High mobility group box 1 (HMGB1) protein是non-histone chromosomal proteins的一種。它可維持nucleosome的結構、調控基因轉錄作用及活化DNA重組、修復與複製的功能。最近的研究指出HMGB1具有細胞激素的作用，可以引發發炎反應，並且會在敗血症的晚期被發現。HMGB-1是經由被活化的巨噬細胞所釋放，可誘導其他前發炎物質的產生，當HMGB-1過量表現時會造成細胞的死亡。本研究的目的是要觀察HMGB-1在敗血症中的表現，進一步利用RNAi的方法抑制HMGB-1來觀察它對於敗血症的影響，以細胞培養方式利用脂多醣(LPS)刺激RAW264.7 細胞釋放HMGB-1並造成敗血症反應，HMGB-1在培養液中的濃度，用西方墨點法偵測，其他發炎相關物質 (如：TNF-α, TGF-β, 及IL-6)則用ELISA分析。本研究發現，透過RNAi去阻斷受LPS刺激的RAW 264.7 細胞所產生的HMGB-1，可以降低LPS所造成的發炎反應。在巨噬細胞的發炎反應中HMGB-1扮演調控發炎因子的角色。

High mobility group box 1 (HMGB-1) protein is a non-histone chromosomal protein. As a DNA binding protein, HMGB-1 is involved in the maintenance of nucleosome structure, regulation of gene transcription and it is active in DNA recombination and repair. It has been known that HMGB-1 is a late mediator of endotoxemia and sepsis. HMGB-1 is released from activated macrophages, induces the release of other proinflammatory mediators, and mediates cell death when overexpressed.
We speculated that the course of sepsis maybe different without the involvement of HMGB-1. The aims of this study are to investigate the role of HMGB-1 in mediating sepsis and to observe the effects by using RNAi to affect the production of HMGB-1. Lipopolysaccharide (LPS) was used to simulate sepsis in culture as well as stimulate the release of HMGB-1 from RAW 264.7 cells. Levels of HMGB-1 in the culture medium were subsequently measured by Western blot. Other proinflammatory cytokines (TNF-α, IL-6 and TGF-β) were measured by ELISA. HMGB-1 could not be detected in the culture medium in the absence of LPS stimuli, but after 0.5 μg/ml LPS treatment HMGB-1 release could be detected. HMGB-1 the amount of released from LPS activated RAW 264.7 cells was in a time- and dose-dependent manner. The present study demonstrated that RNAi in the treatment of LPS-stimulated RAW264.7 cells resulted in the blockade of HMGB-1 and decreased LPS-induced inflammatory response. The results demonstrated that HMGB-1 plays a pivotal role in macrophage inflammatory responses by modulating the production of inflammatory mediators.

Contents……………………………………………………...1
Abbreviations………………………………………………..2
Abstract in Chinese………………………………………….3
Abstract in English…………………………………………..4
Introduction…………………………………………………..5
Materials and Methods……………………………………..11
Results……………………………………………..…..…….15
Discussion……………………………………………....……17
Figures……………………………………………………….29
References………………………………………...…………33
Appendix…………………………………………………….45

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