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研究生:嚴明琦
研究生(外文):Min-Chi Yen
論文名稱:人類C-C趨化激素RANTES/CCL5Promoter區域之單一核苷酸變異情形與僵直性脊椎炎之探討
論文名稱(外文):Single Nucleotide Polymorphism of the Human C-C Chemokine RANTES/CCL5 Promoter Region in Ankylosing Spondylitis
指導教授:陳建和陳建和引用關係華國媛華國媛引用關係
指導教授(外文):Chien-Ho ChenKuo-Yun Hwa
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:生物醫學技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:111
中文關鍵詞:單一核甘酸變異僵直性脊椎炎趨化激素
外文關鍵詞:Single nucleotide polymorphismsAnkylosing spondylitisChemokinesRANTES/CCL5
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背景:僵直性脊椎炎(Ankylosing spondylitis, AS)為慢性發炎性自體免疫疾病,其致病原因至今尚未完全明瞭,但與多重基因以及環境因素有著密切關係。目的:探討C-C趨化激素(chemokines)中RANTES/CCL5[Regulated on Activation Normal T-cell Expressed and Secreted/ Chemokine (C-C motif) ligand 5]基因promoter區域-403及-28位置的單一核苷酸變異(Single nucleotide polymorphism, SNP)與僵直脊椎炎之間的關聯,以及分析此二SNPs在台灣漢人(正常對照組)與台灣原住民種族之間的差異性。方法:1)利用直接定序(direct sequencing)的方式來建立200名正常台灣漢人以及58名台灣原住民的RANTES promoter基因之SNPs基因型資料庫;2)以Real-Time PCR方式來進行118名AS患者RANTES promoter之單一核甘酸變異的確認工作,並以Pearsonχ2 test以及Fisher’s exact test統計學方法評估-28及-403位置之SNPs是否與僵直性脊椎炎有關,以及確認不同種族及不同性別之間的SNPs的分布是否有差異。3)將三種族群之血漿進行ELISA測定血中RANTES蛋白質的濃度分布,並以Student’s t test評估不同種族之間的差異性是否有意義。結果:僵直性脊椎炎患者血漿中RANTES蛋白質(103.58±7.46 ng/ml)的表現比正常對照組(8.04±0.83 ng/ml)濃度高出許多(p=0.001),然而在RANTES基因 promoter區域-403及-28位置之基因型之分布均無統計學上差異(p>0.05)。在台灣漢人與台灣原住民之間RANTES基因 promoter區域-403位置GG(χ2=5.694,p=0.017)及AA(p=0.006)之基因型分布上是有差異的,此外A allele frequency也是有分布差異的(χ2=2.951,p=0.043),然而-28位置之基因型則無分布上的差異。結論:台灣漢人與台灣原住民由於種族上的不同造成RANTES基因 promoter區域-403位置基因型的分布有所差異,但不影響血漿中蛋白質的濃度分布;依目前實驗結果,僵直性脊椎炎與RANTES基因promoter區域SNPs似乎是無關連的,但血漿中RANTES蛋白質的濃度顯著上升顯然是受到其他因子調控而在慢性發炎反應中扮演一定角色。

Background: Ankylosing spondylitis(AS) is a chronic inflammatory autoimmune disease that is found associated with multiple genetic and environment risk factors. The cause for AS is yet to be identified. Objective: To test whether the SNPs in C-C chemokine RANTES/CCL5 [Regulated on Activation Normal T-cell Expressed and Secreted/ Chemokine (C-C motif) ligand 5] gene promoter region —403 and —28 positions are associated with AS or not, and whether the SNP genotypes of Taiwanese population (control subjects) is different from Aboriginal Taiwanese. Methods:i)To establish the RANTES/CCL5 gene promoter region SNP genotype database of 200 Taiwanese population(control subjects) and 58 Aboriginal Taiwanese by auto-sequencing, ii)to validate the SNP genotypes of 118 AS patients using real-time PCR. Pearsonχ2 test and Fisher’s exact test are performed to assess if there is significant genotype distribution between different races, and between normal and disease subjects, iii)to analyze the RANTES/CCL5 protein level in plasma of Taiwanese population (control subjects), Aboriginal Taiwanese, and AS patients by ELISA. Student’s t test is performed to assess if there is statistical significant difference among different populations. Results: The genotype GG(χ2=5.694, p=0.017) and AA(Fisher’s exact test p=0.006)distribution in RNATES/CCL5 gene promoter region -403 position are strikingly different between Taiwanese population (control subjects) and Aboriginal Taiwanese. The A allele frequency (χ2=2.951, p=0.043)is also significantly different. However there is no statistical significant difference(p>0.05) at —28 position. There is also no statistical significant difference in —403 and —28 SNP genotype distributions between Taiwanese population (control subjects) and AS patients. Yet the RANTES/CCL5 protein level of plasma in AS patients(103.58±7.46 ng/ml) is higher than that of Taiwanese population (control subjects) ( 8.04±0.83 ng/ml, Student’s t test =0.001).Conclusion: The RANTES/ CCL5 gene promoter region —403 position genotype distribution is different between Taiwanese population (control subjects) and Aboriginal Taiwanese, but there is no difference in protein level in plasma. Although the SNPs in RANTES/CCL5 gene promoter region is found not likely associated with AS, we suggest that there is/are other factor(s) regulating the RANTES/CCL5 protein secretion, which is possibly playing an important role in this chronic inflammatory disease.

