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研究生:余昆霖
研究生(外文):Kun-Lin Yu
論文名稱:SP110異構蛋白在發炎巨噬細胞中的差異性表現研究
論文名稱(外文):Characterization of Differential Expression of SP110 Isoforms in Inflammatory Macrophages.
指導教授:顏伯勳顏伯勳
指導教授(外文):Bo-Shiun YanBo-Shiun Yan
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:43
中文關鍵詞:SP110發炎巨噬細胞
外文關鍵詞:SP110inflammatorymacrophage
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肺結核是由結核分枝桿菌所引起的一種全球性的感染疾病。每年約有八百萬的人口被感染,而且每年約有大於兩百萬的人們死於此疾病。在先前的研究指出寄主的遺傳因子會影響結核分枝桿菌感染時免疫能力的高低,而在最近的研究發現,老鼠有一個能被干擾素(IFN)刺激而大量表現的基因Ipr1,能夠調控結核分枝桿菌感染時,寄主的先天性免疫反應。目前已經知道在人類的Ipr1同源基因為SP110,另外在一個西非的家族遺傳研究中也發現,不同病人中的基因多樣性的確與人體抵抗結核分枝桿菌的感染是有相關聯的。為了能夠更深入了解SP110在人類先天性免疫反應中所扮演的角色,我們首先利用快速放大cDNA(RACE)和聚合酶連鎖反應(PCR)這兩種方法找出了SP110的異構蛋白(isoforms)。另外從序列分析的結果,推測這些各種結構迥異的異構蛋白為SP110 mRNA選擇性剪接(Alternative splicing)所導致的。再進一步利用定量性聚合酶連鎖反應(qPCR)分析發現,當細胞經過干擾素的刺激後,SP110外顯子(Exon) 的表現具有差異性。因此從這些結果我們可以推測,當結核菌感染細胞後,所引發的干擾素表現,能夠影響SP110 mRNA的選擇性剪接,而導致不同的異構蛋白被轉譯表現。因此,mRNA選擇性剪接可能成為另一種新的參與在先天性免疫反應的機制。
Tuberculosis (TB) is a worldwide infectious disease caused by Mycobacterium tuberculosis (MTB). An estimated eight million people are infected each year and more than two million die annually. Previous studies have been confirmed that genetic factor influence susceptibility and resistance of MTB infection. Among the TB susceptibility associated gene, the Ipr1 has been identified in a mouse model to mediate innate immunity of TB. The closest human homologue of Ipr1, SP110b, also has been identified that polymorphism within this region is associated with TB in human. In order to understand the critical role of SP110 isoforms in innate immunity, rapid-amplified cDNA end (RACE) was used, and we isolated distinct cDNA from this gene, which share discontinuous subsegments of sequence. Subsequently, absolute quantification PCR (Q-PCR) using specific qPCR oligonucleotides for all exons identified that the expression of SP110 isoforms have different induction level after interferon and lipopolysaacharide treatment. These findings imply that the expression of SP110 isoforms may have a possible role in the mediation of gene regulation and result in controlling innate immunity as TB infection.
Content
Signature Page i
Thesis Approval Form ii
Acknowledgments iii
Chinese Abstract iv
Abstract v
Content vi
List of Figures ix
List of Tables x
Chapter 1. Introduction 1
1.1 Mycobacterium tuberculosis 1
1.2 The pathology of tuberculosis 1
1.3 Human genetics and TB susceptibility 2
1.4 Promyelocytic Leukaemia nuclear body 2
1.5 SP110 protein 3
Chapter 2. Aims and Rationales 4
Chapter 3. Materials and Methods 5
3.1 Bacterial strain and culture condition 5
3.2 Cell line and culture condition 5
3.3 Genomic DNA preparation 5
3.4 RNA preparation 6
3.5 Reverse transcription 6
3.6 RT-PCR 7
3.7 Rapid amplified cDNA end (RACE) 7
3.8 Preparation of competent cell 8
3.9 Cloning of cDNA 8
3.10 Quantitative PCR 9
3.11 Bioinformatic 9
Chapter 4. Results 10
4.1 SP110 gene structure 10
4.2 New SP110 isoforms were identified 10
4.3 Pattern of Alternative splicing in SP110 mRNA 11
4.4 Quantification of SP110 expression level 12
Chapter 5. Discussion 14
5.1 Structural difference among SP110 isoforms 14
5.2 Expression of SP110 gene upon IFN treatment 15
Chapter 6. Reference 17
Figures 20
Tables 33
Chapter9. Appendices 42
Abbreviation 42
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