本文關(guān)鍵詞： 職業(yè)性三氯乙烯藥疹樣皮炎 弱陽離子納米磁珠 基質(zhì)輔助激光解析電離飛行時間質(zhì)譜 血清多肽指紋圖譜 診斷模型 生物標(biāo)志 動態(tài)變化　出處：《湖南師范大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景及目的 近年來國內(nèi)外因職業(yè)性接觸三氯乙烯(Trichloroethylene,TCE)而引起中毒的事件屢見報道,其中以職業(yè)性三氯乙烯藥疹樣皮炎(Occupational Medicamentosa-Like Dermatitis induced by Trichloroethylene, OMLDT)居多。自1988年以來廣東省先后在TCE接觸工人中發(fā)現(xiàn)以嚴(yán)重皮損、發(fā)熱、肝功能損害和淺表淋巴結(jié)腫大為表現(xiàn)的病例,至2009年已有394例,并且每年約有20例新發(fā)病例出現(xiàn),被動接觸人群也是逐年增加。該病起病急、易誤診、后果嚴(yán)重,現(xiàn)有預(yù)防措施效果不理想,己成為我國職業(yè)病危害的新問題。但目前其發(fā)病機(jī)制仍不明確,而且缺乏有效的疾病診斷和監(jiān)測的生物學(xué)手段。因此,闡明其發(fā)病機(jī)制、尋找早期診斷生物標(biāo)志以及治療靶標(biāo)具有重要的理論價值和現(xiàn)實(shí)意義。 目前OMLDT已成為研究者關(guān)注的熱點(diǎn)問題。研究內(nèi)容主要集中在臨床病例分析、回顧性職業(yè)流行病學(xué)調(diào)查、疾病易感基因多態(tài)性、免疫損傷機(jī)制等方面,但這些研究結(jié)果尚處于基礎(chǔ)研究階段,尚未在人群中驗(yàn)證,因此還無法作為有效的防治手段應(yīng)用于實(shí)際工作。越來越多的證據(jù)表明血清中的蛋白質(zhì)/多肽常常是疾病的診斷標(biāo)志,是獲得生物標(biāo)志的豐富資源之一。研究疾病與健康狀態(tài)下血清蛋白質(zhì)的差異,將有助于尋找關(guān)鍵蛋白和標(biāo)志蛋白,對于闡明疾病發(fā)生機(jī)制、尋找早期生物監(jiān)測指標(biāo)以及藥物治療靶標(biāo)具有重要意義。 本研究應(yīng)用功能磁珠、基質(zhì)輔助激光解吸電離飛行時間質(zhì)譜(Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry, MALDI-TOF-MS)和ClinProTools生物信息學(xué)方法分離和篩選OMLDT患者發(fā)病各時期血清樣本,建立OMLDT診斷模型,并分析其中蛋白質(zhì)/多肽變化情況以了解整個病程中不同時期血清蛋白/多肽的動態(tài)變化規(guī)律,有望找到差異蛋白/多肽,為臨床早期診斷和篩選高危人群提供參考。 方法 1、研究對象 2009年12月至2011年10月共收集21例(?)OMLDT患者、64例職業(yè)性TCE接觸工人、56例正常人用于臨床調(diào)查研究；篩選18例OMLDT患者、29例職業(yè)性TCE接觸工人、29例正常人用于OMLDT診斷模型的建立和驗(yàn)證；7例OMLDT典型患者用于動態(tài)變化研究。 2、臨床調(diào)查研究 對21例OMLDT患者臨床資料和臨床檢測指標(biāo)進(jìn)行研究,分析其發(fā)病特點(diǎn)和規(guī)律、暴露和防治情況,并與64例職業(yè)性TCE接觸工人以及56例正常人進(jìn)行比較。 3、OMLDT診斷模型的建立和驗(yàn)證 首先將2大影響血清蛋白/多肽指紋圖譜的因素——樣品處理和質(zhì)譜檢測條件進(jìn)行了優(yōu)化,主要包括整個實(shí)驗(yàn)的重復(fù)性評估、蛋白定量對模型的影響。然后按照一定標(biāo)準(zhǔn)選擇OMLDT患者、TCE接觸者和正常人群血清樣品,采用MB-WCX聯(lián)合MALDI-TOF-MS技術(shù)檢測血清多肽指紋圖譜,應(yīng)用ClinProTools軟件建立OMLDT疾病診斷模型,初步篩選OMLDT差異蛋白/多肽。然后用另一部分實(shí)驗(yàn)對象驗(yàn)證模型,評價模型的敏感性和特異性。進(jìn)一步將急性期/接觸者模型和急性期/正常人模型中的差異峰進(jìn)行統(tǒng)計,以找到屬于OMLDT的特征峰。 4、OMLDT典型病例動態(tài)變化研究 根據(jù)OMLDT的特征峰,分析7例患者各個時期血清多肽指紋圖譜,了解整個病程中不同病期血清蛋白組學(xué)的動態(tài)變化規(guī)律,同時進(jìn)一步篩選并初步驗(yàn)證OMLDT血清蛋白/多肽標(biāo)志。 結(jié)果 1、臨床調(diào)查研究結(jié)果 OMLDT發(fā)病主要以年輕人為主,起病時不易引起醫(yī)生和患者的重視而延誤治療。