當細胞受到外界壓力或傷害時會產生大量熱休克蛋白 (heat shock proteins, HSPs) ，一直以來 HSPs 被認為是一種維持細胞生命所必須的蛋白質，平時主要維持正常細胞之生理機能但其生理機制尚未明確，本論文欲探討於大白鼠延腦鼻端網狀腹外側核區 (rostral ventrolateral medulla, RVLM) 中 HSP60、HSP70 及 HSP90 在實驗性內毒素敗血症 (experimental endotoxemia) 過程的生理意義。
來自成熟雄性 Sprague-Dawley 種系大白鼠全身動脈血壓訊號頻譜訊號分析顯示，在 propofol (25 mg/kg/h) 維持麻醉深度下，股靜脈注射 E. coli lipopolysaccharide (LPS, 30 mg/kg; 型號 0111:B4) 使產生 endotoxemia 。在 experimental endotoxemia 過程其 vasomotor components (頻率介於 0-0.8 Hz) 功率密度略受抑制 (稱之為 Phase I) ；爾後 vasomotor components 功率密度大幅增加 (稱之為 Phase II, 也稱之為 pro-life) ；接著再次 vasomotor components 之功率密度持續降低 (稱之為 Phase III, 也稱之為 pro-death) 。如此的 vasomotor component 頻率功率密度改變主要是受到來自於心血管調控中樞，也就是 RVLM 的調控。接續以西方墨點分析 RVLM 中 HSP60、HSP70 及 HSP90 在 experimental endotoxemia 各分期蛋白質表現，結果顯示 HSP60 於 Phase II 及 Phase III蛋白質表現具有統計意義增加； HSP70 於 Phase II 蛋白質表現具有統計意義增加；但 HSP90 蛋白質表現量於 experimental endotoxemia 過程中並無統計意義改變。
進一步探討 RVLM 中 HSP60、HSP70或 HSP90 於experimental endotoxemia 過程扮演的角色，分別微量注射的專一特性 anti-HSP serum (HSPAb; 1:20) 或 antisense oligodeoxy- nucleotide (hsp AODN, 50 pmol) 至雙側 RVLM 中，使拮抗或阻斷特定 HSP 使該 HSP 蛋白質無法發揮其功能作用或不製造，此外以 normal mouse serum (NMS) 、 sense oligodeoxynucleotide (hsp SODN) 以及 scrambled AODN (hsp SC) 為對照組，結果顯示於 RVLM 以 HSP60Ab 或 hsp60 AODN 預處理，使得 experimental endotoxemia 的死亡率升高、存活時間減少、Phase II 時間縮短， Phase II vasomotor component功率密度增加幅度減少，以及 HSP60 蛋白質表現量減少，因此認為在 experimental endotoxemia 過程 RVLM 中 HSP60 扮演 pro-life 的角色。同樣的在 RVLM 以HSP70Ab 或 hsp70 AODN 預處理，結果顯示在 experimental endotoxemia 過程 RVLM 中 HSP70 扮演更重要的 pro-life角色。而在 RVLM 中以 HSP90Ab 或 hsp90 AODN 預處理，不具統計意義影響 experimental endotoxemia 過程，因此 RVLM 中 HSP90 不參與 experimental endotoxemia 致死過程。
在 experimental endotoxemia 致死過程於 RVLM 中的 HSP60 及 HSP70 影響首在 Phase II ，並且 HSP60 及 HSP70是扮演 pro-life 角色，而 RVLM 中的 HSP90 不參與 experimental endotoxemia 致死過程。

