| Summary: | 碩士 === 國立成功大學 === 微生物及免疫學研究所 === 86 === Interleukin 10 (IL-10), expressed mostly in T cells, B cells and macrophages, is a pleiotropic growth and differentiation factor of B cells. It can inhibit the secretion of Th1 cytokines by monocyte, macrophage, and T cells, and exert consequently an important regulatory role in cytokine networks. In order to understand how the expression of IL-10 is regulated, enhanced green fluorescent protein (EGFP) based reporter plasmids under the control of IL-10 promoter sequences were constructed. The DNA fragment spanning from -1212 to -19 region of IL-10 gene was amplified by PCR and confirmed by DNA sequencing. Three IL-10 promtoer clones showing different length in "AC" dinucleotide repeat (32, 36, and 64 bp) were obtained. Two additional clones containing DNA fragment spanning from -746 to -19 or from -558 to -19 were also created. The function of these reporter plasmids had been analyzed in B cell lines: BJAB, and B95/8; T cell lines: Jurkat and Molt-4; and monocyte cell lines: U937 and HL-60. The expression of EGFP was observed directly by fluorescent microscopy and quantitiatively measured by FACScan and fluorescence spectrophotometer. Our results showed that IL-10 promoter was constitutively activated in BJAG, B95/8, and Jurkat but not in Molt-4 and HL-60. DNA transfection caused death of U937 cells. IL-10 reporter activity reduced along with the increasing of the length of the "AC" dinucleotide repeat. The sequences located -1212 to -746 and -746 to -558 showed postitive regulatory activity in BJAB, B95/8, and Jurkat cells. Treatments with phorbol myristate acetate (PMA), calcium ionophore (A23187), or PMA combined A23187 had no significant effects on the IL-10 reporter expression in Jurkat and Molt-4 cells. Lipopolysaccharide (LPS) had no effect on BJAB, B95-8, and HL-60. The expression of EGFP in the tested cell lines was not effected by dehydroepiandrosterone. Although the PMA or LPS could not augment the reporter activities in our tested cell lines, it increased the IL-10 mRNA expression in Jurkat and U937 cells as detected by RT-PCT. These results suggested that there were still some other regulatory elements on IL-10 promoter upstream of -1212 or downstream -of 19.
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