發(fā)病時主要表現(xiàn)為畏寒發(fā)熱、納差厭油膩、皮疹和肝損害,伴有眼損害。B超發(fā)現(xiàn)肝膽脾受損嚴(yán)重。絕大部分患者WBC、EO和CRP明顯升高,RBC和HGB水平顯著降低；大部分都是急性重度中毒性肝病,主要表現(xiàn)為蛋白水平降低,而膽紅素和酶學(xué)指標(biāo)水平明顯升高；腎功能基本穩(wěn)定。甲基強(qiáng)的松龍激素治療有效,有7例出現(xiàn)了病情反跳,1例轉(zhuǎn)院治療,其余均治愈出院。職業(yè)調(diào)查發(fā)現(xiàn)OMLDT患者存在于不同車間不同環(huán)境中,發(fā)病主要原因是防護(hù)措施過于簡單、車間通風(fēng)不良、工作人數(shù)較多以及工作時間長。 2、OMLDT診斷模型的建立和驗(yàn)證結(jié)果 2.1血清多肽指紋圖譜實(shí)驗(yàn)條件優(yōu)化結(jié)果 不管是日內(nèi)重復(fù)性還是日間重復(fù)性,各次實(shí)驗(yàn)的血清多肽指紋圖譜基本一致,絕大部分峰的CV值40%,表明基于MB-WCX的MALDI-TOF-MS技術(shù)能夠得到重復(fù)性高和穩(wěn)定性好的血清多肽指紋圖譜；同樣的樣本經(jīng)過定量得到的血清多肽譜峰數(shù)要明顯多于沒有經(jīng)過定量的,而且模型交互驗(yàn)證和識別能力也要基本高于未定量樣本,但是敏感性和特異性卻低于未定量樣本。我們認(rèn)為,定量樣本用于建模和驗(yàn)證更加嚴(yán)謹(jǐn)、可靠。 2.2急性期和正常人模型 我們對OMLDT急性期患者和正常人血清多肽指紋圖譜進(jìn)行統(tǒng)計共得到158個峰,72個有統(tǒng)計學(xué)意義的峰,其中有52個差異峰。52個差異峰中,有28個相對于正常人是低表達(dá)的,有24個是高表達(dá)的。我們從2D分布圖上明顯看到m/z為2106和2135Da的2個差異峰能夠很好地區(qū)分急性期患者和正常人。ROC曲線分析也表明m/z為2106和2135Da的2個差異峰AUC值最大,幾乎接近1,能夠很好地區(qū)分急性期患者和正常人。我們應(yīng)用SNN算法進(jìn)行識別分析后篩選出由3個m/z分別為2106.3、2134.5和3263.67Da構(gòu)建的診斷模型,交叉驗(yàn)證和識別能力分別是94.44%和100%。進(jìn)一步用另一部分實(shí)驗(yàn)對象對模型進(jìn)行驗(yàn)證,敏感性和特異性分別是100%和66.7%。 2.3急性期和接觸者模型 我們對OMLDT急性期患者和TCE接觸者血清多肽指紋圖譜進(jìn)行檢測共得到157個峰,72個有統(tǒng)計學(xué)意義的峰,其中有35個差異峰。35個差異峰中,有13個相對于TCE接觸者是低表達(dá)的,有22個是高表達(dá)的。進(jìn)一步通過2D峰分布圖發(fā)現(xiàn)m/z為1450和5065Da的2個差異峰可以對兩組樣本進(jìn)行有效區(qū)分,ROC曲線分析也表明這2個差異峰能夠較好地區(qū)分急性期患者和接觸者。我們應(yīng)用SNN算法建立了由m/z為1450.33、1866.16、3262.39、4109.55、5064.85、5248.05、5956.57和6667.04Da共8個多肽峰組成的疾病診斷模型,交叉驗(yàn)證和識別能力分別是97.22%和100%。進(jìn)一步用另一部分實(shí)驗(yàn)對象對模型進(jìn)行驗(yàn)證,敏感性和特異性分別是81.3%和100%,說明建立的OMLDT急性期患者和TCE接觸者診斷模型顯示了良好的區(qū)分能力。 2.4OMLDT特征峰分析結(jié)果 我們將急性期/正常人模型和急性期/接觸者模型中的差異峰進(jìn)行統(tǒng)計,結(jié)果共得到21個相同的差異峰,并且其表達(dá)水平變化也是一致的。其中有9個相對于接觸對照組和正常對照組是低表達(dá)的,有12個是高表達(dá)的。 3、OMLDT典型病例動態(tài)變化研究結(jié)果 在OMLDT21個特征峰中,我們發(fā)現(xiàn)4例病情反跳者在皮疹剝脫期、皮疹反跳期和恢復(fù)正常期3個時期內(nèi)有4個蛋白/多肽峰的動態(tài)變化是基本一致的,m/z分別是4109、4267、5065和9287Da；3例非病情反跳者在急性皮疹期、皮疹剝脫期和恢復(fù)正常期3個時期內(nèi)有2個蛋白/多肽峰的動態(tài)變化是完全一致的,m/z分別是4109和9173Da。而m/z為4109Da的特征峰在反跳組和非反跳組中均有出現(xiàn),并且其表達(dá)水平隨病程的變化呈現(xiàn)升高的趨勢,提示其也許是OMLDT特有的血清學(xué)生物標(biāo)志。 結(jié)論 1、OMLDT主要以畏寒發(fā)熱、納差厭油膩、皮疹和肝損害為主要特征；激素治療有效；由接觸TCE引起,發(fā)病也許跟個體遺傳易感因素有關(guān),為一種職業(yè)性免疫損傷疾�。宦殬I(yè)防護(hù)措施有待進(jìn)一步加強(qiáng)。 2、本研究應(yīng)用MB-WCX聯(lián)合MALDI-TOF-MS技術(shù)結(jié)合ClinProToolsTM軟件首次對OMLDT建立診斷模型并進(jìn)行驗(yàn)證,得到較高的敏感性和特異性,篩選到了特異性差異蛋白/多肽,為臨床早期診斷提供了參考。 3、特征峰峰面積概率分布規(guī)律能夠準(zhǔn)確反映OMLDT患者不同病期蛋白/多肽水平的動態(tài)變化,為監(jiān)測疾病病程提供了一種新的思路。
