臺灣博碩士論文加值系統

多年來靈芝(Ganoderma lucidum)在亞洲被當成藥物用來治療許多人類的疾病。G. lucidum屬靈芝菌絲內的免疫調節蛋白被命名為靈芝-8(LZ-8)並複製重組(rLZ-8)。過去研究指出重組靈芝蛋白可以在體外環境刺激活化T細胞分泌介白素-2、干擾素-γ並且增加T細胞表面介白素-2受體α鏈(CD25)表現，另外有研究指出重組靈芝蛋白可抑制自體免疫性糖尿病之發生。在此我們假設重組靈芝蛋白具免疫調節能力並加以證明。首先結果指出重組靈芝蛋白可以促進人類周邊CD4+ T細胞分泌介白素-2、干擾素-γ及介白素-10，同時重組靈芝蛋白刺激T細胞所分泌出之介白素-2在介白素-10的產生過程中扮演重要的調控角色。此外，體外實驗發現重組靈芝蛋白可以透過T細胞表面之CD45促進原態CD4+ T細胞轉變成調節性T細胞且具有抑制功能，調節性T細胞在避免自體免疫疾病上扮演著重要角色，我們進一步研究重組蛋白誘發出之調節性T細胞在葡聚糖硫酸酯鈉鹽引起之腸道炎症扮演之角色，此為一種自體免疫性腸道炎症。實驗證明重組靈芝蛋白刺激之T細胞(約40%調節性T細胞)打入腸道炎症老鼠體內可以減緩腸道發炎現象。總結，我們研究發現重組靈芝蛋白可以透過增加細胞激素分泌及誘發調節性T細胞達到免疫調節功能，並且靈芝蛋白刺激過之T細胞可以減緩腸道炎症老鼠之發炎症狀。我們認為靈芝重組蛋白有潛力作為免疫調節的藥劑。

Ganoderma lucidum (G. lucidum, Ling-Zhi, 靈芝) has been used as a medicine in Asia for years to treat numerous human diseases. An immunomodulatory protein named Ling Zhi-8 (LZ-8) existing in the mycelia of G. lucidum has been cloned and expressed as a recombinant protein rLZ-8. Previous results indicated that rLZ-8 enhanced IL-2, IFN-γ secretion and up-regulation of CD25 on surface of T cells in vitro. It has been reported that rLZ-8 is able to suppress autoimmune diabetes in mice. Herein we tested the hypothesis of rLZ-8 exerting immunomodulatory capacities. First, we demonstrated that rLZ-8 induced cytokines production including IL-2, IFN-γ and IL-10 in human primary CD4+ T cells. Also, we found that the rLZ-8 induced IL-2 plays an important role in IL-10 production. In addition, rLZ-8 promoted Naïve CD4+ T cells into induced-CD4+CD25+ regulatory T cells (Tregs) and the suppressive activity in vitro. To investigate the mechanism by which rLZ-8 induction of Tregs, we found that rLZ-8-induced Tregs differentiation via a CD45-dependent pathway. Moreover, since Tregs play important roles in the prevention of autoimmune disease, we further tested the role of rLZ-8-induced Tregs in dextrane sulfate sodium (DSS)-proned colitis, one kind of autoimmune inflammatory bowel diseases. We evidenced that ex vivo-generated rLZ-8-induced CD4+ T cells (~40% Tregs) injected into colitis-like mice, and found the colitis symptom was largely reduced as compared to the control mice. In conclusion, we demonstrate that rLZ-8 has immunomodulatory function through induced cytokines production and regulatory T cells differentiation in vitro, as well as that rLZ-8 induced-Tregs could reduce inflammation of colitis-like mice model in vivo. Our current results suggest rLZ-8 has potential as an immunomodulatory agent.

Contents
中文摘要 1
Abstract 2
Introduction 3
Materials and Methods 6
Cells 6
Reagents and antibodies 6
Cytokine measurement 7
Suppression assay 7
Flow cytometric analysis 7
Western Blotting Analysis 8
Mice and induction of colitis 8
Mice CD4+ T cells separated and adoptive transfer 8
Assessment of colonic damage 9
Results 10
Recombinant LZ-8 (rLZ-8) stimulates cytokines secretion and increases CD25 and FoxP3 expression in human peripheral blood mononuclear cells (PBMC) 10
rLZ-8 stimulates cytokines secretion and induces regulatory T cells development in human primary CD4+ T cells. 11
rLZ-8-induced IL-2 plays an important role in IL-10 production. 12
rLZ-8-promoted regulatory T cells differentiation could via CD45-dependent pathway. 12
rLZ-8 promoted IL-2 production via CD18 signaling pathway in human primary CD4+ T cells. 13
Mice treated by ex vivo-generated rLZ-8-induced CD4+ T cells reduced the severity of DSS colitis. 14
Discussion 16
Reference 22
Figure legends 25

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