职业性三氯乙烯药疹样皮炎患者血清多肽指纹图谱的检测及其临床意义

发布时间：2018-01-22 05:53

本文关键词： 职业性三氯乙烯药疹样皮炎弱阳离子纳米磁珠基质辅助激光解析电离飞行时间质谱血清多肽指纹图谱诊断模型生物标志动态变化　出处：《湖南师范大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的近年来国内外因职业性接触三氯乙烯(Trichloroethylene,TCE)而引起中毒的事件屡见报道,其中以职业性三氯乙烯药疹样皮炎(Occupational Medicamentosa-Like Dermatitis induced by Trichloroethylene, OMLDT)居多。自1988年以来广东省先后在TCE接触工人中发现以严重皮损、发热、肝功能损害和浅表淋巴结肿大为表现的病例,至2009年已有394例,并且每年约有20例新发病例出现,被动接触人群也是逐年增加。该病起病急、易误诊、后果严重,现有预防措施效果不理想,己成为我国职业病危害的新问题。但目前其发病机制仍不明确,而且缺乏有效的疾病诊断和监测的生物学手段。因此,阐明其发病机制、寻找早期诊断生物标志以及治疗靶标具有重要的理论价值和现实意义。目前OMLDT已成为研究者关注的热点问题。研究内容主要集中在临床病例分析、回顾性职业流行病学调查、疾病易感基因多态性、免疫损伤机制等方面,但这些研究结果尚处于基础研究阶段,尚未在人群中验证,因此还无法作为有效的防治手段应用于实际工作。越来越多的证据表明血清中的蛋白质/多肽常常是疾病的诊断标志,是获得生物标志的丰富资源之一。研究疾病与健康状态下血清蛋白质的差异,将有助于寻找关键蛋白和标志蛋白,对于阐明疾病发生机制、寻找早期生物监测指标以及药物治疗靶标具有重要意义。本研究应用功能磁珠、基质辅助激光解吸电离飞行时间质谱(Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry, MALDI-TOF-MS)和ClinProTools生物信息学方法分离和筛选OMLDT患者发病各时期血清样本,建立OMLDT诊断模型,并分析其中蛋白质/多肽变化情况以了解整个病程中不同时期血清蛋白/多肽的动态变化规律,有望找到差异蛋白/多肽,为临床早期诊断和筛选高危人群提供参考。方法 1、研究对象 2009年12月至2011年10月共收集21例(?)OMLDT患者、64例职业性TCE接触工人、56例正常人用于临床调查研究；筛选18例OMLDT患者、29例职业性TCE接触工人、29例正常人用于OMLDT诊断模型的建立和验证；7例OMLDT典型患者用于动态变化研究。 2、临床调查研究对21例OMLDT患者临床资料和临床检测指标进行研究,分析其发病特点和规律、暴露和防治情况,并与64例职业性TCE接触工人以及56例正常人进行比较。 3、OMLDT诊断模型的建立和验证首先将2大影响血清蛋白/多肽指纹图谱的因素——样品处理和质谱检测条件进行了优化,主要包括整个实验的重复性评估、蛋白定量对模型的影响。然后按照一定标准选择OMLDT患者、TCE接触者和正常人群血清样品,采用MB-WCX联合MALDI-TOF-MS技术检测血清多肽指纹图谱,应用ClinProTools软件建立OMLDT疾病诊断模型,初步筛选OMLDT差异蛋白/多肽。然后用另一部分实验对象验证模型,评价模型的敏感性和特异性。进一步将急性期/接触者模型和急性期/正常人模型中的差异峰进行统计,以找到属于OMLDT的特征峰。 4、OMLDT典型病例动态变化研究根据OMLDT的特征峰,分析7例患者各个时期血清多肽指纹图谱,了解整个病程中不同病期血清蛋白组学的动态变化规律,同时进一步筛选并初步验证OMLDT血清蛋白/多肽标志。结果 1、临床调查研究结果 OMLDT发病主要以年轻人为主,起病时不易引起医生和患者的重视而延误治疗。发病时主要表现为畏寒发热、纳差厌油腻、皮疹和肝损害,伴有眼损害。B超发现肝胆脾受损严重。绝大部分患者WBC、EO和CRP明显升高,RBC和HGB水平显著降低；大部分都是急性重度中毒性肝病,主要表现为蛋白水平降低,而胆红素和酶学指标水平明显升高；肾功能基本稳定。甲基强的松龙激素治疗有效,有7例出现了病情反跳,1例转院治疗,其余均治愈出院。