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標題: | 荷蘭牛之基因多型性及基因表現與其對結核病感受性之關聯性 Relationship of bovine genetic polymorphisms and gene expression to the risk of bovine tuberculosis in Holstein cattle |
作者: | Ya-fen Cheng 鄭雅芬 |
指導教授: | 蔡向榮 |
關鍵字: | 細胞凋亡,牛型結核病,C型凝集素基因,腫瘤壞死因子α基因,基因表現,牛型分枝桿菌,誘導型一氧化氮合成?基因,基因多型性,巨噬細胞蛋白基因,感受性, apoptosis,bovine tuberculosis,CLEC4E,TNF-α,gene expression,Mycobacterium bovis,NOS2,polymorphism,SLC11A1,susceptibility, |
出版年 : | 2016 |
學位: | 博士 |
摘要: | 許多研究顯示牛隻與人類的基因多型性在宿主抵抗牛型分枝桿菌所引起的人畜共通結核病方面有統計學上的顯著意義。其中,巨噬細胞蛋白、誘導型一氧化氮合成酶和腫瘤壞死因子α等基因於宿主對抗廣泛的傳染性病原體時,扮演著重要的免疫控制作用。本研究目標為瞭解巨噬細胞蛋白、誘導型一氧化氮合成酶和腫瘤壞死因子α等基因多型性對牛型結核病感受性之關聯。本研究取74隻結核病患牛和90隻健康牛之檢體,使用聚合酶鏈式反應及核苷酸定序分型,針對上述基因多型性進行牛型結核病感受性之關聯性分析。結果顯示下列4個基因多型性,分別是g.107117166A > G(位於巨噬細胞蛋白基因第4外顯子,導致第96號胺基酸由丙胺酸轉化為蘇胺酸)、g.107117369C > T(位於巨噬細胞蛋白基因第4內含子)、g.19958101T > G(位於誘導型一氧化氮合成酶基因啟動子)和g.27534932A > C(位於腫瘤壞死因子α第3外顯子,導致第159號胺基酸由精胺酸轉化為絲胺酸)可能與牛型結核病的發病有關,強化荷蘭牛之巨噬細胞蛋白、誘導型一氧化氮合成酶和腫瘤壞死因子α等基因多型性與牛型結核病感受性相關的假設。
其次,瞭解牛型分枝桿菌和宿主細胞間的相互作用對研發牛型結核病之預防、檢測和治療至關重要。基因表達圖譜提供關於這些相互作用的分子機制的大量訊息。本研究取健康牛週邊血液單核細胞與牛型分枝桿菌共同培養4及24小時,使用牛全基因晶片分析比較宿主細胞基因轉錄表達之差異,瞭解該細胞與牛型分枝桿菌作用時之基因表達模式。結果有420個宿主基因表達出至少2倍的顯著差異,經使用生物醫學分析軟體(Ingenuity pathway analysis)及資料庫(Kyoto Encyclopedia of Genes and Genomes)分析比對發現這些基因主要與免疫系統、訊息傳遞、吞噬作用、炎症反應和細胞凋亡相關,其中與免疫系統相關基因有84.85%表現量下降。研究發現牛型分枝桿菌會干擾宿主腫瘤壞死因子α基因表現,致使其介導之細胞凋亡訊息傳遞路徑相關基因的表達被抑制。另發現C-型凝集素基因在感染後4小時即能經由Syk/ CARD9訊息傳遞路徑引發促炎性細胞激素相關基因表達呈負調控現象。本研究首次證實C型凝集素基因參與牛型分枝桿菌感染初期宿主細胞訊息傳遞路徑,這些發現支持牛型分枝桿菌會抑制宿主抗分枝桿菌防禦訊息傳遞路徑的觀點。同時,牛週邊血液單核細胞與牛型分枝桿菌作用24小時後,巨噬細胞蛋白、誘導型一氧化氮合成酶、腫瘤壞死因子α等基因表達分別顯著下降3.03、1.68、5.66倍,證實結核病發病機制早在牛型分枝桿菌與宿主細胞作用最初24小時即開始。 本研究針對牛型分枝桿菌引發荷蘭牛基因表現提供有用的信息,並提出巨噬細胞蛋白、誘導型一氧化氮合成酶、腫瘤壞死因子α等基因多型性與牛型結核病之感受性有關,具有作為結核病防治輔助標記之潛力。 Many studies suggest significant genetic polymorphisms in the resistance of cattle and humans to infection with Mycobacterium bovis (M. bovis), the causative agent of zoonotic tuberculosis. The natural resistance associated macrophage protein 1 (NRAMP1 which is encoded by the SLC11A1 gene), inducible nitric oxide synthase (iNOS which is encoded by the NOS2 gene) and tumor necrosis factor α (TNF-α which is encoded by the TNF-α gene) play important roles in the immunological control of a broad spectrum of infectious agents. The aim of the present study was to investigate the influence of genetic polymorphisms in SLC11A1, NOS2 and TNF-α gene on bovine tuberculosis (bTB) susceptibility. In this study, the above-mentioned genes of 74 bTB-infected Holstein cows and 90 healthy controls were genotyped using PCR followed by nucleotide sequencing. The influence of the foregoing genetic polymorphisms on bTB susceptibility was subsequently investigated by association analysis. Our data revealed that genetic polymorphisms in SLC11A1 exon 4 (g.107117166A > G which results in an alanine to threonine amino acid change at position 96) and intron 4 (g.107117369C > T), in NOS2 promoter (g.19958101T > G) and in TNF-α exon 3 (g.27534932A > C which results in an arginine to serine amino acid conversion at position 159) may contribute to the occurrence and development of bTB, strengthening the hypothesis that polymorphisms of the SLC11A1, NOS2, and TNF-α genes are associated with risk of bTB in Holstein cattle. Understanding the interactions between M. bovis and host cells is essential in developing tools for the prevention, detection and treatment of M. bovis infection. Gene expression profiles provide a large amount of information regarding the molecular mechanisms underlying these interactions. The present study analyzed changes in gene expression in bovine peripheral blood mononuclear cells (PBMC) at 0, 4 and 24 h following exposure to M. bovis. Using bovine whole-genome microarrays, a total of 420 genes were identified that exhibited significant alterations in expression (≥2‑fold). Significantly enriched genes were identified using the Ingenuity Pathway Analysis and the Kyoto Encyclopedia of Genes and Genomes database, of which the highest differentially expressed genes were associated with the immune system, signal transduction, endocytosis, cellular transport, inflammation and apoptosis. Of the genes associated with the immune system, 84.85% displayed downregulation. Our results reveal that during M. bovis infection, apoptosis of PBMCs was suppressed by interfering with TNF-α signaling. We also discovered the likely involvement of C-type lectin domain family 4, member E (CLEC4E) as early as 4 h post- infection triggering a series of negative intracellular signaling events via the Syk/CARD9 pathway for cytokines. This is the first report confirming induction of CLEC4E and the Syk/CARD9 pathway in PBMCs in response to M. bovis infection, and these findings support the view that M. bovis inhibits signaling pathways of antimycobacterial host defense in bovine PBMCs. In addition the comparison of the SLC11A1, NOS2, TNF-α mRNA level pre- and post-exposure to M. bovis show statistically significant decreased 3.03, 1.68, 5.66-fold at the 24-h time point, individually. These in vitro data demonstrated that the molecular alterations underlying the pathogenesis of tuberculosis begin early, during the initial 24 h following M. bovis infection. The current study provides valuable information for further characterization of host responses to M. bovis infection and suggests that genetic marker-based selection for resistance to bTB has the potential to make a significant contribution to bTB control. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51536 |
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