[Abstract]:Background and purpose of research
In recent years, the domestic occupation of external exposure to trichloroethylene (Trichloroethylene, TCE) caused by poisoning incidents have been reported, the occupation of DMLT (Occupational Medicamentosa-Like Dermatitis induced by Trichloroethylene, OMLDT) majority. Since 1988 in Guangdong province has TCE contact with severe skin lesions, fever found workers, liver function damage and superficial lymph nodes for the performance of the case, to 2009 had 394 cases, and each year about 20 new cases, passive exposure population is increasing year by year. The disease of acute onset, easy misdiagnosis, serious consequences, the prevention effect is not ideal, has become a new problem in our country occupation disease harm but. At present, the pathogenesis is still not clear, and the lack of biological means of disease diagnosis and monitoring effectively. Therefore, to clarify the pathogenesis, early diagnosis for biological It is of great theoretical and practical significance to mark and treat targets.
At present, OMLDT has become the focus of the researchers. The research mainly focused on analysis of clinical cases, retrospective epidemiological investigation of occupation susceptible gene polymorphism, disease, immune injury mechanism, but the results are still in the basic research stage, has not been verified in the crowd, so is not as effective means of prevention in the actual work. More and more evidence that serum protein / peptide is often the indicators for the diagnosis of disease, is one of the biomarkers of abundant resources. Difference of serum protein of disease and health condition, will help find the key protein and protein markers, for clarifying the mechanism of disease, it is significant to find early biological monitoring indexes and drug target.
