紅斑性狼瘡患者多形核嗜中性白血球與單核性細胞表面Toll-like receptors 2及4的表現及血清中可溶性受體sTLR2及sCD14之研究

余家利臺灣大學：分子醫學研究所劉韋博Liu, Wei-BoWei-BoLiu2007-11-262018-07-092007-11-262018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/51322近年來發現的模式辨識受體(pattern recognition receptors)家族中，Toll-like受體(TLRs)在脊椎動物先天與後天免疫的調節活化上，扮演著相當重要的角色。TLRs可表現在許多不同的細胞及組織上，其中包含樹突細胞、巨噬細胞及顆粒球細胞，主要為辨識革蘭氏陽性與陰性細菌、病毒的RNA、非甲基化CpG DNA、及黴菌等病原分子。研究顯示，TLRs從多個途徑影響生物體的免疫反應，與慢性炎症疾病及系統性自體免疫疾病發展有著密切的關係。全身性紅斑狼瘡(systemic lupus erythematosus, SLE)為全身性的自體免疫疾病，其致病機轉至今尚未完全清楚；本實驗我們透過正常人與SLE病人多形核嗜中性白血球(PMN)與單核性細胞(MNC)上TLR2及TLR4表現量的差異與否，進一步了解是否有其他協同作用分子也參與TLR2及TLR4訊息傳導，藉此找出與SLE疾病相關的TLRs訊息傳遞機轉。實驗結果發現，TLR2及TLR4在正常人與SLE病人PMN和MNC上的表現量並無顯著差異，反倒是TLR4與LPS的訊息傳遞協同作用分子sCD14，在SLE病人血清中卻顯著的提高；sTLR2為TLR2免疫反應的調節分子，我們的研究發現SLE病人血清中sTLR2的表現量較正常人低。另外，以anti-TLR2及anti-TLR4抗體來模擬免疫活化刺激PMN與MNC，觀察各項刺激對於sTLR2與sCD14表現的影響；結果顯示，各項刺激能使MNC產生的sCD14減少，sCD14可能參與SLE相關的免疫反應，而sTLR2的產生不受各別刺激影響，推估sTLR2是普遍存在人體血清中的。儘管sTLR2和sCD14在SLE疾病發展所扮演的角色仍有待釐清，希望本實驗結果對於慢性免疫性炎症疾病的發病機制與預防治療能提供一個嶄新的思考策略。Toll-like receptors (TLRs), a recently discovered family of pattern recognition receptors, play an important role in mediating the activation of innate and adaptive immunity in vertebrate animals. TLRs express on a diverse variety of cells and tissues especially on dendritic cells, macrophages and granulocytes, recognizing pathogens like Gram-positive and -negative bacteria, viral RNA, unmethylated CpG DNA and fungi. Recent studies show that TLRs signaling is involved in either the initiation or the progression of systemic autoimmune disease and chronic inflammatory diseases. Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease and its aetiology remains unclear. In this study, we investigate the expression of TLR2 and TLR4 on polymorphonuclear neutrophil (PMN) and mononuclear cell (MNC) in SLE patients and healthy subjects. We also try to identify the synergistic molecule related to TLR2 or TLR4 signaling and the mechanism involved in the pathogenesis of SLE. Our results show that the expression of TLR2 or TLR4 on PMN and MNC are not significantly different between healthy subjects and SLE patients. Nevertheless, we detect that the expression level of soluble CD14 (sCD14), the mediator of LPS-TLR4 signaling, in serum are significantly elevated in SLE patients. Moreover, we show that the expression level of soluble TLR2 (sTLR2), the modulator of TLR2 signaling, in serum are reduced in SLE patients. In addition, we utilize anti-TLR2 and anti-TLR4 antibodies to mimic immune stimulations to examine the immune response on PMN and MNC. These findings demonstrate that the production of sTLR2 seems unaffected by those stimulations. Only MNC is down- regulated by those stimulations in sCD14 expression. Likely sTLR2 is an existed molecule in normal serum and sCD14 is related to the immunity in SLE. Although further studies elucidating the detailed mechanisms of sTLR2 and sCD14 are required, this study provides a new direction for SLE treatment and prevention.目錄口試委員會審定書..........................................i 誌謝.....................................................ii 英文縮寫及中英文關鍵字..................................iii 中文摘要..................................................1 英文摘要..................................................2 第一章緒論...............................................3 1.Toll-like受體(TLRs).....................................3 1.1 TLRs的分佈及信號傳導..................................4 1.2 TLRs對適應性免疫的影響................................6 1.3 TLRs與慢性免疫性炎症..................................8 2.全身性紅斑狼瘡(SLE)....................................10 2.1 SLE致病機轉..........................................11 2.2 SLE活動性評估與治療預後..............................11 2.3 TLRs在SLE疾病所扮演之角色............................12 第二章研究動機..........................................14 第三章儀器與試藥........................................15 第四章材料與方法........................................18 第五章結果..............................................23 第六章討論..............................................27 第七章展望..............................................32 參考文獻.................................................33 圖表.....................................................40 圖表圖1.TLRs家族成員及其相對應配體...........................41 圖2.以流式細胞儀測定正常人與SLE病人其PMN上TLR2及TLR4的表現.......................................................42 圖3.正常人與SLE病人其PMN上TLR2及TLR4表現之分佈情形.......43 圖4.以流式細胞儀測定正常人與SLE病人其MNC上TLR2及TLR4的表現.......................................................44 圖5.正常人與SLE病人其MNC上TLR2及TLR4表現之分佈情形.......45 圖6.正常人與SLE病人血清中sCD14之表現量...................46 圖7.正常人血清中sTLR2 polypeptide的測定..................47 圖8.正常人與SLE病人血清中sTLR2之表現量...................48 圖9.血清中高濃度sCD14表現的SLE病人其sTLR2之表現情形......49 圖10.免疫細胞經由anti-TLR2/anti-TLR4 Ab刺激後其sCD14表現之變化情形.................................................50 圖11.免疫細胞經由anti-TLR2/anti-TLR4 Ab刺激後其sTLR2表現之變化情形.................................................512103180 bytesapplication/pdfen-USToll-like受體全身性红斑狼瘡可溶性受體sCD14及sTLR2模式辨識受體Toll-like receptorsystemic lupus erythematosussCD14sTLR2PAMPs紅斑性狼瘡患者多形核嗜中性白血球與單核性細胞表面Toll-like receptors 2及4的表現及血清中可溶性受體sTLR2及sCD14之研究Studies on the expression of TLR2 and TLR4 on polymorphonuclear neutrophils and mononuclear cells and their soluble receptors, sTLR2 and sCD14, in the serum of patients with systemic lupus erythematosusotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51322/1/ntu-96-P93448009-1.pdf