Heat shock proteins (HSPs) are abundantly produced in cells that are under stress or injury by acting as a chaperone or promoting folding, unfolding, packing, degradation or denaturing of proteins or peptides. This study evaluated the role of HSP60, HSP70 or HSP90 in the rostral ventrolateral medulla (RVLM), in experimental endotoxemia in the rat.
Adult, male Sprague-Dawley rats maintained by i.v. infusion propofol (25 mg/kg/h) were used. During experimental endotoxemia induced by intravenous administration of E. coli lipopolysaccharide (LPS, 30 mg/kg; serotype 0111:B4), the power density of the vasomotor component of systemic arterial pressure (SAP) spectrum underwent a decrease (Phase I), followed by an increase (Phase II; “pro-life”) and a secondary decrease (Phase III; “pro-death”). Western blot analysis revealed that HSP60 expression in the RVLM was significantly increased during Phase II and Phase III endotoxemia; and HSP70 expression was maximally increased during Phase II. HSP90 protein expression in the RVLM was not significantly changed during endotoxemia.
We further studied the role of HSP60, HSP70 or HSP90 at the RVLM in experimental endotoxemia by pretreating animals with bilaterally microinjection of an anti-HSP serum (HSPAb, 1:20), normal mouse serum, antisense oligodeoxynucleotide (hsp AODN, 50 pmol), sense oligodeoxynucleotide (hsp SODN) or scrambled AODN (hsp SC). Pretreatment with HSP60Ab or hsp60 AODN resulted in significantly higher mortality, shorter survival time and shorter Phase II duration. In addition, the augmented power density of the vasomotor component of SAP signals during Phase II endotoxemia was significantly reduced. Even more detrimental effects were obtained on local application of HSP70Ab or hsp70 AODN into the RVLM. Pretreatment with HSP90Ab or hsp90 AODN was ineffective.
We conclude that the expression of HSP60 and HSP70 in the RVLM may play a “pro-life” role in fatal experimental endotoxemia; and HSP90 may not be involved.

中文摘要 2 – 4
英文摘要 5 – 7
第一章 緒論 8 – 17
第二章 研究動機與目的 18 - 19
第三章 實驗材料與方法 20 – 26
第四章 實驗結果 27 – 35
第五章 討論 36 – 48
第六章 結論 49 – 50
第七章 未來展望 51 – 52
參考文獻 53 – 61
實驗圖表 62 –140

Akselrod S (1988) Spectral analysis of fluctuations in cardiovascular parameters: a quantitative tool for the inverstigation of autonomic control. Trends Pharmacol Sci 9:6-9.

Araujo GC, Lopes OU, and Campos RR (1999) Importance of glycinergic and glutamatergic synapses within the rostral ventrolateral medulla for blood pressure regulation in conscious rats. Hypertension 34:752-755.

Becker J, and Craig EA (1994) Heat-shock proteins as molecular chaperones. Eur J Biochem 219:11-23.

Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana T, Tailor P, Morimoto RI, Cohen GM, and Green DR (2000) Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol 2:469-475.

Benjamin IJ, and McMillan DR (1998) Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res 83:117-132.

Bergamaschi CT, Campos RR, and Lopes OU (1999) Rostral ventrolateral medulla : A source of sympathetic activation in rats subjected to long-term treatment with L-NAME. Hypertension 34:744-777.

Bergamaschi CT, Campos RR, Schor N, and Lopes OU (1995) Role of the rostral ventrolateral medulla in maintenance of blood pressure in rats with goldblatt hypertension. Hypertension 26:1117-1120.

Brown DL, and Guyenet PG (1984) Cardiovascular neurons of brain stem with projections to spinal cord. Am J Physiol 247:R1009-R1016.

Calaresu FR, and Yardley CP (1988) Medullary basal sympathetic tone. Ann Rev Physiol 50:511-524.

Cerutti C, Barrès C, and Paultre C (1994) Baroreflex modulation of blood pressure and heart rate variabilities in rats: assessment by spectral analysis. Am J Physiol 266:H1993-H2000.

Chan JYH, Wang SH, and Chan SHH (2001) Differential roles of iNOS and nNOS at rostral ventrolateral medulla during experimental endotoxemia in the rat. Shock 15:65-72.

Chan SHH, Chan JYH, and Ong BT (1986) Anatomic connections between nucleus reticularis rostroventrolateralis and some medullary cardiovascular sites in the rat. Neurosci Lett 71:277-282.

Chan SHH, Chang KF, Ou CC, and Chan JYH (2002) Up-regulation of glutamate receptors in nucleus tractus solitarii underlies potentiation of baroreceptor reflex by heat shock protein 70. Mol Pharmacol 61:1097-1104.

Chan SHH, Wang LL, Chang KF, Ou CC, and Chan JYH (2003) Altered temporal profile of heat shock factor 1 phosphorylation and heat shock protein 70 expression induced by heat shock in nucleus tractus solitarii of spontaneously hypertensive rats. Circulation 107:339-345.

Dampney RA, Goodchild AK, Robertson LG, and Montgomery W (1982) Role of ventrolateral medulla in vasomotor regulation: a correlative anatomical and physiological study. Brain Res 249:223-235.

