【摘要】：職業(yè)接觸三氯乙烯(Trichloroethylene,TCE)引起的藥疹樣皮炎是我國東南沿海地區(qū)嚴(yán)重影響作業(yè)工人健康的重要問題。TCE藥疹樣皮炎病因復(fù)雜,它是由環(huán)境和基因等多種因素共同引起的一種自身免疫性疾病,與人類白細(xì)胞抗原(Human leukocyte antigen,HLA)的多態(tài)性存在明顯關(guān)聯(lián),對于其發(fā)病機(jī)制的研究目前還處于未知狀態(tài)。本課題組在分子流行病學(xué)研究中發(fā)現(xiàn)HLA-B*13:01等位基因與TCE藥疹樣發(fā)病存在強(qiáng)關(guān)聯(lián)。提示HLA-B*13:01分子在TCE藥疹樣皮炎發(fā)病過程中可能發(fā)揮著重要作用。HLA分子具有明顯的多態(tài)性,主要功能是抗原的提呈和加工,在免疫反應(yīng)中,通過形成HLA-抗原肽復(fù)合物將抗原特異性地呈遞給T細(xì)胞受體,進(jìn)而激活T細(xì)胞,啟動免疫應(yīng)答。因此,我們提出如下假說：HLA-B*13:01分子通過提呈特異性抗原肽參與了TCE藥疹樣皮炎的發(fā)生過程。為了驗(yàn)證該假說我們設(shè)計(jì)的試驗(yàn)方案如下：(1)選擇HLA-B*13:02作為對照,分析HLA-B*13:01提呈抗原肽的特異性及其與疾病發(fā)生的關(guān)系。因?yàn)镠LA-B*13:01和HLA-B*13:02的一級結(jié)構(gòu)僅存在3個氨基酸的差別,但HLA-B*13:02等位基因與TCE藥疹樣皮炎無顯著關(guān)聯(lián),因此,我們研究HLA-B*13:01結(jié)合抗原肽,可以將HLA-B*13:02抗原肽作為對照研究組。(2)利用已經(jīng)構(gòu)建的表達(dá)HLA-B*13:01 及 HLA-B*13:02的B淋巴母細(xì)胞HMy2.C1R細(xì)胞系,通過免疫親和層析法,分離HLA Ⅰ類分子與抗原肽復(fù)合物,然后經(jīng)酸解離和分子篩過濾,獲得純化后的小分子抗原肽；采用反相高效液相色譜-質(zhì)譜技術(shù)來分離并測定抗原肽的一級結(jié)構(gòu)；比較HLA-B*13:01與HLA-B*13:02提呈抗原肽的一級結(jié)構(gòu)差異,分析特定HLA分子結(jié)合抗原肽的共同基序。(3)根據(jù)抗原肽與HLA分子結(jié)合的關(guān)鍵錨定位點(diǎn),即抗原肽第二位(Position 2,P2)和羧基末端(PΩ),高頻率出現(xiàn)氨基酸的情況,合成HLA-B*13:01/HLA-B*13:02的不同類型抗原肽,驗(yàn)證這些抗原肽與HLA.B分子的結(jié)合力,并同時(shí)觀察TCE以及三氯乙醇(Trichloroethanol,TCOH)對抗原肽與HLA-B分子結(jié)合的影響,揭示TCE通過改變HLA分子與抗原肽結(jié)合而誘導(dǎo)疾病發(fā)生的機(jī)制。研究結(jié)果如下：(一)HLA分子結(jié)合抗原肽結(jié)構(gòu)分析：本研究最終獲得57條HLA-B*13:01抗原肽序列和88條HLA-B*13:02抗原肽序列。通過分析抗原肽每個位點(diǎn)氨基酸出現(xiàn)的頻率,發(fā)現(xiàn)在HLA-B*13:01抗原肽中,P2位錨定氨基酸為亮氨酸(Leucine,L)和谷氨酰胺(Glutamine,Q),頻率分別為39%和33%；PΩ位錨定氨基酸為L(35%),苯丙氨酸(Phenylalanine,F).異亮氨酸(Isoleucine,I)(15%)和色氨酸(Tryptophan,W)(11%).HLA-B*13:02抗原肽中,P2位錨定氨基酸為Q(26%)和L(16%)；PΩ位為L(26%)、纈氨酸(Valine,V)(21%)和Ⅰ(14%)。對比HLA-B*13:01和HLA-B*13:02抗原肽P2和PΩ位錨定氨基酸的特點(diǎn),發(fā)現(xiàn)P2位二者錨定氨基酸一致,均為L和Q；PQ位錨定氨基酸存在差異,除L外HLA-B*13:01抗原肽的PΩ位優(yōu)勢氨基酸還包括F,W和Ⅰ,HLA-B*13:02則為V和I.HLA-B*13:01與HLA-B*13:02一級結(jié)構(gòu)僅在第94位(I,HLA-B*13:01; T,HLA-B*13:02).第95位(I,HLA-B*13:01;W,HLA-B*13:02)和第97位(R,HLA-B*13:01;T,HLA-B*13:02)3個氨基酸存在差別,其中95和97位氨基酸構(gòu)成HLA分子與抗原肽結(jié)合的F口袋,F口袋剛好與抗原肽PΩ位氨基酸側(cè)鏈結(jié)合,F口袋的形狀及特性決定了與之結(jié)合的抗原肽氨基酸側(cè)鏈類型,提示HLA-B*13:01與HLA-B*13:02抗原肽在PQ位置氨基酸存在的差別與HLA分子中參與構(gòu)成F口袋的分子結(jié)構(gòu)有關(guān)。(二)抗原肽的功能分析：通過HLA-抗原肽結(jié)合力試驗(yàn)發(fā)現(xiàn),在HLA-B*13:01抗原肽中,RQDDPSYRST(Q-F,即P2位為Q,PΩ位為F,下同)、AGDGTFQKW(G-W)、ALLNLAERL(L-L)、KLYEEKTGNAW(L-W)、 LFDHAVSKF(F-F)、RMDEEFTKI(M-I)和FLSKIEEKLT(L-T)等與HLA-B*13：01結(jié)合力較高,結(jié)合力分別為54%、50%、45%、44%、36%、36%和32%；在HLA-B*13:02抗原肽中,KLKGEENTV(L-V)的結(jié)合力較高,結(jié)合力達(dá)到73%。