated in the emergency department. Crit Care Med 2010, 38: 1-9
32. Bernard et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. NEJ. Med. 2001, 344:699-709
33. Baltch AL et al. Effect of recombinant human activated protein C on the bactericidal activity of human monocytes and modulation of pro-inflammatory cytokines in the presence of antimicaobial agents. Antimicrob. Chemother. 2007, 59(6):1177-81.
34. Riewald M, Petrovan RJ, Donner A, Ruf W. Activated protein C signals through the thrombin receptor PAR1 in endothelial cells. J Endotoxin Res. 2003; 9:317–321. [PubMed: 14577849]
35. Htiffany M Osborn. Early goal-directed therapy in the treatment of severe sepsis and septic shock. Emergency Med & Crit Care Review.2008:23-28
36. Alan D.Pemberton et al. Proteomic identification of interactions between histones and plasma proteins: Implications for cytoprotection. Proteomics 2010,10:1484-1493
37. Daniel Rittirsch et al. Immunodesign of experimental sepsis by cecal ligation and puncture. Nature protocols, 2009, 4: 31-36
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