臺灣博碩士論文加值系統

敗血症是加護病房中最常見及最嚴重的死亡原因之一，而且死亡率仍居高不下。敗血症的致病機轉很複雜，其中最常引起病人死亡的原因為多重器官衰竭症候群(MODS)。肝臟已被認為是在敗血症時會被影響的重要器官之一，因此肝臟功能的喪失是敗血症病程中多重器官衰竭進行的指標之一。最近，細胞凋亡已被認為是在敗血症動物模式中造成細胞功能喪失的一個重要機制。
細胞激素是敗血症時重要的早期調控者，且發炎性細胞激素的增加與病情進展到敗血性休克則有高度的相關性。特別是腫瘤壞死因子α(TNFα)已經被認為是敗血症的一個主要病理性調控者，且會誘發多種細胞發生細胞凋亡的現象。在許多種類的細胞中，調控腫瘤壞死因子α接受體的因子為一些脂質性的次級訊息傳遞者如sphingosine及/或ceramide，其會抑制蛋白激酶Cα(PKCα)的活性。我們先前的研究結果亦顯示，於敗血症晚期細胞膜相關的蛋白激酶C同功酶α的活性顯著降低。因此蛋白激酶Cα的受抑制作用，在敗血症晚期肝臟功能衰竭上可能扮演重要的角色。
當細胞或生物體受到脅迫或環境溫度的升高時，會誘發合成一群具高保留性的蛋白質，稱為熱休克蛋白質(heat shock proteins; Hsps)。熱休克蛋白質已被認為具有保護細胞的功能，且其中在分子量72的蛋白質則最具有誘導性。至今已有足夠的實驗可以顯示熱休克蛋白質72能有效的抑制許多不同的刺激所誘發的細胞凋亡。然而，在敗血症脅迫下，大白鼠卻無法合成表現熱休克蛋白質72，而這合成表現的失敗被認為是促成細胞凋亡及不良的病程進展之部份原因。因此，熱休克蛋白質對細胞凋亡所調控的保護機制仍有待被探討。在本實驗室先前的研究結果顯示，敗血症期間當細胞凋亡顯著的增加時，蛋白激酶Cα有被不活化且熱休克蛋白質72並無被誘導出。所以，我們假定敗血症期間肝細胞凋亡的發生可能是由於必須經由PKC磷酸化熱休克因子而產生的熱休克蛋白質72誘導合成失敗所致。
為了瞭解PKCα在熱休克前處理時對TNFα所誘發的肝細胞凋亡保護效應之角色，我們利用一個生體外(in vitro)的TNFα誘發細胞凋亡系統，設計實驗去證實熱休克前處理在肝細胞抗細胞凋亡的效應。另外，PKCα在熱休克蛋白質72表現的角色及熱休克前處理的保護效應也將被研究。細胞死亡是以細胞計數，in situ TUNEL染色法及流式細胞儀偵測。蛋白激酶Cα及熱休克蛋白質72的表現以西方墨點法來量化分析。實驗結果顯示：(1) 腫瘤壞死因子α所誘發的細胞死亡，包括細胞凋亡，在熱休克前處理後有減少的現象。 (2) 熱休克前處理後8小時熱休克蛋白質72的表現最多。 (3) 蛋白激酶Cα蛋白質被活化的現象在腫瘤壞死因子α處理後有減少的現象，但於熱休克前處理後其不活化的現象即被改善。(4)在以PKCαantisense處理阻斷PKCα的再合成後，則熱休克前處理的保護作用即消失。 (5) 熱休克蛋白質72的表現於PKCαantisense存在下即被顯著的抑制。
以上結果顯示，蛋白激酶Cα在熱休克蛋白質72的表現及熱休克前處理於腫瘤壞死因子α所誘發的肝細胞凋亡機制中扮演一重要的角色。

Sepsis is the most frequent and serious course of death in intensive care units and associated with a high mortality rate. The mechanisms of sepsis are complex and the most common cause of death in patients with sepsis is the multiple organ failure (MODS). The liver is a central organ in response to sepsis. Loss of hepatic function is one of the indicators of the development of multiple organ failure associated with sepsis. Recently, apoptosis (programmed cell death) has been shown to be an important mechanism of cellular dysfunction in animal models of sepsis.