在抗原肽特異性交叉驗(yàn)證中,HLA-B*13:01抗原肽Q-F和G-W與HLA-B*13:01的結(jié)合力分別為54%和50%,但與HLA-B*13:02的結(jié)合力分別下降到24%和0.09%,提示這兩條抗原肽是HLA-B*13:01的特異性抗原肽；在HLA-B*13:02抗原肽中,L-V與HLA-B*13:02的結(jié)合力為73%,但與HLA-B*13:01的結(jié)合力下降到27%,提示L-V是HLA-B*13:02的特異性抗原肽。(三)TCE/TCOH對HLA-B分子結(jié)合抗原肽的影響：研究發(fā)現(xiàn)TCE可以明顯降低Q-F、L-T和G-W等抗原肽與HLA-B*13:01的結(jié)合力,它們與HLA-B*13:01的結(jié)合力分別為54%、32%和50%,經(jīng)TCE處理后,其結(jié)合力分別下降為11%、11%和32%,降低值分別為43%、21%和18%。Q-F這條抗原肽與HLA-B*13:01結(jié)合受TCE影響最大。TCOH使Q-F和L-W與HLA-B*13:01的結(jié)合力分別由54%和44%降低到43%和33%,降低值分別為11%和11%。但是TCE/TCOH對抗原肽與HLA-B*13:02的結(jié)合力并不產(chǎn)生影響。以上結(jié)果提示TCE確實(shí)可以通過HLA-B*13:01提呈的特異性抗原肽介導(dǎo)疾病的發(fā)生。綜上所述,本研究得出以下結(jié)論：(1)研究發(fā)現(xiàn)HLA-B*13:01與HLA-B*13:02提呈抗原肽的差別主要在于抗原肽的PΩ位氨基酸,而該位點(diǎn)氨基酸主要結(jié)合于HLA-B分子的F口袋,提示TCE藥疹樣皮炎的發(fā)生與HLA-B*13:01分子F口袋的結(jié)構(gòu)特異性有關(guān)；(2)TCE和TCOH可以影響某些抗原肽與HLA-B*13:01的結(jié)合,使HLA-B*13:01對所結(jié)合的抗原肽的偏好發(fā)生改變,而不改變抗原肽與HLA-B*13:02的結(jié)合,這與HLA-B*13:02基因與TCE藥疹樣皮炎無關(guān)聯(lián)的結(jié)果相吻合。當(dāng)抗原肽PQ位氨基酸為F(苯丙氨酸)時(shí),其與HLA-B*13:01的結(jié)合受TCE和TCOH的影響最大,提示TCE或TCOH可以改變HLA-B*13:01抗原肽的結(jié)構(gòu)特征,通過提呈致病性抗原肽,進(jìn)一步引起機(jī)體免疫應(yīng)答。本研究從HLA分子功能角度闡述了HLA-B*13:01與疾病關(guān)聯(lián)的分子機(jī)制,不僅豐富了國際抗原肽譜的數(shù)據(jù)庫,而且為進(jìn)一步研究TCE藥疹樣皮炎的發(fā)病機(jī)制提供了實(shí)驗(yàn)依據(jù)。
[Abstract]:Drug Eruption-like dermatitis caused by occupational exposure to trichloroethylene (TCE) is an important problem that seriously affects the health of workers in southeastern coastal areas of China. TCE drug Eruption-like dermatitis is an autoimmune disease caused by environmental and genetic factors. It is associated with human leukocyte antigen (HLA). HLA-B*13:01 allele was found to be strongly associated with TCE drug Eruption-like pathogenesis. HLA-B*13:01 may play an important role in the pathogenesis of TCE drug Eruption-like dermatitis. Molecules have obvious polymorphism and their main function is to present and process antigens. In the immune response, HLA-antigen peptide complexes are formed to present antigens specifically to T cell receptors, thereby activating T cells and initiating immune responses. Therefore, we propose the following hypothesis: HLA-B*13:01 molecules participate in TC by presenting specific antigen peptides. To validate this hypothesis, we designed the following protocols: (1) HLA-B*13:02 was selected as the control to analyze the specificity of HLA-B*13:01 antigen-presenting peptides and their relationship with the pathogenesis of the disease. There was no significant association between HLA-B*13:01 binding antigen peptide and TCE Eruption-like