臺灣博碩士論文加值系統

背景　敗血症，一種全身性發炎反應以及多重器官衰竭的嚴重疾病。在衛生署民國94年台灣地區死亡原因的統計公報中，敗血症佔國人主要死因的第12 位。敗血症的高死亡率使美國平均每年仍花費16.7億美金在敗血症的治療上，但其死亡率仍舊是逐年增加。由本實驗室及其他已發表的研究結果顯示，經熱休克前處理誘發細胞內大量熱休克蛋白質72（Heat shock protein72; Hsp72）的合成，明顯降低敗血症動物模式之死亡率。在近十多年，陸續有文獻提出這個在細胞內具保護作用的壓力蛋白(stress protein)被釋放到細胞外。在創傷、發炎及敗血症等病人血液中皆測得高量的胞外Hsp72 (Extracellular Heat shock protein72;eHsp72)，且其血液中的含量與病人癒後具相關性。經研究顯示被釋放到細胞外的Hsp70在免疫功能上參與重要的角色。但胞外Hsp72在敗血症中所扮演的角色及其功能仍有待釐清。本研究將針對於此分為二個部分:一、探討在敗血症中刺激Hsp72釋放的因子及釋放到Hsp72所扮演的角 色為何?二、探討Hsp72在敗血症中的保護作用為何?

方法　針對實驗一、我們以盲腸結紮及穿孔手術來建立敗血症動物模式，因此將實驗大鼠分為4組:分別為熱休克前處理組、熱休克前處理及盲腸結紮及穿孔手術組、&;#20702;手術組及盲腸結紮及穿孔手術組；以ELISA分析其血液中胞外Hsp72的濃度；以西方墨點法偵測胞內Hsp72的表現；以細胞激素抗體列陣偵測細胞激素的變化。針對實驗二、將實驗大鼠分為３組:分別為&;#20702;手術組、盲腸結紮及穿孔手術組、及盲腸結紮及穿孔手術後皮下給予人類重組Hsp72組(20mg/kg)；偵測血液中麥胺丙酮酸轉&;#37238;（glutamic-pyruvic transaminase, GPT）與麥胺草醋酸轉&;#37238;（glutamic-oaa transaminase, GOT）來分析肝功能；以TUNEL染色分析肝臟組織細胞凋亡；以西方墨點法偵測肝臟組織中Bcl-2, Bax, cleaved-caspasese-3 and -9 及cleaved-PARP蛋白質表現。

結果：　　本研究第一部分的實驗結果顯示僅經熱休克前處理的敗血症大鼠血液中Hsp72的濃度會隨著時間而增加，其餘三組實驗動物血中Hsp72的濃度則維持不，且Hsp72在血液中的濃度與老鼠的存活率成正相關。熱休克前處理刺激非敗血症老鼠(+535%, p<0.01)及敗血症老鼠(+116%, p<0.01%)白血球中Hsp72的蛋白質表現增加。在誘發敗血症後第三小時老鼠的血清可刺激經熱休克前處理的巨噬細胞細胞株(NR8383)釋放Hsp72。經細胞激素抗體列陣分析顯示，敗血症老鼠在早期第3小時血液中CINC-3 (+211.3%, p<0.05), IL-10 (+147%,p<0.05), MCP-1 (+49.6%, p<0.05), and TNF-?? (+51.8%, p<0.05) 濃度顯著增加。MCP-1以及LPS可誘發經熱休克前處理的NR8383細胞株釋放Hsp72 。第二部分的實驗結果顯示，與對照組相比在敗血症老鼠中血中GOT，GPT的濃度顯著增加，但經皮下給予rhHsp72 (20?慊/kg)的敗血症老鼠其血中GOT，GPT的濃度則與對照組不具差異。在肝臟組織染色中顯示， 敗血症老鼠的細胞凋亡數目明顯高於與對照組6.6倍(+665.7%)，然而rhHsp72給予的敗血症老鼠其與對照組相比則不具差異。在肝臟組織中的蛋白質層面顯示，敗血症老鼠的Bcl-2/Bax比值明顯低於對照組(-26%; p<0.01)，但rhHsp72皮下注射的敗血症老鼠與對照組相比則不具差異；而 cleaved-capase-3, caspase-9, and PARP的蛋白質表現在敗血症老鼠組亦顯著上升(+103.3%; **p<0.001, +40.9%; **p<0.001 and +1106%; **p<0.001)，rhHsp72皮下注射的敗血症老鼠組與對照組相比亦不具差異。

結論 在本研究的結果，我們發現在熱休克前處理的敗血症老鼠，主要透過LPS及MCP-1刺激，使Hsp72釋放到血液中，而eHsp72增加敗血症老鼠的存活率。而eHsp72對老鼠的保護作用，可透過抑制粒腺體所啟動細胞凋亡之訊息傳遞路徑，降低肝組織的細胞凋亡， 進而減少敗血症對肝功能的損傷。 我們希望透過本研究，對將來進一步的敗血症藥物開發，提供重要的參考依據。