Cytokines are critical early mediators of inflammation, and the severity of sepsis highly correlated with increased levels of inflammatory cytokines. Particularly, tumor necrosis factorα(TNFα), an apoptosis trigger, has been proposed to be one of the key pathological mediators in sepsis. In many cell models, TNFα receptor-mediated apoptosis is modulated by some lipid second messengers, such as sphingosine and /or ceramide, which inhibits PKC/PKCα activity. Our previous results showed that membrane-associated PKCαactivity was significantly decreased at late stage of sepsis. Thus, the inactivation of PKCαmay play an important role in liver dysfunction during late sepsis.
Stress events and elevated temperature induce synthesis of a set of highly conserved proteins known as heat shock proteins (Hsps). Hsps have been reported to be cytoprotective, and this function has been attributed to one of the major stress-inducible member, heat shock protein72 (Hsp72). There are increasing number of studies to show that heat shock protein72 could be a potent inhibitor of apoptosis induced by a wide range of stimuli. Previous work from our laboratory has indicated that PKCαwas down regulated and the heat shock protein72 (Hsp72) was not induced during sepsis when the apoptosis was markedly increased. From these results, it could be inferred that apoptosis of hepatocytes during sepsis may be due to the inducing failure of Hsp72 expression, which is correlated with PKCαinactivation.
In order to know the role of the PKCα in the protective effect of heat shock pretreatment on TNFα-induced apoptosis in hepatocyte, studies were designed to justify the anti-apoptotic effect of heat shock pretreatment in hepatocytes by using an in vitro TNFα-induced apoptosis system. In addition, the role of PKCαin Hsp72 expression and its subsequent protective effect was investigated. Clone-9 cell line was used in the study. Cell death was evaluated by cell count, in situ TUNEL stain and flow cytometry. The protein expressions of PKCαand Hsp72 were quantified by Western blot analysis. The results showed that: (1) The Hsp72 was highly expressed at 8 hrs after heat shock pretreatment. (2)The increase of TNFα-induced cell death including apoptosis was diminished by heat shock pretreatment. (3) The protein expression of PKCαwas decreased by TNFαtriggering, while it was increased by heat shock pretreatment. (4) In the presence of PKCαantisense, which blocks the PKCαresysthesis, no protective effect of heat shock pretreatment in TNFα-triggered apoptosis was observed. (5)The protein expression of HSP72 was significantly suppressed in the treatment of PKCαantisense.
These results suggested that sufficient PKCα is essential for the expression of Hsp72, and heat shock preconditioning prevents hepatocytes from TNFα-induced apoptosis through PKCα preservation and subsequent Hsp72 synthesis.

目 錄
致謝
一、 中文摘要 …………………………………………………… 1
二、英文摘要 …………………………………………………… 3
三、緒論 ………………………………………………………‥ 6
四、研究目的及實驗設計……………………………………… 14
五、實驗材料與方法…………………………………………… 16
六、結果………………………………………………………… 26
七、討論………………………………………………………… 47
八、參考文獻…………………………………………………… 52
九、發表著作…………………………………………………… 65
十、作者簡歷…………………………………………………… 66

Adamson GM, Billings RE (1993) Tumor necrosis factor: receptor binding and expression of receptors in cultured mouse hepatocytes. J Pharmacol Exp Ther 269: 367-373.
Ahn JH, Ko YG, Park WY, Kang YS, Chung HY, Seo JS (1999) Suppression of ceramide-mediated apoptosis by HSP70. Mol Cell 9: 200-206.
Backmann RP, Mizzen LA, Welch WJ (1990) Interaction of Hsp70 with newly synthesized proteins: implications for protein folding and assembly. Science 248: 850-854.
Baxter GT, Miller DL, Kuo RC, Wada HG, Owicki JC (1992) PKCε is involved in granulocyte-macrophage colony-stimulating factor signal transduction: evidence from microphysiometry and antisense oligonucleotide experiments. Biochem 31: 10950-10954.