dermatitis, so HLA-B*13:02 antigen peptide could be used as a control group. (2) HLA-B*13:01 and HLA-B*13:02 B mother cells HMy2.C1R cell lines were constructed to express HLA-B*13:01 and HLA-B*13:02. The purified peptide was obtained by acid dissociation and molecular sieve filtration. The primary structure of the peptide was separated and determined by RP-HPLC-MS. The primary structure of the peptide was compared between HLA-B*13:01 and HLA-B*13:02, and the common motif of the peptide was analyzed. The key anchoring sites for binding of antigenic peptides to HLA molecules, i.e. the second position 2 (P2) and the carboxyl terminal (P_) of antigenic peptides, occur at high frequencies. Different types of antigenic peptides of HLA-B*13:01/HLA-B*13:02 were synthesized to verify the binding ability of these antigenic peptides to HLA.B molecules. TCE and trichloroethanol (TCO) were also observed. The results are as follows: (1) Structural analysis of HLA-binding antigen peptides: 57 HLA-B*13:01 antigen peptides and 88 HLA-B*13:02 antigen peptides were obtained in this study. The frequencies of amino acids were 39% and 33% for leucine (L) and glutamine (Q), 35% for P_, 15% for phenylalanine (F), and 11% for tryptophan (W), respectively. Among 02 antigen peptides, P2 anchored amino acids were Q (26%) and L (16%); P_anchored amino acids were L (26%), Valine (21%) and I (14%). Compared with HLA-B*13:01 and HLA-B*13:02 antigen peptides P2 and P_anchored amino acids, the anchored amino acids at P2 were both L and Q, and there were differences between them except L, HLA-B*13:01 antigen peptides. The preponderant amino acids at position P_include F, W and I, while HLA-B * 13:02 is V and I. HLA-B * 13:01 and HLA-B * 13:02 are only at position 94 (I, HLA-B * 13:01; T, HLA-B * 13:02). The preponderant amino acids at position 95 (I, HLA-B * 13:01; W, HLA-B * 13:02) and 97 (R, HLA-B * 13:01; T, HLA-B * 13:02) are different from those at position 97 (R, HLA-B * 13:01; T, HLA-B * 13:02). The shape and characteristics of F pocket determine the type of amino acid side chain of antigenic peptides bound to P_site. It is suggested that the difference between HLA-B * 13:01 and HLA-B * 13:02 antigenic peptides in PQ site is related to the molecular structure of HLA molecules involved in forming F pocket. Functional analysis of peptides: HLA-B*13:01 antipeptide binding assay showed that in HLA-B*13:01 antipeptide, RQDDDPSYRST (Q-F, P2 Q Q, P2 Q, P96position F, the same below), AGDGTFQKW (G-W), ALLNLAERL (L-L), KLYEEKTGNAW (L-W), LFDHAVSKF (DEF-F), RMEFTKI (M-I) and FLSKIEKLT (L-L T) and HLA-B*13:15:15:45% of the binding power of RQA-B*15:45%, respectively, the binding power of LFDDDDDDDDDDDDDDDDDDDDDPSSYSYRSSY44% Among the HLA-B*13:02 antigenic peptides, the binding power of KLKGEENTV (L-V) was higher and reached 73%. In the cross-validation of antigenic peptide specificity, the binding power of HLA-B*13:01 antigenic peptide Q-F and G-W to HLA-B*13:01 were 54%, 36% and 32%, respectively, but the binding power to HLA-B*13:02 decreased to 24% and 0.09% respectively, suggesting that the two antigenic peptides were HLA-B*13:01 antigenic peptides. In HLA-B*13:02 antigen peptide, the binding force of L-V to HLA-B*13:02 antigen peptide was 73%, but that of L-V to HLA-B*13:01 decreased to 27%, suggesting that L-V is a specific antigen peptide of HLA-B*13:02. (3) The effect of TCE/TCOH on HLA-B binding antigen peptide: TCE can significantly reduce the binding ability of Q-F, L-T, G-W and other antigen peptides to HLA-B*13:02. The binding power of Q-F to HLA-B*13:01 decreased from 54%, 32% and 50% respectively. After TCE treatment, the binding power of Q-F to HLA-B*13:01 decreased from 54% and 44% to 43% and 33% respectively. The low values were 11% and 11%, respectively. However, TCE / TCOH had no effect on the binding ability of antigen peptides to HLA-B*13:02. These results suggest that TCE can indeed mediate the occurrence of diseases through HLA-B*13:01 specific antigen peptides. In conclusion, the following conclusions were drawn: (1) The difference between HLA-B*13:01 and HLA-B*13:02 antigen peptides was found. It is suggested that the occurrence of TCE eruption dermatitis is related to the structural specificity of HLA-B*13:01 molecular F pocket; (2) TCE and TCOH can influence the binding of some antigenic peptides to HLA-B*13:01, which makes HLA-B*13:01 preferred to binding antigenic peptides. The binding of HLA-B*13:02 antigen peptide to HLA-B*13:02 was not altered, which was consistent with the result that HLA-B*13:02 gene was not associated with TCE Eruption-like dermatitis. In this study, the molecular mechanism of HLA-B*13:01 associated with disease was elucidated from the molecular function of HLA, which not only enriched the database of international antigen peptide spectrum, but also provided experimental basis for further study on the pathogenesis of TCE drug Eruption-like dermatitis.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R135.7

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