This study used functional magnetic beads, matrix assisted laser desorption ionization time-of-flight mass spectrometry (Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry, MALDI-TOF-MS ClinProTools) and bioinformatics methods for the isolation and screening of OMLDT patients serum samples in each period, the establishment of OMLDT diagnosis model, and analysis the changes of protein / peptide in order to understand the dynamic changes of different during the period of serum protein / peptide in the whole course, is expected to find differential protein / peptide, provide reference for clinical early diagnosis and screening of high-risk population.
Method
1, the object of research
From December 2009 to October 2011 a total of 21 cases (?) OMLDT patients, 64 cases of occupation of workers exposed to TCE, 56 cases of normal people for clinical investigation; screening of 18 OMLDT patients, 29 cases of occupation of workers exposed to TCE, 29 cases of normal people used to establish and verify the OMLDT diagnosis model; 7 cases of OMLDT patients for the study of typical dynamic change.
2, clinical investigation and research
Methods the clinical data and clinical detection indexes of 21 patients with OMLDT were studied. The characteristics and rules of exposure, prevention and treatment were analyzed, and compared with 64 occupational TCE contacting workers and 56 normal persons.
3, establishment and verification of OMLDT diagnostic model
The 2 - sample processing and mass spectrometric detection conditions of serum protein / peptide fingerprints were optimized, including the experimental repeatability evaluation, the quantitative effect of protein to the model. Then according to certain criteria for the selection of OMLDT patients, TCE contacts and normal group serum samples, using MB-WCX combined with MALDI-TOF-MS detection technology serum peptide fingerprint, using ClinProTools software to establish the OMLDT model of disease diagnosis, preliminary screening of OMLDT proteins / peptides. Then another part of experiment to verify the model, the sensitivity and specificity of evaluation model. Further acute / contact model and acute / normal differences in the model peak statistics, to find the peaks belong to OMLDT.
4, study on dynamic changes of typical OMLDT cases
According to the characteristic peaks of OMLDT, the serum polypeptide fingerprints of 7 patients were analyzed, and the dynamic changes of serum proteomics in different stages of disease were analyzed. At the same time, OMLDT serum protein / polypeptide markers were further screened and preliminarily verified.
Result
1, the results of clinical investigation
The incidence of OMLDT in young people, at the onset of disease is not easy to cause the attention of doctors and patients and delayed treatment. When the disease is mainly manifested as fever chills, anorexia, tired greasy, skin rash and liver damage, accompanied by eye damage. Were found in liver and spleen were severely damaged. Most of the patients of WBC, EO and CRP increased significantly, RBC and HGB the level decreased significantly; most of them are severe acute poisoning liver disease, mainly for protein levels decreased and bilirubin levels were significantly increased and enzymology; renal function remained stable. The effective methylprednisolone methylprednisolone hormone therapy, 7 patients had disease rebound, 1 cases of hospital treatment, the others were cured. The survey found that occupation OMLDT patients exist in different workshops in different environment, the main reason is the onset of protective measures are too simple, the workshop ventilation, work number and long working time.
2, the establishment and verification of the OMLDT diagnostic model
Optimization results of experimental conditions of 2.1 serum polypeptide fingerprint
Whether it is day or the day repeatability reproducibility, the fingerprint of serum peptides of every experiment are basically the same, most of the CV peak value of 40%, indicating that MB-WCX MALDI-TOF-MS technology can obtain high repeatability and good stability of serum peptide fingerprint based on the same sample; after quantitative serum peptide peaks number was obtained without more than quantitative, and the model of cross validation and recognition ability to basic higher than the quantitative sample, but the sensitivity and specificity was lower than non quantitative samples. We believe that the quantitative sample used for modeling and verification is more precise and reliable.