Beal AL and FB Cerra (1994) Multiple organ failure syndrome in the 1990s: systemic inflammatory response and organ dysfunction. JAMA 271:226-233.
Beg AA and Baltimore D (1996) An essential role for NF-κB in preventing TNF-α-induced cell death. Science 274: 782-784.
Bird GL, Sheron N, Goka AK, Alexander GL, Williams RS (1990) Increased plasma tumor necrosis factor is severe hepatitis. Ann Intern Med 112: 917-920.
Bone RC, Fisher CJ Jr, Clemmer TP, Slotmqn GJ, Metz CA, Balk RA (1989) Sepsis syndrome: a valid clinical entry. Methylperdnisolone server sepsis study group. Crit Care Med 17: 389-393.
Brun-Buisson C (1995) Incidence, risk factors, and outcome of sever sepsis and septic shock in adults. J Am Med Assoc 274: 968-974.
Buckley AR, Crowe PD, Russell DH (1988) Rapid activation of protein kinase C in isolated rat liver nuclei by prolactin, a known hepatic mitogen. Proc Nat Acad Sci USA 85: 8649-8653.
Casey LC, Balk RA, Bone RC (1993) Plasma cytokine and endotoxin levels correlate with survival in patients with the sepsis syndrome. Ann Intern Med 119: 771.
Chen GH, Reddy RC, Newstead MW, Tateda K, Kyasapura BL, Standiford TJ (2000) Intrapulmonary TNF gene therapy reverses sepsis-induced suppression of lung antibacterial host defense. J Immunol 165: 6496-6503.
Chen HW, Hsu C, Hsu HK, Lu TS, Wang SJ, Yang RC (2001) Heat shock pretreatment influences the expression of PKC isoforms during sepsis. J Surg Res 101: 202-209.
Choi HS, Li B, Lin Z, Huang LE, Liu AY-C (1991) c-AMP and c-AMP-dependent protein kinase regulate the human heat shock protein 70 gene promoter activity. J Biol Chem 266: 11858-11865.
Clemens MS, Chaudry IH, Baue AE (1984) Alterations in hepatic water and electrolyte balance in sepsis. Arch Surg 119: 44-48.
Creagh EM, Sheehan D, Cotter TG (2000) Heat shock proteins-modulators of apoptosis in tumor cells. Leukemia 14: 1161-1173.
Currie RW, Ross, BM, Davis TA (1990) Induction of the heat shock response in rats modulates heart rate, creatine kinase and protein synthesis after a subsequent hyperthermic treatment. Cardiovasc Res 24: 87-93.
Deitch EA (1992) Multiple organ failure: pathophysiology and potential future therapy. Ann Surg 216: 117-134.
Dhainaut JF, Marin N, Mignon A, Vinsonneau C (2001) Hepatic responses to sepsis: interaction between coagulation and inflammatory processes. Crit Care Med 29: S42-S47.
Dienel GA, Kiessling M, Jacewice M, Pulsinelli WA (1986) Synthesis of heat shock protein in rat brain cortex after transient ischemia. J Cereb Blood Flow Metab 6: 505-510.
Ebong S, Call D, Nemzek J, Bolgos G, Newcomb D, Remick D rt(1999) Immunopathologic alterations in murine models of sepsis of increasing severity. Infect Immun 67: 6603-6610.
Elist M, Gantner F, Naumann H, Bluethmann H, Vogt K, Regina BF, Nicotera P, Volk HD (1997) Tumor necrosis factor-induced apoptosis during the poisoning of mice with hepatotoxins. Gastroenterology 112: 923-934.
Engelmann H, Holtmann H, Brakebush C, Avni YS (1990) Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity. J Biol Chem 265: 14497-14504.
Ertel W, Morrison MH, Wang P, BA Z, Ayala A, Chaudry IH (1990) The complex pattern of cytokines in sepsis. Ann. Surg 214: 141-148.
Espevik T, Brockhaus M, Loetscher H, Nonstad U, Shalaby R (1990) Characterization of bonding and biological effects of monoclonal antibodies against a human tumor necrosis factor. J Exp Med 171: 415-426.