致 謝………………………………………………………..……...…i
中文摘要…………………………………………………….……….…..ii
英文摘要………………………………………………………………...iv
縮 寫 表………………………………………………………………...vi
目 錄……………………………………………………...…...……vii
圖 目 錄……………………………………………………….....….….xi
表 目 錄…………………………………………………………….…xiii
附錄目錄………………………………………………………….….…xv
第一章 緒論…………………………………………………….……….1
第一節 趨化激素總論…………………………………………1
第二節 C-C 趨化激素”RANTES/CCL5”基因組成………….4
第三節 C-C 趨化激素”RANTES/CCL5”蛋白質之功能及作
用………………………………………………………5
第四節 單一核甘酸變異………………………………………6
第五節 珍貴的台灣原住民樣本………………………………6
第六節 僵直性脊椎炎…………………………………………8
第七節 僵直性脊椎言之分子機轉研究探討……………..…..9
第八節 研究動機……………………………………………..12
第二章 實驗材料及方法………………………………………………16
第一節 實驗材料……………………………………………..16
第二節 實驗方法及步驟……………………………………..23
第三章 實驗結果………………………………………………………31
第一節 台灣漢人(正常對照組)與台灣原住民之間血漿濃度
之RANTES蛋白質表現無差異;然而僵直性脊椎炎患
者血漿濃度之RANTES蛋白質表現是顯著上升…..31
第二節 台灣漢人(正常對照組)與台灣原住民之RANTES/
CCL5 promoter基因片段-469~+33區域定序結果..32
第三節 僵直性脊椎炎患者之RANTES/CCL5基因promoter區
域—403及-28位置之基因型分析結果………...…….33
第四節 台灣漢人(正常對照組)與台灣原住民之RANTES/ CCL5基因 promoter之-403位置基因型分布有差異; 然而-28位置基因型分布則無差異…………………34
第五節 台灣漢人(正常對照組)與僵直性脊椎炎患者之RANTES / CCL5基因promoter區域-403及-28 位置基因型分布無差異…………………………………..36
第六節 台灣漢人(正常對照組)與台灣原住民之RANTES/CCL5基因promoter區域-403 A allele frequency分布有差異;台灣漢人(正常對照組)與僵直性脊椎炎患者-403 A allele及-28 G allele frequency分布則均無差異…………………………..37
第七節 僵直性脊椎炎男女患者之間其RANTES/CCL5基因promoter區域-403位置出現A allele頻率之分布無差異……………………………………………………..38
第八節 台灣原住民男女之間其RANTES/CCL5基因promoter區域-28位置出現G allele頻率之分布有差異;台灣漢人(正常對照組)男女以及僵直性脊椎炎男女患者之間則無差異………………………………………..39
第九節 台灣漢人(正常對照組)與台灣原住民RANTES/CCL5基因promoter區域-403/-28 haplotype之分布有差異;台灣漢人(正常對照組)與僵直性脊椎炎患者之間分布則無差………………………………..………40
第四章 討論……………………………………………………………41
第一節 RANTES/CCL5基因promoter區域SNPs資料的建
立………………………………………………..……..41
第二節 RANTES/CCL5基因promoter區域SNPs基因型分布
之探討…………………………………………………42
第三節 僵直性脊椎炎患者血漿中RANTES/CCL5濃度與疾病
之關係…………………………………………………43
第四節 僵直性脊椎炎相關之基因研究報告探討……………45
第五節SNPs genotyping方法與生物資訊……………………49
第五章 參考文獻………………………………………………………50
第六章 圖表………………………………………………..…………..63
第七章 附錄…………………………………………………………....87
圖 目 錄
圖一 台灣漢人(正常對照組)之RANTES/CCL5基因promoter區域PCR電泳圖結果………………………………………………..63
圖二 台灣原住民族群之RANTES/CCL5基因promoter區域PCR電泳圖結果………………………………………………………..64
圖三 RANTES/CCL5基因promoter區域-403及-28之基因型
(GG/CC)定序結果………………………………………...….65
圖四 RANTES/CCL5基因promoter區域-403及-28之基因型(GG/GC)定序結果……………………………………………66
