出版年:

研究生:

黃俊淵

研究生(英文姓名):

Huang, Jun-Yuang

論文名稱:

利用增強型綠色螢光蛋白研究介白素十號的起動子調控

英文論文名稱:

Study on the regulation of IL-10 promoter by using the reporter system of green fluorescent protein (EGFP)

指導教授:

楊倍昌

指導教授(英文姓名):

Yang, Bei-Chung

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

S46861131

學年度:

87

語文別:

中文

論文頁數:

62

關鍵詞:

介白素十號 ; 綠色螢光蛋白 ; 報告基因 ; 起動子

英文關鍵詞:

IL-10 ; GFP ; reporter gene ; promoter

被引用次數:

0

[ 摘要 ]

介白素十號，interleukin-10 (IL-10)，是體內一種重要的細胞激素，在免疫反應中扮演抑制發炎反應與抑制免疫能力的角色。它主要由體內的T淋巴球細胞、B淋巴球細胞與巨噬細胞所製造分泌。它能抑制T cells、NK cells與macrophages的活性；也可以增強B cells與mast cells的增生與分化。而文獻也指出IL-10和自體免疫疾病與腫瘤的形成有關。因此我們想要了解IL-10的調控情形，尤其是在IL-10啟動子上。所以我們選殖了含有不同IL-10 promoter片段，並將它們接入載體：-19到-1212 (pEGFP10p)、-19到-746 (pEGFPd3)與-19到-558(pEGFPd)，使IL-10 promoter片段下游接著綠色螢光蛋白(enhanced green fluorescent protein，EGFP)報告基因。我們建立起帶有質體並穩定表現EGFP的Jurkat與BJAB穩定細胞株，並以多種藥物處理穩定細胞株，如：mitogen，forskolin，dexamethasone，cyclosporine A，和staurosporine，或送入activated Ha-Ras mutant gene，或與不含質體的Jurkat、BJAB，與U-937細胞，肝癌細胞，腦膠質瘤細胞，與SLE病人和健康人的周邊血單核球細胞和血漿，進行混合培養(coculture)，來觀察對於三種不同長度的IL-10 promoter片段Jurkat與BJAB穩定細胞株內啟動螢光蛋白表現的影響。結果得知在多種藥物處理下Jurka與BJAB的IL-10 promoter activities受到調控的情形是不同的。在混合培養實驗中，活化的Jurkat、BJAB，與U-937細胞可以增強BJAB穩定細胞株的IL-10 promoter的活性，但在Jurkat穩定細胞株中則都沒有影響。腦膠質瘤細胞可以增強pEGFP10p的IL-10 promoter 在Jurkat穩定細胞株的活性，卻抑制了其在BJAB穩定細胞株的活性。肝癌細胞抑制了pEGFP10p的IL-10 promoter在BJAB穩定細胞株的活性，而不影響pEGFPd3與pEGFPd的IL-10 promoter的活性。而SLE病人與健康人的PBMC和plasma對於IL-10 promoter活性也有不同的影響。因此，穩定細胞株可以反應出Jurkat與BJAB的IL-10 promoter有不同的調控機制，是一個方便有效的偵測系統。

[ 英文摘要 ]

IL-10 is an important immunosuppressive and anti-inflammatory cytokine. It is mainly produced by T cells, B cells and macrophages in vivo. It can inactivate macrophages and dendritic cells to loss the ability in T and NK cells activation due to inhibition of cytokines synthesis, MHC class II presentation, and costimulator expression. In constract, it promotes B cells and mast cells differentiation and proliferation. Besides, it involves in the tumorgenesis of comman tumors and in the pathogenesis of autoimmune diseases. To understand how it is regulated, especially on IL-10 promoter, we constructed reporter plasmids using the enhanced green fluorescent protein (EGFP) under the control of IL-10 promoter sequences spanning from -1212 to -19 (pEGFP10p), from -746 to -19(pEGFPd3), and from -558 to -19 (pEGFPd) regions. We established Jurkat and BJAB cell lines stably containing EGFP-reporter plasmids under the selection by G418. EGFP expressions driven by the three different fragments of IL-10 promoter were analyzed in the stable cells upon treatments with mitogens, forskolin, cyclosporine A, dexamethasone, staurosporine, and active Ras overexpression. Besides, we also analyzed dynamic EGFP-expression in stable cells when cocultured with Jurkat, BJAB, U-937 cells, hepatoma cells, glioma cells, and peripheral mononuclear cells and plasma of healthy people or SLE patients. We found that the IL-10 promtoer activities were differentially regulated in BJAB and Jurkat cells upon mitogen and chemical agents. In coculture experiments, activated Jurkat, BJAB and U-937 enhanced the activities of IL-10 promoter in BJAB stable cells but not that in Jurkat stable cells. Glioma cells enhanced the IL-10 promoter activity of pEGFP10p in Jurkat stable cells and suppressed that in BJAB stable cells. Hepatoma cells suppressed the IL-10 promoter activity of pEGFP10p but not that of pEGFPd3 and pEGFPd in BJAB stable cells. PBMC and plasma of SLE patients had various effects on IL-10 promoter activity. In conclusion, the activities of IL-10 promoter in Jurkat and BAJB cells were differently regulated. The stable cells with EGFP reporter plasmids may serve as a convenient biosensor system to study the gene regulation.