Gabai VL and Kabakov AE (1993) Rise in heat-shock protein level confers tolerance to energy deprivation. FEBS 327: 247-250.
Harkema JM, Dean RE, Stephan RN, Chaudry IH (1990) Cellular dysfunction in sepsis. J Crit Care 5: 62-69.
Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381: 571-580.
Himiramastu M, Hotchkiss RS, Karl IE, Buckman TG (1997) Cecal ligation and puncture (CLP) induced apoptosis in thymus, spleen, lung and gut by endotoxin and TNF-independent pathway. Shock 7: 247-253.
Hotchkiss RS, Swanson PE, Cobb JP (1997) Apoptosis in lymphoid and parenchymal cells during sepsis: finding in normal and T-and B-cell-deficient mice. Crit Care Med 25:1298-1307.
Hsu C, Hsieh YC, Hsu HK, Yang RC (2002) Alteraction of protein kinase C isoforms in the liver of septic rat. Shock 17: 41-46.
Ishizuki S, Kisa S, Fujihira E (1985) Hypersensitivity to endotoxin hepatotoxicity in rats with inflammatory lesion. Experientia 41: 661-664.
Jäättelä M, Saksela K, Saksela E (1989) Heat shock protects WEHI-164 target cells from the cytolysis by tumor necrosis factor αandβ. Eur J Immunol 19: 1413-1417.
Jäättelä M, Wissing D, Bauer PA, Li GC (1992) Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity. EMBO J 11: 3507-3512.
Jäättelä M (1999) Heat shock proteins as a cellular lifeguards. Ann Med 31: 261-271.
James G, Olson EN (1992) Deletion of the regulatory domain of protein kinase Cαexpose regions in the hinge and catalytic domains that mediated nuclear targeting. J Cell Biol 166: 863-8
Jefferson AB, Schulman H (1988) Sphingosine inhibits calmodulin-dependent enzymes. J Biol Chem 263:15241-15244.
Kim SH, Kim JH, Erdos G, Lee YJ (1993) Effect of starurosporine on suppression of heat shock gene expression and thermotelerance development in HT-29 cells. Biochem Biophys Res Commun 193:759-763.
Kobayashi D, Watanabe N, Yamauchi N, Tsuji N, Sato T, Sasaki H, Okamoto T, Niitsu Y (1997) Protein kinase C inhibitors augment tumor necrosis factor-induced apoptosis in normal human diploid cells. Chemotherapy 43: 415-423.
Lee KJ, Hahn GM (1988) Abnormal proteins as the trigger for the induction of stress response: heat , diamide and sodium arsenite. J Cell Physiol 136: 411-420.
Leist, M, Gantner F, Jilg S, and Wendge A (1995) Activation of the 55kDa TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release. J Immunol 154:1307-1316.
Leist M, Gantner F, Bohlinger I, Tiegs G, Germann PG, Wendel A (1995) Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models. Am J Pathol 146: 1220-1234.
Leist M, Gantner F, Naumann H, Bluethmann H, Vogt K, Brigelius-Flohe R, Nicotera P, Volk HD, Wendel A (1997) Tumor necrosis factor-induced apoptosis during the poisoning of mice with hepatotoxins. Gastroenterology 112: 923-934.
Lindquist S (1992) Heat-shock proteins and stress tolerance in microorganism. Cure Opin Genet Dev 2: 748-755.
Maio AD (1999) Heat shock proteins: facts, thoughts, and dreams. Shock 11: 1-12.
Maloyan A, Palmon A, Horowitz M (1999) Heat acclimation increases the basal HSP72 level and alters its production dynamic during heat stress. Regulatory Intergrative Com Physiol 45: R1506-R1515.
Mosser DD, Caron AW, Bourget L, Denis-Larose C, Massie B (1977) Role of human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol Cell Biol 17: 5317-5327.
Muto Y, Nouri-Aria KT, Meager A, Alexander GJ, Eddleston AL, Williams R (1988) Enhanced tumor necrosis factor in interleukin-1 in fulminant hepatic failure. Lancent 2: 72-74.
