臺灣博碩士論文加值系統

人類MxA蛋白質是干擾素誘導的蛋白質之一，具有對抗influenz病毒及其它RNA病毒的抗病毒性，被認為在寄主對抗某些特定的病毒中扮演重要的角色。76-kDa的MxA蛋白質已被確認其結構與功能和鼠科Mx1蛋白質類似。一個共同的特性是在某些器官中MxA蛋白質被干擾素-α或干擾素-β刺激反應而合成，基因表現的實驗也已顯示MxA基因起動子中至少有一區域含有對干擾素有反應的類似干擾素刺激反應序列(interferon stimulated response element like sequence；ISRE-like sequence)。本實驗隨機選取了30位南台灣閩南人進行MxA基因起動子核苷酸序列分析。而為了瞭解MxA基因起動子核酸苷序列之多型性是否和慢性C型肝炎(chronic hepatitis C)病人治療效果有相關性，共有108位接受完整療程的慢性C型肝炎病人之MxA基因起動子被分析。實驗結果顯示，MxA基因起動子無TATA盒子(TATA-box)和CAAT盒子(CAAT-box)，但有一富含GC核苷酸之區域。南台灣閩南族群之MxA基因起動子核苷酸序列與NCBI基因庫(NCBI GenBank)中MxA基因起動子之核苷酸序列相同度為98%，其中，在-2 nt(-2位置之核苷酸)與-3 nt間有一插入核苷酸，即Guanylic acid（G），此位置恰位於一般所謂的起始子(initiator)區域內。在-32 nt與-33 nt之間插入的G核苷酸（Guanylic acid），恰位於所推測的SP1結合位置內，而-41 nt位置缺失之C核苷酸（Cytidylic acid）恰緊鄰於ISRE1之下游一個核苷酸位置。在起動子-89 nt及-124 nt位置上則發現分別為G/T與A/C的對偶單核苷酸多型性。此兩個核苷酸位置使用限制片斷長度多型性分析(restriction fragment length polymorphism assay；RFLP assay)，共確認了80位無反應者(nonresponder；NR)及28位持續反應者(sustained responder；SR)所屬的合子(zygote)種類。為了探究單核苷酸多型性(single nucleotide polymorphism；SNP)與病人各變項或因子間的關係，病人則按相同治療背景其資料被分群如下:男性與女性、NR與SR、治療期間C型肝炎病毒RNA(hepatitis C virus RNA；HCV RNA)曾經陰性(含陰性)與陽性、治療前AST及ALT值正常與異常、治療期間ALT值曾正常(含正常)與異常、肝硬化與非肝硬化、干擾素處理後G型肝炎病毒RNA陰性與陽性、G型肝炎病毒感染帶原與G型肝炎病毒anti-E2抗體陽性。而治療前AST及ALT值及治療前病毒RNA濃度亦被比較，結果顯示，在不同合子間其AST及ALT值及治療前病毒RNA濃度無統計學上的明顯差異(p>0.05)，而所有分群病人中其合子之分佈亦無明顯差異，即無統計學上強烈的關連性(p>0.05)。然而C/C同型合子與G/G同型合子彼此間卻顯示高度的關連性(P<0.001)。此外，兩個SNP間亦顯示高度的關連性，所有108個個體中，其中在—124 nt位置上具有C核苷酸(Cytidylic acid )的106個個體中，有97.2 %(103/106)的個體本身在—89 nt位置上有G核苷酸(Guanylic acid)；而在—89 nt位置上具有T核苷酸(Thymidylic acid)的49個個體中，有63.3 %(31/49)的個體本身在—124 nt位置上有A核苷酸(Adenylic acid)。若將此兩個SNP結合並分析其9種排列組合所得合子之分佈，亦無發現任何統計學上有意義的顯著差異。

