胡忠怡臺灣大學:醫學檢驗暨生物技術學研究所詹承儒Chan, Cheng-JuCheng-JuChan2010-05-112018-07-062010-05-112018-07-062008U0001-2107200812130500http://ntur.lib.ntu.edu.tw//handle/246246/182946生長停滯特異性基因6(Gas6)產物屬於vitamin K-dependent蛋白家族之一員，為一廣泛表現於人體內多種組織細胞(如內皮細胞、巨噬細胞、肺臟、小腸與骨髓等)之分泌性蛋白。Gas6蛋白之N端可與凋亡細胞表面之phosphatidylserine (PtdSer)結合，C端可為吞噬細胞表面的Gas6之接受器Mer所辨識，故Gas6於吞噬過程中具有橋接分子之功能。全身性紅斑性狼瘡(SLE)是一種系統性、慢性發炎的自體免疫疾病，起因於病人體內存在有會對抗自身抗原的淋巴球、自體抗體以及免疫複合物。在小鼠模型中發現，清除凋亡細胞之過程若有缺陷，會增加自體抗原暴露之機會，因而與自體免疫疾病(如SLE)之發生相關，本研究即希望探討橋接分子Gas6之異常表現與人類SLE發生或疾病進展之可能關聯。研究針對83名全身性紅斑性狼瘡病人以及40名非狼瘡對照組進行血清Gas6含量以及Gas6基因單點核甘酸變異之基因型分析。結果顯示40.96%的SLE病患相較於非狼瘡對照組有較高的血清Gas6含量(SLE: 24.51±14.06ng/mL, non-lupus control: 18.44±6.29 ng/mL, p<0.0001)，進一步分析血清Gas6量與發炎指標ESR、CRP之關聯，初步認定與發炎關聯不大。此外在高疾病活性(SLEDAI>6)、有狼瘡性腎炎或有血管炎之SLE病患均比其他SLE病患有較高的血清Gas6含量(高SLEDAI組: 25.65±14.77ng/mL, 低SLEDAI組: 20.89±8.70ng/mL, p=0.0092；有腎炎組: 27.40±14.70ng/mL, 無腎炎組: 23.52±13.51ng/mL, p=0.0286；有血管炎組: 28.55±14.54ng/mL, 無血管炎組: 23.71±13.69ng/mL, p=0.0287)。本研究亦分析Gas6基因intronic單點核苷酸變異(SNP)834+7G/A與同義性SNP1332C/T。基因分型結果顯示，Gas6 834+7A對偶基因型與SLE病患發生血小板減少有正相關(odds ratio=3.05, 95% CI: 1.02-9.13, p= 0.047)，而Gas6 1332之T對偶基因與SLE病患發生血管炎有正相關(odds ratio=3.07, 95% CI: 1.25-7.56, p=0.013)。此外帶Gas6 834+7GA/AA基因型的SLE病患血清中Gas6含量較帶Gas6 834+7GG基因型的SLE病患高(834+7GA/AA group: 27.03±16.44ng/mL, 834+7GG: 22.94±11.43ng/mL, p=0.0168)。結論：1. 相較於非狼瘡對照組，SLE病患血清Gas6量較高，且並非全由發炎造成，血清中較高的Gas6量可能與SLE的發生或疾病進展有關。2. 有腎炎與血管炎的SLE病患有較高的血清Gas6量。3. Gas6 SNP 1332C/T 與834+7G/A基因型分別與SLE病患血管炎、血小板減少有正相關。Growth arrest specific gene 6, a member of vitamin K-dependent protein family, is expressed in endothelial cells, macrophage, lung, intestine and bone marrow in a secreted form. Gas6 can function as bridging molecule between phosphatidylserine (PtdSer) on the apoptotic cells and Mer receptor on macrophages.Systemic lupus erythematosus(SLE) is an autoimmune disease characterized by chronic systemic inflammation resulted from autoreactive lymphocytes, autoantibodies and immune complexes. Ineffective clearance of apoptotic cells which leads to the release of self-antigen, may be important in the development of lupus-like autoimmune disease in mice. In this study, we intend to investigate whether serum Gas6 level and genetic variation in Gas6 are associated with prevalence or disease activity of human SLE.eral Gas6 level and the single nucleotide polymorphisms (SNPs) on Gas6 gene were investigated in 83 SLE patients and 40 non-lupus control subjects. Elevated serum Gas6 level was noted in 40.96% of SLE patients as compared with the non-lupus control.(SLE: 24.51±14.06ng/mL versus non-lupus control: 18.44±6.29 ng/mL, p≦0.0001). Correlation between serum Gas6 and ESR/CRP was also analysed. Serum Gas6 level is unlikely to be associated with inflammatory states in SLE. Furthermore, SLE patients with high disease activity index (SLEDAI>6), lupus nephritis or vasculitis showed higher serum Gas6 level than the other SLE patients. (high SLEDAI group: 25.65±14.77ng/mL versus low SLEDAI group: 20.89±8.70ng/mL, p=0.0092; nephritis group: 