Neidhardt FC, VanBogelen RA (1981) Positive regulatory gene for temperature-controlled proteins in Escherichia coli. Biochem Bioph Res Comm 110: 894-900.
Neuman MG (2001) Apoptosis in disease of the liver. Crit Rev Clin Sci 38: 109-66.
Nishimura H, Emoto M, Kimura K, Yoshikai Y (1997) HSP70 protection macrophages infected with Salmonella choleraesuid against TNFα-induced cell death. Cell stress & chaperones 2: 50-59.
Nollen EA, Brunsting JF, Roelofens H, Weber LA, Kampinga HH (1999) In vivo chaperone activity of heat shock protein 70 and thermotoleance. Mol Cell Biol 10: 2069-2079.
Oberholzer C, Oberholzer A, Michael CS, Moldawer LL (2001) Apoptosis in sepsis: a new target for therapeutic exploration. FASEB J 15: 879-892.
Osaki J, Haneda T, Kashiwagi T, Oi S, Fukuzawa J, Sakai H, Kikuchi K (1998) Pressure-induced expression of heat shock protein 70 mRNA in adult rat heats is coupled both to protein kinase A-dependent and protein kinase C-dependent system. J Hypertens 16: 1193-1200.
Parrillo FE (1993) Pathogenetic mechanisms of septic shock. New England J Med 328: 1471-1477.
Patel T, Steer CJ, Gores GJ (1999) Apoptosis and the liver: A mechanism of disease, growth regulation, and carcinogenesis. Hepatology 30: 811-815.
Pinsky MR (2001) Sepsis: a pro-and anti-inflammatory disequilibrium syndrome. Contrib Nephrol 132: 354-366.
Rackow EC, Astiz ME (1991) Pathophysiology and treatment of septic shock. JAMA 266: 548-554.
Regel G, Grotz M, Weltner T, Strum JA, Tscherne H (1996) Pattern of organ failure following severe trauma. World J Surg 20:422-429.
Richard H (1999) Organ apoptosis in the septic patient: A potential therapeutic target? Crit Care Med 27: 1381-1382.
Ring A, Stremmel W (2000) The hepatic microvascular responses to sepsis. Semin Thromb Hemost 26: 589-594.
Rogers HW, Callery MP, Deck B (1996) Listeria monocytogenes induces apoptosis of infected hepatocytes. J Immunol 156: 679-684.
Roitt IM (1994) Essential immunology, Owford: Blackwell Scientific Publications, 12-13.
Rubin BY, Smith LJ, Hellermann GR, Lunn RM, Richardson NK (1988) Correlation between the anticellular and DNA fragmenting activities of tumor necrosis factor. Cancer Res 48: 6006-6010.
Rudiger HA, Clavien PA (2002) Tumor necrosis factor alpha, but not Fas, mediates hepatocellular apoptosis in the murine ischemic liver. Gastroenterology 122: 202-210.
Ruvolo PP, Deng X, May WS (2001) Phosphorylation of Bcl-2 and regulation of apoptosis. Leukemia 15: 515-522.
Seigel JH, Cerra FB, Peters D, Moody E, Brown D, McMenamy RH, Border JR (1979) The physiologic recovery trajectory as the organizing principle for the quantification of hormone metabolic adaptation to surgical stress and server sepsis. Adv Shock Res 2: 177-203.
Shi Y, Thomas JO (1992) The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate. Mol Cell Biol 12: 2186-2192.
Stone R (1994) Search for sepsis drugs dose on despite failure. Science 264: 365-367.
Streetz K, Leifeld L, G rundmann D, Ramakers J, Eckert K, Spengler U, Brenner D, Manns M, Trautwein C (2000) Tumor necrosis factorαin the pathogenesis of human and murine fulminant hepatic failure. Gastroenterology 119: 446-460.
Tartaglia LA, Weber RF, Figari IS, Reynolds C, Palladino MA, Goedddel DV (1991) The two different receptors for tumor necrosis factor mediate distinct cellular responses. Proc Natl Acad Sci USA 88: 9292-9296.
