臺灣博碩士論文加值系統

多年以來靈芝已經在亞洲國家廣泛的被用作治療藥物去治療免疫相關的疾病。我們已經證實靈芝多醣體萃取物透過產生發炎細胞激素包括介白素-1而具有免疫調節的功能。我們最近發現靈芝多醣體萃取物在老鼠和人類巨噬細胞中大量增加可以和具生物活性的介白素-1競爭的介白素-1受體拮抗因子。為了繼續研究靈芝多醣體萃取物誘導介白素-1受體拮抗因子表現的機制，首先我們用濃度和時間點的實驗來討論，發現介白素-1受體拮抗因子會在人類單核球和單核球細胞株THP-1中表現。另外，發現靈芝多醣體萃取物也會提升介白素-1受體拮抗因子的訊息核糖核酸表現。有趣的是透過靈芝多醣體萃取物去刺激有基因變異的老鼠巨噬細胞株HeNC2和GG2EE還有利用類鐸受體2和4受到具中合力抗體影響的人類單核球，都讓我們發現類鐸受體2和4會參與在靈芝多醣體萃取物刺激介白素-1受體拮抗因子的產生。利用類鐸受體缺乏的老鼠，我們發現不論在類鐸受體2或4缺乏的老鼠中在靈芝多醣體萃取物刺激下，介白素-1受體拮抗因子的產生都有降低的情形，表示類鐸受體2和4會參與在靈芝多醣體萃取物刺激介白素-1受體拮抗因子的表現。我們已經發現靈芝多醣體萃取物會去調控不同的蛋白激化的傳遞路徑並與抗發炎的細胞激素，介白素-1受體拮抗因子表現有關。特別的是靈芝多醣體萃取物會持續的刺激蛋白酪氨酸激磷酸化進而誘發蛋白激化如磷脂酸肌醇3激、磷酸酯解C、不同類型的蛋白激化和活化致裂原活化蛋白激(心室肌組織中細胞外調節激、c-Jun氨基端激和p38)活化。在kinase活性偵測試驗、西方點墨實驗和介白素-1受體拮抗因子酵素免疫分析法中配合蛋白激化抑制劑去證明不同的蛋白激化在靈芝多醣體萃取物刺激介白素-1受體拮抗因子的分泌中所扮演的角色。我們發現靈芝多醣體萃取物透過與類鐸受體2和4鍵結後產生蛋白激化訊息路徑去使介白素-1受體拮抗因子產生。

For years, Ganoderma lucidum (Reishi or Ling-zhi), has been widely used in Asian countries as therapeutic medicines to treat abnormality related to immunity. We have demonstrated that an extract of Reishi polysaccharides (EORP) exerting an immuno-modulating activities by stimulating inflammatory cytokines including interleukin-1 (IL-1). We recently reported that EORP dramatically increased interleukin-1 receptor antagonist (IL-1Ra), which compete with biological functions of IL-1, expression in mice model and human primary macrophages. In order to continue to investigate the mechanism of EORP induction of IL-1Ra expression, initially, we conducted the experiments of dose and time-course studies, and found IL-1Ra expression in both human primary monocytes and THP-1 monocytic cell lines. In addition, EORP was found to up-regulate IL-1Ra message RNA expression. Interestingly, via responding to EORP and LPS in genetic variation of murine macrophage HeNC2 and GG2EE cell lines, as well as using toll-like receptor-2 (TLR2) and -4 (TLR4) neutralizing antibodies blockage in human monocytes, we found that both TLR2 and TLR4 involved in the EORP-mediated IL-1Ra secretion. Using TLR-/- knockout mice, we found that reduction of EORP-induced IL-1Ra production in either TLR2-/- or TLR4-/- mice, indicating that incorporated of TLR2 and TLR4 involved in EORP-mediated IL-1Ra expression. We have found that EORP differentially modulates the protein kinase (PK)-mediated signal transduction pathways associated with anti-inflammatory cytokine, IL-1Ra expression. Specifically, EORP rapidly stimulated protein tyrosine kinase (PTK)-mediated phosphorylation, followed by induction of PKs such as phosphoinostitide 3-kinase (PI3K), phospholipase C (PLC), various PKC isoforms and activation of MAPKs: ERK, JNK and p38. Using PK inhibitors in the kinase activity assays, Western blot analyses and IL-1Ra ELISA, we have extensively examined and dissected the role of individual PK in the regulation of IL-1Ra. Our findings establish that initially, EORP binds to TLR2 and TLR4, followed by EORP-mediated signaling pathway: PTK/PI3K/PLC/PKCs/MEK1/ERK, which involved in the IL-1Ra regulation.

Contents ………………………………………………………………………… 1
中文摘要 ………………………………………………………………………… 3 Abstract (English)………………………………………………………………………… 5 Introduction ………………………………………………………………………… 7
Materials and Methods
………………………………………………………………………… 11
l Cell cultures and cell stimulation
l Materials
l Preparation of EORP
l RNA Isolation, Reverse Transcription Reaction
and Polymerase Chain Reaction Amplification for
detecting the expression of IL-1Ra, Western blot
Analysis and Assay Activity of ERK
l Enzyme-Linked Immunosorbent Assay (ELISA) for
measurement of human IL-Ra
l Enzyme-Linked Immunosorbent Assay (ELISA) for
measurement of mouse IL-Ra
l Preparation of membrane protein
l Flow cytometric analysis
l siRNA Transfections
l Preparation of peritoneal macrophages from
Knockout mice
Statistical Analysis
Results………………………………………………………………… 19
l Extract of Reishi Polysaccharides (EORP) induces
interleukin-1 receptor antagonist (IL-1Ra)
secretion and message expression within human
THP-1 monocytes and human primary monocytes
l TLR2 and TLR4-mediated signaling pathways are
cooperated in the EORP-induced IL-1Ra secretion
within THP-1 monocytes and mouse macrophages
l Phosphatidylinositol-3-OH kinase (PI3-kinase) and
phospholipase C (PLC), but not Src family kinases
regulate EORP-stimulated IL-1Ra secretion within
THP-1 monocytes
l EORP activates phosphorylation of protein kinase
C (PKC) isoforms within THP-1 monocytes
l EORP activates phosphorylation and activity of
mitogen activated protein kinases (MAPKs) within
THP-1 monocytes and human primary monocytes
l Role of EORP-induced protein kinase (PK) in the
regulation of IL-1Ra secretion within THP-1
monocytes
l Role of PI3-kinase and PLC in EORP-induced
PKC/MEK1/ERK pathway and in the EORP-mediated
regulation of IL-1Ra secretion
l EORP regulates the expression of surface markers
and receptors on human monocytes
l Ca2+ and G protein-coupled receptors (GPCRs) play
an important role on EORP-induced IL-1Ra secretion
within THP-1 monocytes
Discussion…………………………………………………………… 33
References…………………………………………………………… 43
Figure Legends
………………………………………………………………………… 52

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