The human MxA protein is one of the interferon-inducible proteins that confers resistance to influenza virus and other RNA viruses. It is known to play an important role in the host defense against certain viruses. The human 76-kDa MxA protein was identified as a structural and functional homologue of the murine Mx1 protein. A common trait is that MxA proteins are synthesized in response to stimulation by IFN-α or β(interferon-α or β)in some organs. Gene expression test had been suggested that at least one region of nucleotide sequence of MxA gene promoter that contains ISRE-like sequence (interferon stimulated response element like sequence) which responds to interferon. The nucleotide sequences of MxA gene promoters of 30 randomly selected individuals of Southern Min group in southern Taiwan were analyzed. In order to investigate if any genetic polymorphism in the promoter region of the MxA gene is associated with the IFN responsiveness of hepatitis C virus (HCV)-infected patients, MxA gene promoters of 108 chronic hepatitis C patients who accepted integrated interferon-αtherapy were analyzed. There was no apparent TATA box or CAAT box but a GC-rich region in the MxA gene promoter. Identity of nucleotide sequence of MxA gene promoter from Southern Min group of southern Taiwanese was 98% in comparison with those of from NCBI GenBank. The result showed that a Guanylic acid and a Cytidylic acid were inserted between nt (nucleotide) positions —2 and —3 as well as —32 and —33 in the initiator region and putative SP1 binding site of MxA gene promoter, respectively. A deleted Cytidylic acid was found at nt position —41 which was located at one nucleotide downstream of the ISRE1 region. G/T and A/C allelic single nucleotide polymorphisms (SNPs) were found at nt positions —89 and —124, respectively. A RFLP assay (restriction fragment length polymorphism assay) was used for identifying the type of nucleotide or zygote of these two sites, including 80 NRs (nonresponders) and 28 SRs (sustained responders). In order to investigate if any variate or factor was associated with the SNP (single nucleotide polymorphism), patients who had the same therapeutic background were divided into different groups, including male and female、NR and SR、HCV RNA ever negative and positive during therapy、normal and abnormal AST or ALT value patients before therapy、ever normal and abnormal AST or ALT during therapy、cirrhosis and non-liver cirrhosis、HGV RNA negative and positive after interferon treatment、HGV infected and HGV anti-E2 antibody positive. AST、ALT value and HCV RNA concentration of different zygotes before therapy were analyzed. Difference of average of AST、ALT value and HCV RNA concentration between different zygotes before therapy were insignificant(p>0.05). Distributions of zygotes among different groups were also insignificant difference (p>0.05). However, the C/C homozygotes and G/G homozygotes showed a high linkage to each other (P<0.001). Furthermore, among 108 individuals, two SNPs showed a high linkage. 106 and 103 of 108 individuals had C at nt position —124 and C at nt position —124 as well as G at nt position —89, respectively. The linkage reached 97.2 ﹪(103/106). 49 and 31 of 108 individuals had T at nt position —89 and T at nt position —89 as well as A at nt position —124, respectively. The linkage reached 63.3 ﹪(31/49). Statistical analysis of the 9 combined zygotes in the MxA gene promoter showed no insignificant difference.

目錄
中文摘要………………………………………………………………Ι
英文摘要………………………………………………………………Π
壹、前言…………………………………………………………….1
一、起動子(promoter)與基因表現……………………………1
二、Mx基因簡介……………………………………………...2
三、干擾素與MxA基因之關係………………………………3
四、C型肝炎與干擾素治療…………………………………..6
五、C型肝炎與MxA基因……………………………………7
六、研究動機與目的…………………………………………..8
貳、材料與方法…………………………………………………….10
一、病人與血液樣本之來源………………………………….10
二、病人白血球去氧核糖核酸(DNA)之萃取………………..10
三、聚合酶連鎖反應(PCR)………………………………..….11
四、聚合酶連鎖反應產物水平膠體電泳分析……………….12
五、聚合酶連鎖反應產物之純化…………………………….12
六、MxA基因起動子核苷酸序列分析與比較…………………..…..12
七、限制片斷長度多型性分析(restriction fragment length polymorphism assay；RFLP assay)………………………13
八、統計分析…………………………………………………..14
(一)、比率變項(proportional variable)之統計分析……..14
(二)、t-檢定(Student-test)………………………………..14
(三)、費氏精確檢定法(Fisher exact test)………………..14
(四)、卡方檢定(Chi-Square test)…………………………14
參、結果………………………………………………………………16
一、聚合酶連鎖反應產物膠體電泳分析……………………...16
二、MxA基因起動子核苷酸序列分析………………………..16
(一)、MxA基因起動子核苷酸序列與NCBI GenBank MxA基因起動子核苷酸序列之比較…………………..16
(二)、MxA基因起動子存在之單核苷酸多型性……….21
(三)、核苷酸序列中潛在可能之調節要素 (potential
regulatory lement )……………………………….21
三、限制片斷長度多型性分析(restriction fragment length polymorphism assay；RFLP assay)………………………26
四、MxA基因起動子對偶單核苷酸多型性衍生之各種合
子之分析…………………………………………………..29
(一)、各合子分佈與性別之關連性分析……………..….29
(二)、不考慮病人治療背景下，各合子在分群病人
中分佈之分析……………………………………..32
(三)、考慮病人治療背景下，各合子在分群病人中
分佈之分析………………………………………..37
(四)、各合子在G型肝炎病人中之分佈………………..48
(五)、-89 nt與-124 nt位置上核苷酸種類之關連性分析48
(六)、結合合子之分佈……………………………………51
肆、討論……………………………………………………………..55
伍、參考文獻………………………………………………………..64
陸、附錄 (本實驗所用之統計方法簡介)……………………….…73

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