Background: Sepsis, the leading cause of death in intensive care units, annually
affects more than 500,000 patients in the United States; despite advances in treatment
and supportive care, the mortality rate remains higher than 20%. Our previous study
revealed that heat shock reduces the sepsis-related mortality rate by increasing the
expression of heat shock protein 72 (Hsp72, also known as Hp70). Hsp72, a
molecular chaperone intracellularly induced by stress, exhibits antiinflammatory and
antiapoptotic effects. Hsp72 protects cells and is released into the circulation by
various cells in response to stress and toxic treatments. However, the precise role of
extracellular Hsp72 (eHsp72) during sepsis remains unclear . The present study was
divided into two parts; the first part was conducted to clarify the effect of eHsp72 on
the sepsis-related survival rate and to determine the underlying factors. The second
part was conducted to assess the hypothesis that eHsp72 is involved in reversing
sepsis-induced liver dysfunction.
Methods: Part 1: Sepsis was induced by cecal ligation and puncture (CLP). Changes
in serum levels of Hsp72 and cytokines were determined during sepsis, and the results
were correlated with the survival rate. The effects of heat pretreatment on Hsp72
expression in septic rat leukocytes and those of septic rat serum, lipopolysaccharide
(LPS), and certain cytokines on Hsp72 expression in macrophage NR8383 cells were
7
determined. Part II: Liver function was determined on the basis of changes in the
enzymatic activities of serum glutamic oxaloacetic transaminase (GOT) and glutamic
pyruvic transaminase (GPT). Apoptosis was measured using terminal
deoxynucleotidyl transferase dUTP nick end labeling staining. The expression of
Bcl-2, Bax, cleaved caspase-3 and -9, and cleaved poly(ADP ribose) polymerase
(PARP) in liver tissue was analyzed using W estern blotting.
Results: Part 1: Circulating Hsp72 levels increased during sepsis (0, 5.5, 6.5, 10, and
6.5 ng/mL at 0, 3, 6, 9, and 18 h after CLP), positively correlating with survival rates.
LPS triggered Hsp72 expression in heat-pretreated rats. Heat pretreatment also
increased Hsp72 expression in nonseptic (535%, p < 0.01) and septic (116%, p < 0.01)
rat leukocytes. Furthermore, incubating the serum of septic rats with NR8383 cells
increased eHsp72 levels in a cultured medium. Cytokine profiling revealed that
among the 19 cytokines screened, the levels of cytokine-induced neutrophil
chemoattractant 3 (211.3%, p < 0.05), interleukin-10 (147%, p < 0.05), monocyte
chemotactic protein 1 (MCP-1; 49.6%, p < 0.05), and tumor necrosis factor alpha
(51.8%, p < 0.05) increased. MCP-1 and LPS released Hsp72 from NR8383 cells.
Part II: The results revealed that GOT and GPT activities increased by 126% and
121%, respectively, during sepsis and returned to the control level following the
administration of recombinant human Hsp72 (rhHsp72). During sepsis, apoptotic cells
8
in liver tissue were augmented (665.7%, p < 0.01); however, the effect was reversed
on treatment with rhHsp72. Furthermore, during sepsis, Bcl-2/Bax protein expression
in liver tissue was downregulated (&;#8722;26%; p < 0.01), and the downregulation was
diminished after rhHsp72 treatment. Moreover, during sepsis, expression of cleaved
capase-3, cleaved caspase-9, and PARP in liver tissue was upregulated by 40.9%,
103.3%, and 1106%, respectively, and the upregulation was reversed after treatment
with rhHsp72.
Conclusion: These results demonstrate that increases in levels of circulating Hsp72
improved the survival rate during sepsis. The increases in circulating Hsp72 may be
mediated through MCP-1 and/or LPS. Moreover, during sepsis, eHsp72 restored liver
function by ameliorating apoptosis through the mitochondria-initiated caspase
pathway . Our findings provide a biochemical basis for the development of rhHsp72 as
a therapeutic agent for sepsis management.

中文摘要 --------------------------------------------------------------------------------------- 3

Abstract ------------------------------------------------------------------------------------------ 6

Abbreviation ------------------------------------------------------------------------------------ 9

Chapter.1

Introduction--------------------------------------------------------------------------------------11
1.1. Sepsis ---------------------------------------------------------------------------------------11
1.1.1 The definition of sepsis

1.2. Heat Shock protein (Hsp) ----------------------------------------------------------------12
1.2.1. Heat shock protein 70 family
1.2.2. Heat shock protein 72
1.2.2-1 The functions of intracellular Hsp 72
1.2.2-2 The functions of extracellular Hsp 72(eHSP72)
1.2.2-3 Mechanism of eHsp72 releasing

Chapter.2
Purpose and Motive---------------------------------------------------------------------------20

Chapter.3--- Release of endogenous Hsp72 on the survival of sepsis in rats----------24

3.1. Abstract ------------------------------------------------------------------------------------25

3.2. Introduction -------------------------------------------------------------------------------27

3.3. Materials and Methods ------------------------------------------------------------------30

3.4. Results -------------------------------------------------------------------------------------37

3.5. Discussion ---------------------------------------------------------------------------------41

3.6. Figures -------------------------------------------------------------------------------------47

Chapter.4---Role of exogenous Hsp72 on liver dysfunction during sepsis------------54

4.1. Abstract ------------------------------------------------------------------------------------55

4.2. Introduction -------------------------------------------------------------------------------56

4.3. Materials and Methods -------------------------------------------------------------------63

4.4. Results --------------------------------------------------------------------------------------63

4.5. Discussion ---------------------------------------------------------------------------------66

4.6. Conclusion ---------------------------------------------------------------------------------69

4.7. Figures -------------------------------------------------------------------------------------70

Chapter 5- Conclusion and perspective------------------------------------------------------74

References---------------------------------------------------------------------------------------76

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