臺灣博碩士論文加值系統

介白素-12 ( Interleukin-12 )為抗原呈現細胞受到病原菌刺激後產生之細胞激素之一。主要之功能為活化自然殺手細胞及促使輔助型T細胞成熟。活化的介白素-12由p35及p40兩個次單元以雙硫鍵連接構成p70的異構雙體，其在先天性和後天性適應免疫反應上扮演很重要的橋樑。在抗原呈現細胞中，IL-12 p40基因表現之分子機轉已經廣泛被研究，而且已經找到一些重要的轉錄因子參與調控；相反的，在研究p35基因調控的領域上卻尚未明朗。然而，根據近期的一些研究指出，p35這個次單元的形成是決定產生活化的介白素-12的重要決定因素，而不是傳統認知的p40。因此在我們研究中，選殖了人類介白素12 p35啟動子區間-1183至+41，此區間之序列經由軟體比對發現除了目前已知之轉錄因子辨識位置之外，仍有一些與免疫相關之轉錄因子辨識區域可能存在。我們將啟動子區域序列自5’端選擇性截斷消去，再將這些不同長度之啟動子區域序列放置於蟲螢光酵素基因之前，來建構一系列的啟動區截斷型報導質體系統，以電穿孔方法將這些質體分別送入巨噬細胞中，再以干擾素�蚺峇漪r素刺激細胞做研究。
目前研究初步發現以IFN-�蚰�處理再以LPS作用處理細胞發現可以協同性地激活啟動子區間-595至-552及-440至-392，經由軟體比對分別可能為NF�羠，或 C/EBP (-579至-562)以及Oct-1(-410至-396)的辨識區間，所以顯示這些區間在IFN-r/LPS作用處理下會激活IL-12 p35基因的表現。往後，我們將進一步利用DNA結合親合性分析與電膠體位移實驗進而去探究在外來病原菌的刺激之下IL-12 p35基因作用的分子機轉。

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.

中文摘要 i
Abstract ii
Acknowledgment iv
Contents v
Table contents vii
Figure Contents viii
Appendix Contents x
Introduction 1
Materials and Methods 7
I. Materials 7
Reagents 7
Primers and plasmids 8
II. Methods 8
Cell line and cell culture 8
Preparation of genomic DNA 9
Promoter subcloning and plasmids construction 10
Preparation of reporter plasmids stably trancfected cell lines 11
Luciferase Assay 13
Preparation of cell lysate 13
Luciferase assay 13
Protein assay 14
Western Blotting 14
DNA sequencing 15
Results 17
Discussion 23
Reference 28

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