Tomei LD, Cope FO (eds) (1994) ApoptosisⅡ: the molecular basis of apoptosis in disease. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Tsugane K, Keiko TK, Nagino M, Nimura Y, Yoshida S (1999) A possible role of nuclear ceramide and sphingosine in hepatocyte apoptosis in rat liver. J Hepal 31:8-17.
Vandenabeele P, Declercq W, Beyaert R, Fiers W (1995) Two tumor necrosis factor receptors: structure and function. Trends In Cell Biology 5: 392-399.
Vayssier M, Banzet N, Francois D, Bellmann K, Polla BS. Tobacco smoke induces both apoptosis and necrosis in mammalian cells: differential effects of HSP70. Am J Physiol 275: 1771-1779.
Ven der Poll T and Lowry SF (1995) Tumor necrosis factor in sepsis mediator of multiple organ failure or essential part of host defense? Shock 3: 1.
Villar J, Ribeiro SP, Mullen JB, Kuliszewski M, Post M, Slutsky AS (1994) Induction of the heat shock response reduced mortality rate and organ damage in a sepsis-induced acute lung injury model. Crit Care Med 22: 914-921.
Volpes R, Oord VD, De Vos JJ, Desmet VJ (1992) Hepatic expression of type A and type B receptors for tumor necrosis factor. J Hepatol 14: 361-369.
Wang P, Ayala A, Ba ZF, Zhou M, Perrin MM, Chaudry IH (1993) Tumor necrosis factor-αproduces hepatocellular dysfunction despite normal cardic output and hepatic microcirculation. Am J Physiol 256: G126-G132.
Wang P, Chaudry IH (1996) Mechanism of hepatocellular dysfunction during hyperdynamic sepsis. Am. J Physiol 270: R927-938.
Wang P, Thomas J, Wood BS, Zhou M, Zheng F, Cgaudry IH (1996) Inhibition of the biologic activity of tumor necrosis factor maintains vascular endothelial cell function during hyperdynamic sepsis. J Trauma 40: 694-701.
Willingham MC (1999) Cytochemical methods for the detection of apoptosis. J Histochem cytochem 47: 1101-1109.
Williams GT (1994) Apoptosis in the immune system. J Pathol 173: 1-4.
Wong GH, Tartaglia LA, Lee MS, Goeddel DV (1992) Antiviral activity of tumor necrosis factor is signaled through the 55-kDa type 1 TNF receptor. J Immunol 149: 3550-3553.
Wong HR (1998) Potential protective role of the heat shock response in sepsis. New Horiz 6: 194-200.
Wong GH and Goeddel DV (1989) Tumor necrosis factor. In Human Monocytes, edited by Asherson GL and Zembala M, 195-215.
Woo KR, Shu WP, Kong L, Lin BCS (1996) Tumor necrosis factor mediates apoptosis via Ca++/Mg++ dependent endonuclease with protein kinase C as a possible mechanism for cytokine resistant in human renal carcinoma cells. J Urol 155: 1779-1783.
Wyllie AH (1987) Apoptosis: cell death under homeostasis control. Arch Toxicol 11: 3-10.
Yamada Y, Kirillova I, Peschon JJ, Fauste N (1997) Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type 1 TNF receptor. Proc Natl Acad Sci USA 94: 1441-1446.
Yang XU, Bialik S, Jones BE, Iimuro Y, Kitsis RN, Srinivasan A, Brenner DA, Czaja MJ (1998) NFκB inactivation converts a hepatocyte cell line TNFα response from proliferation to apoptosis. Am J Physiol 275: C1058-C1066.
Yang RC, Wang CI, Chen HW, Chou FP, Lue SI, Hwang KP (1998) Heat shock treatment decreases the mortality of sepsis in rats. Kaohsiung J Med Sci 14: 664-672.
Zhang YH, Takahashi K, Jiang GZ, Kawai M, Fukada M, and Yokochi T (1993) In vivo induction of apoptosis (programmed cell death) in mouse thymus by administration of lipopolysaccharide. Infect Immun 61: 5044-5048.
Zhang XM, Morikawa A, Takahashi K (1994) Localization of apoptosis (programmed cell death)in mice by administration of lipopolysaccharide. Microbiol Immunol 38: 669-671.