职业调查发现OMLDT患者存在于不同车间不同环境中,发病主要原因是防护措施过于简单、车间通风不良、工作人数较多以及工作时间长。 2、OMLDT诊断模型的建立和验证结果 2.1血清多肽指纹图谱实验条件优化结果不管是日内重复性还是日间重复性,各次实验的血清多肽指纹图谱基本一致,绝大部分峰的CV值40%,表明基于MB-WCX的MALDI-TOF-MS技术能够得到重复性高和稳定性好的血清多肽指纹图谱；同样的样本经过定量得到的血清多肽谱峰数要明显多于没有经过定量的,而且模型交互验证和识别能力也要基本高于未定量样本,但是敏感性和特异性却低于未定量样本。我们认为,定量样本用于建模和验证更加严谨、可靠。 2.2急性期和正常人模型我们对OMLDT急性期患者和正常人血清多肽指纹图谱进行统计共得到158个峰,72个有统计学意义的峰,其中有52个差异峰。52个差异峰中,有28个相对于正常人是低表达的,有24个是高表达的。我们从2D分布图上明显看到m/z为2106和2135Da的2个差异峰能够很好地区分急性期患者和正常人。ROC曲线分析也表明m/z为2106和2135Da的2个差异峰AUC值最大,几乎接近1,能够很好地区分急性期患者和正常人。我们应用SNN算法进行识别分析后筛选出由3个m/z分别为2106.3、2134.5和3263.67Da构建的诊断模型,交叉验证和识别能力分别是94.44%和100%。进一步用另一部分实验对象对模型进行验证,敏感性和特异性分别是100%和66.7%。 2.3急性期和接触者模型我们对OMLDT急性期患者和TCE接触者血清多肽指纹图谱进行检测共得到157个峰,72个有统计学意义的峰,其中有35个差异峰。35个差异峰中,有13个相对于TCE接触者是低表达的,有22个是高表达的。进一步通过2D峰分布图发现m/z为1450和5065Da的2个差异峰可以对两组样本进行有效区分,ROC曲线分析也表明这2个差异峰能够较好地区分急性期患者和接触者。我们应用SNN算法建立了由m/z为1450.33、1866.16、3262.39、4109.55、5064.85、5248.05、5956.57和6667.04Da共8个多肽峰组成的疾病诊断模型,交叉验证和识别能力分别是97.22%和100%。进一步用另一部分实验对象对模型进行验证,敏感性和特异性分别是81.3%和100%,说明建立的OMLDT急性期患者和TCE接触者诊断模型显示了良好的区分能力。 2.4OMLDT特征峰分析结果我们将急性期/正常人模型和急性期/接触者模型中的差异峰进行统计,结果共得到21个相同的差异峰,并且其表达水平变化也是一致的。其中有9个相对于接触对照组和正常对照组是低表达的,有12个是高表达的。 3、OMLDT典型病例动态变化研究结果在OMLDT21个特征峰中,我们发现4例病情反跳者在皮疹剥脱期、皮疹反跳期和恢复正常期3个时期内有4个蛋白/多肽峰的动态变化是基本一致的,m/z分别是4109、4267、5065和9287Da；3例非病情反跳者在急性皮疹期、皮疹剥脱期和恢复正常期3个时期内有2个蛋白/多肽峰的动态变化是完全一致的,m/z分别是4109和9173Da。而m/z为4109Da的特征峰在反跳组和非反跳组中均有出现,并且其表达水平随病程的变化呈现升高的趋势,提示其也许是OMLDT特有的血清学生物标志。结论 1、OMLDT主要以畏寒发热、纳差厌油腻、皮疹和肝损害为主要特征；激素治疗有效；由接触TCE引起,发病也许跟个体遗传易感因素有关,为一种职业性免疫损伤疾病；职业防护措施有待进一步加强。 2、本研究应用MB-WCX联合MALDI-TOF-MS技术结合ClinProToolsTM软件首次对OMLDT建立诊断模型并进行验证,得到较高的敏感性和特异性,筛选到了特异性差异蛋白/多肽,为临床早期诊断提供了参考。 3、特征峰峰面积概率分布规律能够准确反映OMLDT患者不同病期蛋白/多肽水平的动态变化,为监测疾病病程提供了一种新的思路。
[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.

【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R135.7

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