圖五 RANTES/CCL5基因promoter區域-403及-28之基因型
(GA/CC)定序結果……………………………………………67
圖六 RANTES/CCL5基因promoter區域-403及-28之基因型
(GA/GC)定序結果……………………………………………68
圖七 RANTES/CCL5基因promoter區域-403及-28之基因型
(AA/CC)定序結果……………………………………………69
圖八 RANTES/CCL5基因promoter區域-403及-28之基因型(AA/GC)定序結果……………………………………………70
圖九 RANTES/CCL5基因promoter區域-403及-28之基因型(AA/GG)定序結果…………………………………………...71
圖十 RT-PCR(TaqMam assay)分析-403位置三種不同基因型之SNP結果…………………………………………………….72
圖十一 RT-PCR(TaqMam assay)分析-28位置三種不同基因型之SNP結果…………………………………………………………..73
圖十二 台灣漢人(正常對照組)、台灣原住民與僵直性脊椎炎患者之血漿中RANTES濃度分布柱狀圖………………………74
表 目 錄
表一 台灣漢人(正常對照組)與台灣原住民之RANTES/CCL5基因
promoter區域- 403位置之基因型分布差異比較………………75
表二 台灣漢人(正常對照組)與台灣原住民之RANTES/CCL5基因
promoter區域- 28位置之基因型分布差異比較………………76表三 台灣漢人(正常對照組)與台灣原住民之RANTES/CCL5基因
promoter區域— 403及-28位置之基因型分布差異比較………77
表四 台灣漢人(正常對照組)與僵直性脊椎炎病人之RANTES/CCL5基因promoter區域- 403位置之基因型分布差異比較………78
表五 台灣漢人(正常對照組)與僵直性脊椎炎病人之RANTES/CCL5基因promoter區域- 28位置之基因型分布差異比較…………79
表六 台灣漢人(正常對照組)與僵直性脊椎炎病人之RANTES/CCL5基因promoter區域-403與-28位置之基因型分布差異比較
…………………………………………………………………..80
表七 台灣漢人(正常對照組)、台灣原住民及僵直性脊椎炎患者之RANTES/ CCL5基因promoter區域-403位置出現A allele的頻率分布差異比較………………………………………………..81
表八 台灣漢人(正常對照組)、台灣原住民及僵直性脊椎炎患者之RANTE/CCL5基因promoter區域-28位置出現G allele的頻率分布差異比較…………………………………………..82
表九 不同性別之台灣漢人(正常對照組)、台灣原住民及僵直性脊椎炎患者之RANTES/CCL5基因promoter區域-403位置出現A allele的頻率分布差異比較 ………………………83
表十 不同性別之台灣漢人(正常對照組)、台灣原住民及僵直性脊椎炎患者之RANTES/CCL5基因promoter區域-28位置出現G allele的頻率分布差異比較…………………………….84
表十一 台灣漢人(正常對照組)與台灣原住民之間RANTES/CCL5基因 promoter區域—403/-28位置haplotype之分布差異比較……………………………………………………………..85
表十二 台灣漢人(正常對照組)與僵直性脊椎炎病患之間RANTES/ CCL5基因promoter區域—403/-28位置haplotype之分布差異比較………………………………………………………..86
附 錄 目 錄
附錄一 趨化激素四種subfamilies圖示……………………………….87
附錄二 人類RANTES/CCL5基因promoter序列…………………….88
附錄三 DNA sequence and consensus elements of the RANTES immediate upstream region…………………………………..89
附錄四 RANTES/CCL5基因promoter重要轉錄因子結合位置簡圖..90
附錄五 Major Histocompatibility Complex (MHC) Class I分子組成..91
附錄六 MHC Class I 分子之摺疊與組裝程序圖…………………….92
附錄七 Human Leukocyte Antigen-B27(HLA-B27)錯誤摺疊之
結果圖示……………………………………………………..93
附錄八 TaqMan 方法分析SNP genotypes之原理圖………………..94
附錄九 RANTES/CCL5基因promoter區域-403/-28位置四種haplotypes 和血漿中RANTES濃度之關係柱狀圖…..……95

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