27.40±14.70ng/mL versus non-nephritis group: 23.52±13.51ng/mL, p=0.0286; vasculitis group: 28.55±14.54ng/mL versus non-vasculitis group: 23.71±13.69ng/mL, p=0.0287).Two single nucleotide polymorphisms (SNPs), intronic Gas6 834+7G/A and synonymous Gas6 1332C/T were studied. The genotyping results indicate that Gas6 834+7 A allele is positively associated with thrombocytopenia in SLE patients (odds ratio=3.05, 95% CI: 1.02-9.13, p= 0.047) and Gas6 1332 T allele was positively associated with vasculitis in SLE patients (odds ratio=3.07, 95% CI: 1.25-7.56, p=0.013). Furthermore, SLE patients with Gas6 834+7GA/AA genotype showed higher serum Gas6 level as compared to the SLE patients with a GG genotype (834+7GA/AA group: 27.03±16.44ng/mL, 834+7GG: 22.94±11.43ng/mL, p=0.0168). Conclusion: 1. Elevated serum Gas6 level was noted in some of SLE patients and did not completely result from inflammation. Elevated serum Gas6 level may be associated with SLE development. 2. SLE patients with lupus nephritis or vasculitis showed higher serum Gas6 level than the other SLE patients. 3. Gas6 SNP 1332C/T and 834+7G/A are positively associated with vasculitis and thrombocytosis, respectively.目錄………………………………………………………..………….…….I文摘要………………………………………………………………….IV文摘要………………………………………………………………….VI一章 序論…………………………………………….………………..1一節 全身性紅斑性狼瘡簡介…………………..…………………1 壹 自體免疫疾病簡介…………………………………….…….1 貳 全身性紅斑性狼瘡簡介……………………………….…….1 參 全身性紅斑性狼瘡臨床症狀與疾病診斷………………..…2 肆 SLE流行病學資料與可能之罹病風險因子………………..2二節 凋亡細胞清除與全身性紅斑性狼瘡之關聯…………..……4三節 生長停滯特異性基因6產物與其接受器簡介………...……6 壹 生長停滯特異性基因產物6………………….……………..6 貳 Gas6之接受器---Tyro3 family…………..…………………..7 參 Gas6與吞噬清除功能…………………….…………………9四節 研究目的與實驗設計………………….…………………...10二章 材料與方法……….……………………………………………11 一節 材料…………………………………………...…………….11 壹 檢體收集…………………………...……………………….11 貳 試劑清單………………………………...………………….12 參 試劑組/酵素………………………..…………………….…13 肆 聚合酶鏈鎖反應引子………………………………………14 伍 實驗儀器……………………………...…………………….15 陸 軟體與網路工具……………………………………………16二節 試劑配方……………………...…………………………….17三節 方法………………………………...……………………….20 壹 檢體處理……………………………...…………………….20 貳 Genomic DNA純化……………..………………………….20 參 聚合酶鏈鎖反應……………………...….…………………21 肆 洋菜膠電泳分析…………………………………………....25 伍 PCR產物純化與定序前處理………….…………………...25 陸 限制酶片段長度多型性分析……….……………………...26 柒 酵素結合免疫吸附法………………………………………27 捌 統計分析……………………………………………………28三章 實驗結果……………………………………………………….29一節 SLE病人臨床資料與疾病活性、發炎程度、症狀分析…..29二節 血清中Gas6蛋白量分析…………………………………..30 壹 SLE病人血清Gas6蛋白量顯著高於非狼瘡對照組…..….30 貳 血清Gas6蛋白量與發炎指標之關聯性分析…………..….30 參 血清Gas6蛋白量與SLE疾病活性之關聯性分析……...…31 肆 血清Gas6蛋白量與臨床症狀之關聯性分析…………...…31三節 Gas6基因之候選SNPs篩選……………………………….32四節 Gas6基因SNPs基因型分析…………….…………………33五節 Gas6基因SNPs與血清Gas6蛋白量相關性分析…………35四章 討論…………..…………………………...……………………36考文獻…………………………………………………………….……41 表…………………………………………..…………………………...47 錄……………………………………………..………………………...65application/pdf1933666 bytesapplication/pdfen-US全身性紅斑性狼瘡生長停滯特異性基因6凋亡細胞清除血清生長停滯特異性蛋白6含量單點核苷多型性systemic lupus erythematosus (SLE)Growth arrest specific gene 6(Gas6 gene)clearance of apoptotic cellsserum Gas6 levelsingle nucleotide polymorphism(SNP)[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/182946/1/ntu-97-R95424020-1.pdf