出版年:

研究生:

陳盈杰

研究生(英文姓名):

論文名稱:

發展及運用增強型綠色螢光報告基因組以研究IL-10起動子

英文論文名稱:

Development and Utilization Enhanced Green Fluoresent Protein (EGFP) as a Reporter to Study IL-10 Promoter

指導教授:

楊倍昌

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物及免疫學研究所

學年度:

86

語文別:

中文

論文頁數:

87

被引用次數:

0

[ 摘要 ]

IL-10由T細胞、B細胞或是巨噬細胞所分泌。它可作用於B細胞導致活化及分化，亦可抑制免疫細胞進行細胞性反應（Th1反應），因而被歸類為Th2細胞激素。IL-10可抑制細胞分泌IL-1、TNF-α、IL-6、IL-8、IL-12、INF-γ以及IL-2，是一種細胞激素合成抑制因子，扮演著細胞激素平衡之重要調節因子。為了瞭解IL-10表現之調控機制，本論文構築一系列以EGFP為基礎的IL-10起動子／報告基因組，轉染於免疫細胞中，觀察由IL-10起動子所調控之報告基因的表現。從Jurkat細胞染色體DNA中，利用PCR法複製IL-10基因前端-1212/-19序列，選殖於質體－pGFP-1中，再次選殖於質體－pEGFP。取其中三選殖株進行DNA序列分析，發現"AC" dinucleotide repeat區長度各有不同（分別為32 bp、36 bp
、64 bp）。此外，又另構築了二含有不同長度之IL-10起動子之EGFP報告質體（分別包含IL-10基因之-746/-19及-558/-19序列）。將此些IL-10/EGFP報告基因，轉染於T細胞（Jurkat和Molt-4）、單核球細胞（HL-60和U-937）及B細胞（BJAB和B95-8）後，分析報告基因的活性。比較不同測量方法如螢光顯微鏡、流體細胞分析儀及螢光分光光度計分析，發現流體細胞分析儀使用上較方便、靈敏度較高並可進行定量分析。分析結果指出，EGFP可持續性的表現於Jurket、BJAB及B95-8細胞中，而不表現於Molt-4及HL-60細胞中。大量之質體DNA轉染入U937細胞後，會導致細胞進行計劃性死亡而無法分析。於細胞中，EGFP表現程度依起動子增長而有增加，而因"AC" dinucleotide repeat增長而減少。此結果顯示於IL-10起動子-1212/-746及-746/-558間有正調控因子存在，又"AC" repeat可能是一基因表現之結構上的障礙。細胞以phorbolmyristate acetate (PMA)、calcium ionophore (A23187)、PMA＋A23187（T細胞）、lipopolysaccharide (LPS)（B細胞及單核球）及DHEA（所有的細胞）處理，並無法影響報告基因活性。以RT-PCR分析，發現Jurkat細胞因PMA的加入導致表現IL-10 mRNA而U937也因LPS的加入而微量增加IL-10 mRNA表現。可能LPS及PMA作用於-1221/-19區域外，而影響細胞表現IL-10 mRNA。

[ 英文摘要 ]

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.