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

• Main Authors: Huang, Jun-Yuang, 黃俊淵
• Other Authors: Yang, Bei-Chung
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/69896919975039680549

碩士 === 國立成功大學 === 微生物暨免疫學研究所 === 87 === 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.