Delogu G, Lo Bosco L, Marandola M, Famularo G, Lenti L, Ippoliti F, and Signore L (1997) Heat shock protein (HSP70) expression in septic patients. J Crit Care 12:188-192.

Granata AR, Ruggiero DA, Park DH, Joh TH, and Reis DJ (1985) Brain stem area with C1 epinephrine neurons mediates baroreflex vasodepressor responses. Am J Physiol 248:H547-H567.

Gupta S, and Knowlton AA (2002) Cytosolic heat shock protein 60, hypoxia, and apoptosis. Circulation 106:2727-2733.

Guyenet PG, Haselton RJ, and Sun MK (1989) Sympathoexcitatory neurons of the rostroventrolateral medulla and the origin of the sympathetic vasomotor tone. Prog Brain Res 81:105-115.

Huang YH, Chang AYW, Huang CM, Huang SW, and Chan SHH (2002) Proteomic analysis of lipopolysaccharide-induced apoptosis in PC12 cells. Proteomics 2:1220-1228.

Jaattela M (1999) Heat shock proteins as cellular lifeguards. Ann Med 31:261-271.

Jaattela M, Wissing D, Kokholm K, Kallunki T, and Egeblad M (1998) Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J 17:6124-6134.

Keeler R, Barrientos A, and Lee K (1981) Circulatory effects of acute or chronic endotoxemia in rats. Can J Physiol Pharmacol 59:204-208.

Kirchhoff SR, Gupta S, and Knowlton AA (2002) Cytosolic Heat Shock Protein 60, Apoptosis, and Myocardial Injury. Circulation 105:2899-2904.

Knowlton AA, and Sun L (2001) Heat-shock factor-1, steroid hormones, and regulation of heat-shock protein expression in the heart. Am J Physiol 280:H455-H464.

Kuo TBJ, and Chan SHH (1993) Continuous, on-line, real-time spectral analysis of systemic arterial pressure signals. Am J Physiol 264:H2208-H2213.

Kuo TBJ, Yang CCH, and Chan SHH (1997a). Selective activation of vasomotor components of SAP spectrum by nucleus reticularis ventrolateralis in rats. Am J Physiol 272:H485-H492.

Kuo TBJ, Yien HW, Hseu SS, Yang CCH, Lin YY, Lee LC, and Chan SHH (1997b) Diminished vasomotor components of systemic arterial pressure signals and baroreflex in brain death. Am J Physiol 273:H1291-H1298.

Latchman DS (2001) Heat shock proteins and cardiac protection. Cardiovasc Res 51:637-646.

Len WB, and Chan JYH (2001) Rostral ventrolateral medulla suppresses reflex bradycardia by the release of gamma-aminobutyric acid in nucleus tractus solitarii of the rat. Synapse 39:23-31.

Li CY, Lee JS, Ko YG, Kim JI, and Seo JS (2000) Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation. J Biol Chem 275:25665-25671.

Li PL, Chao YM, Chan SHH, and Chan JYH (2001) Potentiation of baroreceptor reflex response by HSP70 in nucleus tractus solitarii confers cardiovascular protection during heatstroke. Circulation 103:2114-2119.

Lin KM, Lin B, Lian IY, Mestril R, Scheffler IE, and Dillmann WH (2001) Combined and individual mitochondrial HSP60 and HSP10 expression in cardiac myocytes protects mitochondrial function and prevents apoptotic cell deaths induced by simulated ischemia-reoxygenation. Circulation 103:1787-1792.

Malliani A, Pagani M, Lombardi F, and Cerutti S (1991) Cardiovascular neural regulation explored in the frequency domain. Circulation 84:482-492.

Miller CM, Smith NC, and Johnson AM (1999) Cytokines, nitric oxide, heat shock proteins and virulence in Toxoplasma. Parasitol Today 15:418-422.

Morrison SF, Gallaway J, Milner TA, and Reis DJ (1991) Rostral ventrolateral medulla: a source of the glutamatergic innervation of the sympathetic intermediolateral nucleus. Brain Res 562:126-135.

Pandey P, Saleh A, Nakazawa A, Kumar S, Srinivasula SM, Kumar V, Weichselbaum R, Nalin C, Alnemri ES, Kufe D, and Kharbanda S (2000) Negative regulation of cytochrome c-mediated oligomerization of Apaf-1 and activation of procaspase-9 by heat shock protein 90. EMBO J 19:4310-4322.

