The Molecular Mechanism Studies of the IFN-γ and Endotoxin-induced Up-regulation of Human IL-12 p35 Gene

• Main Authors: Yi-Fang Lu, 呂宜芳
• Other Authors: Chiun-Jye Yuan
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/83872331230134845491

碩士 === 國立交通大學 === 生物科技系所 === 93 === Interleukin-12 (IL-12) is a pro-inflammatory cytokine secreted by antigen-presenting cells (APCs) in response to pathogen infections. The major functions of IL-12 are to induce the production of interferon-Interleukin-12 (IL-12) is a pro-inflammatory cytokine secreted by antigen-presenting cells (APCs) in response to pathogen infections. The major functions of IL-12 are to induce the production of interferon-Interleukin-12 (IL-12) is a pro-inflammatory cytokine secreted by antigen-presenting cells (APCs) in response to pathogen infections. The major functions of IL-12 are to induce the production of interferon-γ �� (IFN-γ ��), to activate natural killer cells, and to promote naïve T helper cells to functional type 1 T helper cells. Active IL-12 is a 70-kDa heterodimer of two subunits, p40 (40 kDa) and p35 (35 kDa), linked by disulfide bonds to form a p70 protein, and forms a link between innate resistance and adaptive immunity. The molecular mechanisms that regulate IL-12 p40 gene expression have been studied extensively. However, the regulation of the p35 gene expression is still obscure. Recent studies suggest that the expression of the p35 is a rate-limiting step for IL-12 heterodimer production. In our studies, we has cloned the promoter region -1183 to +41 of human IL-12 p35 gene containing several putative transcription factor binding elements with their DNA sequence confirmed. A set of luciferase reporter plasmids containing various length of human IL-12 p35 promoter were constructed and transfected into RAW264.7 cells to generate stable cell clones, which were futher used to study the molecular mechanisms underlying IFN-γ/LPS induced IL-12 p35 gene activation. The preliminary study shows that the promoter region -595 to-552 and -440 to -392 are important for the IFN-γ-priming LPS-induced response. The regions, -595 to-552 and -440 to -392, may contain a NF�羠, or C/EBP binding site and Oct-1 binding site upon searching with MatInspector® and TESS, and may be essential for the up-regulation of IL-12 p35 gene in response to treatment of IFN-γ��/LPS. Further, we will confirm these results with DNA binding affinity assay and electrophoretic mobility shift assay to understand more about the regulation of IL-12 p35 gene, in response to pathogenic stimulation. �� (IFN-γ��), to activate natural killer cells, and to promote naïve T helper cells to functional type 1 T helper cells. Active IL-12 is a 70-kDa heterodimer of two subunits, p40 (40 kDa) and p35 (35 kDa), linked by disulfide bonds to form a p70 protein, and forms a link between innate resistance and adaptive immunity. The molecular mechanisms that regulate IL-12 p40 gene expression have been studied extensively. However, the regulation of the p35 gene expression is still obscure. Recent studies suggest that the expression of the p35 is a rate-limiting step for IL-12 heterodimer production. In our studies, we has cloned the promoter region -1183 to +41 of human IL-12 p35 gene containing several putative transcription factor binding elements with their DNA sequence confirmed. A set of luciferase reporter plasmids containing various length of human IL-12 p35 promoter were constructed and transfected into RAW264.7 cells to generate stable cell clones, which were futher used to study the molecular mechanisms underlying IFN-γ/LPS induced IL-12 p35 gene activation. The preliminary study shows that the promoter region -595 to-552 and -440 to -392 are important for the IFN-γ��-riming LPS-induced response. The regions, -595 to-552 and -440 to -392, may contain a NF�羠, or C/EBP binding site and Oct-1 binding site upon searching with MatInspector® and TESS, and may be essential for the up-regulation of IL-12 p35 gene in response to treatment of IFN-γ/LPS. Further, we will confirm these results with DNA binding affinity assay and electrophoretic mobility shift assay to understand more about the regulation of IL-12 p35 gene, in response to pathogenic stimulation. �� (IFN-γ), to activate natural killer cells, and to promote naïve T helper cells to functional type 1 T helper cells. Active IL-12 is a 70-kDa heterodimer of two subunits, p40 (40 kDa) and p35 (35 kDa), linked by disulfide bonds to form a p70 protein, and forms a link between innate resistance and adaptive immunity. The molecular mechanisms that regulate IL-12 p40 gene expression have been studied extensively. However, the regulation of the p35 gene expression is still obscure. Recent studies suggest that the expression of the p35 is a rate-limiting step for IL-12 heterodimer production. In our studies, we has cloned the promoter region -1183 to +41 of human IL-12 p35 gene containing several putative transcription factor binding elements with their DNA sequence confirmed. A set of luciferase reporter plasmids containing various length of human IL-12 p35 promoter were constructed and transfected into RAW264.7 cells to generate stable cell clones, which were futher used to study the molecular mechanisms underlying IFN-γ/LPS induced IL-12 p35 gene activation. The preliminary study shows that the promoter region -595 to-552 and -440 to -392 are important for the IFN-��-priming LPS-induced response. The regions, -595 to-552 and -440 to -392, may contain a NF�羠, or C/EBP binding site and Oct-1 binding site upon searching with MatInspector® and TESS, and may be essential for the up-regulation of IL-12 p35 gene in response to treatment of IFN-γ/LPS. Further, we will confirm these results with DNA binding affinity assay and electrophoretic mobility shift assay to understand more about the regulation of IL-12 p35 gene, in response to pathogenic stimulation.