2.2 acute and normal human model
We statistics on the acute phase of OMLDT patients and normal human serum peptide fingerprint were obtained 158 peaks, 72 significant peaks, of which there are 52 different peaks of.52 differential peaks, 28 of them are low relative to normal expression, 24 were high expression. We from the 2D distribution figure 2 m/z peak clearly see the difference between 2106 and 2135Da can well distinguish the normal people and patients with acute.ROC curve analysis indicated that m/z 2 AUC 2106 2135Da and the difference of peak value of the maximum, nearly 1, could be well separated in acute stage patients and normal people. We use the SNN algorithm to analyze after screened by 3 m/z respectively for 2106.32134.5 and 3263.67Da to establish the diagnosis model, cross validation and recognition ability were 94.44% and 100%. to further validate the model with the other part of the experimental object, the sensitivity and specificity were 100% And 66.7%.
2.3 acute phase and contact model
We were used to detect OMLDT and TCE in patients with acute contact serum peptide fingerprint were obtained 157 peaks, 72 significant peaks, of which there are 35 different peaks of.35 differential peaks, 13 relative to the TCE contact is low expression, 22 were high expression in. Step through the 2D peak m/z peak distribution found 2 differences between the 1450 and 5065Da can effectively distinguish between the two groups, ROC curve analysis showed that the difference between the 2 peaks could distinguish patients and contacts. We applied SNN algorithm established by m/z for the disease diagnosis model of 1450.331866.163262.394109.555064.855248.055956.57 and 6667.04Da 8 a peptide peak, cross validation and recognition ability were 97.22% and 100%. to further validate the model with the other part of the experimental object, the sensitivity and specificity were 81.3% and 100%, that building The diagnostic model of patients with acute OMLDT and TCE contacts showed a good distinction.
Analysis results of 2.4OMLDT characteristic peak
We will difference acute / normal model and acute phase / contact model of the peak statistics, results of 21 identical differential peaks were obtained, and the expression is the same. There are 9 contact relative to the control group and normal control group is low expression, there are 12 high expression.
3, research results of dynamic changes in OMLDT typical cases
In the OMLDT21 peaks, we found 4 cases of anti jumping in exfoliative rash, the rash rebound period of dynamic change and restore the normal period in 3 periods, 4 protein / peptide peak is basically the same, m/z is respectively 410942675065 and 9287Da; 3 cases of non disease rebound in acute skin rash, rash, exfoliative dynamic changes and restore the normal period in 3 periods, 2 protein / peptide peak is completely consistent with that of m/z were 4109 and 9173Da. and m/z as the characteristic peaks of 4109Da in the anti jump group and non rebound groups have emerged, and its expression level with the course of change is the increasing trend, suggesting that the OMLDT may be the biological specific marker. Serological
conclusion
1, OMLDT mainly chills fever, anorexia, tired greasy, skin rash and liver damage as the main character; hormone therapy; disease caused by TCE exposure, perhaps with individual genetic susceptibility factors, as a kind of occupation of immune injury disease; occupation protection measures should be further strengthened.
2, MB-WCX and MALDI-TOF-MS technology combined with ClinProToolsTM software were used to establish diagnostic models for OMLDT for the first time. The sensitivity and specificity of the diagnosis models were obtained. Specific differentially expressed proteins / peptides were screened, which provided a reference for early diagnosis in clinic.
3, the probability distribution law of the characteristic peak area can accurately reflect the dynamic change of protein / polypeptide level in different stages of OMLDT patients, and provide a new idea for monitoring the course of disease.

【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R135.7

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5 吳昆;深圳市1993～1997年三氯乙烯中毒分析[J];職業(yè)與健康;2002年05期

6 ;花季少女鐘表廠打工化學(xué)中毒[J];健康必讀;2005年12期

7 賈孟冬;;用血和尿中代謝產(chǎn)物的分析監(jiān)測三氯乙烯的接觸[J];鐵道勞動安全衛(wèi)生與環(huán)保;1978年01期

8 張壽林;職業(yè)病防治知識講座(十二) 急性職業(yè)性有機(jī)溶劑中毒(一)——苯、三氯乙烯[J];勞動保護(hù);2002年10期

9 謝國輝,黃振輝,張錦周,黃海雄;三氯乙烯對肝抗氧化酶活性與脂質(zhì)過氧化的影響[J];中國衛(wèi)生工程學(xué);2003年03期

10 徐新云;職業(yè)接觸三氯乙烯的皮膚損害特點(diǎn)及診斷探討[J];中國公共衛(wèi)生;2004年02期

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