Pirkkala L, Nykanen P, and Sistonen L (2001) Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB J 15:1118-1131.

Riabowol KT, Mizzen LA, and Welch WJ (1988) Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70. Science 242:433-436.

Robertson JD, and Orrenius S (2002) Role of mitochondria in toxic cell death. Toxicology 181-182:491-496.

Ross CA, Ruggiero DA, Park DH, Joh TH, Sved AF, Fernandez-Pardal J, Saavedra JM, and Reis DJ (1984) Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing C1 adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin. J Neurosci 4:474-494.

Ross CA, Ruggiero DA, and Reis DJ (1985) Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla. J Comp Neurol 242:511-534.

Ritossa F (1962) A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 19:571-573.

Sam II AD, Sharma AC, Law WR, and Ferguson JL (1997) Splanchnic vascular control during sepsis and endotoxemia. Front Biosci 2:e72-e92.

Sato K, Saito H, and Matsuki N (1996) HSP70 is essential to the neuroprotective effect of heat-shock. Brain Res 740:117-123.

Snoeckx LH, Cornelussen RN, Van Nieuwenhoven FA, Reneman RS, and Van Der Vusse GJ (2001) Heat shock proteins and cardiovascular pathophysiology. Physiol Rev 81:1461-1497.

Soltys BJ, and Gupta RS (1996) Immunoelectron microscopic localization of the 60-kDa heat shock chaperonin protein (Hsp60) in mammalian cells. Exp Cell Res 222:16-27.

Sun MK, and Reis DJ (1995) NMDA receptor-mediated sympathetic chemoreflex excitation of RVL-spinal vasomotor neurons in rats. J Physiol 482:53-68.

Sun MK, and Reis DJ (1996) Excitatory amino acid-mediated chemoreflex excitation of respiratory neurons in rostral ventrolaterals medulla in rats. J Physiol 492:559-571.

Tissieres A, Mitchell II K, and Tracy U (1974) Protein synthesis in salivary glands of Drosophilla melanogaster：relation to chromosome puff. J Mol Bio 84:389-398.

Van Der Straten A, Rommel C, Dickson B, and Hafen E (1997) The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila. EMBO J 16:1961-1969.

Welch WJ (1993) Heat shock proteins functioning as molecular chaperones：their roles in normal and stressed cells. Phil Trans R Soc Lond B Biol Sci 339:327-333.

Wick G, Perschinka H, and Millonig G (2001) Atherosclerosis as an autoimmune disease: an update. Trends Immunol 22:665-669.

Willette RN, Punnen S, Krieger AJ, and Sapru HN (1984) Interdependence of rostral and caudal ventrolateral medullary areas in the control of blood pressure. Brain Res 321:169-174.

Wu CC, and Yen MH (1997) Beneficial effects of dantrolene on lipopolysaccharide-induced haemodynamic alterations in rats and mortality in mice. Eur J Pharmacol 327:17-24.
Yahara I, Minami Y, and Miyata Y (1998) The 90-kDa stress protein, Hsp90, is a novel molecular chaperone. Ann N Y Acad Sci 851:54-60.

Yang CC, Kuo TBJ, and Chan SHH (1996) Auto- and cross-spectral analysis of cardiovascular fluctuations during pentobarbital anesthesia in the rat. Am J Physiol 270:H575-582.

Yang MW, Kuo TBJ, Lin SM, Chan KH, and Chan SHH (1995) Continuous, on-line, real-time spectral analysis of SAP signals during cardiopulmonary bypass. Am J Physiol 268:H2329-2335.

Yang RC, Wang CI, Chen HW, Chou FP, Lue SI, and Hwang KP (1998) Heat shock treatment decreases the mortality of sepsis in rats. Kaohsiung J Med Sci 14:664-672.

Yin K, Hock CE, Tahamont M, and Wong PY (1998) Time-dependent cardiovascular and inflammatory changes in acute endotoxemia. Shock 9:434-442.

Zucchi I, Bini L, Albani D, Valaperta R, Liberatori S, Raggiaschi R, Montagna C, Susani L, Barbieri O, Pallini V, Vezzoni P, and Dulbecco R (2002) Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland. Proc Natl Acad Sci U S A 99:8660-8665.