三氯乙烯药疹样皮炎细胞免疫学指标及发病机制研究大学本科毕业论文.doc

目 录
中文摘要 1
英文摘要 6
英文缩写词表 12
前 言 14
第一部分 TCE药疹样皮炎细胞免疫学指标及发病机制研究 19
第一节 TCE药疹样皮炎患者外周血淋巴细胞抗原特异性增殖功能测定 21
第二节 TCE药疹样皮炎患者外周血免疫细胞检测分析 26
第三节 Fas/FasL与PF/GrB介导的TCE免疫毒作用机制探讨 37
小 结 55
第二部分 8例TCE药疹样皮炎患者病例报告分析 56
小 结 71
总 结 72
致 谢 74
参考文献 76
附录1 生物标志物的验证及其在健康危险预警中的作用 86
附录2 个人简历 96

三氯乙烯药疹样皮炎细胞免疫学指标及发病机制研究
中文摘要
职业接触三氯乙烯（Trichloroethylene，TCE）引起的TCE药疹样皮炎是近年来我国职业卫生领域令人关注的问题，但目前关于该病的发病机制尚不清楚，对TCE职业接触工人缺乏特异性健康监护指标，对TCE药疹样皮炎患者缺乏特异性临床治疗措施。
根据该病的发病特点一般认为其属于Ⅳ型超敏反应的可能性最大，Ⅳ型超敏反应发生的机制与抗体和补体无关，主要是T细胞介导的免疫损伤。T细胞介导的免疫反应发生的过程涉及到记忆性T淋巴细胞的生成和效应性T细胞的活化增殖，效应性T细胞通过分泌一系列细胞因子发挥作用，并调节B细胞、NK细胞以及其他各群免疫细胞的活化增殖。另外，细胞毒性T细胞（CTL）在T细胞介导的超敏反应中发挥重要的作用，其主要通过两条途径杀伤靶细胞，其一是穿孔素-颗粒酶途径，其主要参与免疫防御；其二是Fas-FasL途径，Fas/FasL主要介导活化的效应性细胞凋亡，清除激活的外周CTL，下调免疫应答。这两条途径对于机体对外来抗原进行适度的免疫应答，维持免疫自稳发挥重要作用。正常状态下，特异性淋巴细胞在外周免疫器官接受抗原刺激而大量增殖，在免疫应答末期，机体通过Fas/FasL细胞凋亡机制使发生活化的T细胞凋亡，从而对免疫过程进行严密调控，以维持免疫自稳。因此，TCE药疹样皮炎的的发生可能是由于机体免疫自稳状态被打破，发生过强的免疫应答反应所致。
为验证上述假说，本研究采用成组设计的多个样本比较分析以及TCE药疹样皮炎患者入院治疗以及肝功能恢复正常时机体细胞免疫学指标的比较分析，进一步探讨机体细胞免疫学指标的变化与TCE药疹样皮炎的关系。研究结果如下：
一、TCE药疹样皮炎细胞免疫学指标及发病机制研究
本研究选择三组研究对象，病例组为16例TCE药疹样皮炎患者。接触对照组为32例来自与病例同车间的从事TCE作业的健康职业工人；非接触对照组为28例从未接触过TCE及其他职业性有害因素的的健康就业前体检工人。三组研究对象按年龄性别进行匹配后，结果如下：
1.淋巴细胞抗原特异性增殖功能
16例病人除皮疹外，还伴有不同程度的肝损害，谷丙转氨酶（ALT）水平升高者15例（93.33%）；谷草转氨酶（AST）升高者12例（73.33%）。三组研究对象外周血淋巴细胞分别与不同浓度TCE（0.2mmol/L、1 mmol/L、5 mmol/L）共培养，在各组内随着TCE染毒浓度的增加外周血淋巴细胞的存活率逐渐降低，差异有统计学意义(p<0.05)，相同染毒浓度下淋巴细胞的存活率组间差异无统计学意义(p>0.05)，提示TCE本身具有细胞毒性，而在TCE药疹样皮炎患者以及TCE接触对照体内并不存在针对TCE原形的特异性记忆性淋巴细胞。
2.外周血淋巴细胞亚群分析
研究对象血常规检测结果发现，16例病人中有6例（37.5%）病人外周血淋巴细胞（LYMP）高于临床正常参考值范围，1（6.25%）例低于临床正常参考值范围；6例（37.5%）单核细胞（MONO）高于临床正常参考值范围；14例（87.5%）中性粒细胞（NEUT）高于临床正常参考值范围；8例（50%）嗜酸性粒细胞（EO）高于临床正常参考值范围，2例（12.5%）低于临床正常参考值范围，8例（50%）嗜碱性粒细胞（BASO）高于临床正常参考值范围。在接触对照组和非接触对照组体内上述指标均在临床正常参考值范围内。提示TCE药疹样皮炎患者体内免疫细胞的数量发生异常，由于外周血免疫细胞的改变与组织内免疫细胞的浸润密切相关，尤其是嗜酸性粒细胞主要分布于人体内呼吸道、消化道和泌尿生殖道粘膜组织中，其水平的升高可能与TCE药疹样皮炎病人出现的眼、口、生殖器等处的粘膜损伤有关。
结合血常规与流式细胞术检测的结果，比较各组之间淋巴细胞亚群在外周血中的变化情况。结果发现病例组外周血淋巴细胞总数、T细胞、CD4+T细胞计数均明显高于接触对照组和非接触对照组(p<0.05)，在接触对照组和非接触对照组之间无统计学差异(p>0.05)，提示这三个指标可作为TCE药疹样皮炎的疾病标志； CD8+T细胞和CD8+CD28-T细胞三组之间比较为病例组>接触对照组>非接触对照组，各组间差异有统计学意义(p<0.05)，CD3-CD56+细胞三组之间比较为病例组<接触对照组<非接触对照组，各组之间有统计学差异(p<0.05)。提示这三个指标可作为TCE接触工人的免疫学效应指标。
3.外周血Fas/FasL、PF/GrB的测定
病例组外周血CD8+T细胞表面Fas阳性表达的百分比高于接触对照组和非接触对照组，差异有统计学意义（p<0.05），在接触对照组和非接触对照组之间差异无统计学意义（p>0.05）；病例组与接触对照组外周血CD4+T细胞表面FasL阳性表达的百分比均高于非接触对照组（p<0.05），在病例组与接触对照组之间均无显著性差异（p>0.05）；CD8+T细胞表面FasL阳性表达的百分比呈现病例组>接触对照组>非接触对照组，组间差异有统计学意义（p<0.05）。PF蛋白在外周血淋巴细胞中表达高于接触对照与非接触对照组(p<0.05)，而在接触对照组与非接触对照组之间无显著性差异(p>0.05)。GrB在病例组的表达高于非接触对照组 (p<0.05)，在病例组和接触对照组之间差异无统计学意义(p>0.05)。提示在病例组工人体内活化的CTL通过Fas/FasL途径清除了一部分，但由于同时表达FasL的CD8+CTL随血液循环迁移至皮肤、肝脏、肾脏等组织器官，通过与这些组织细胞表面的FAS蛋白结合介导靶细胞的凋亡。同时病例组体内通过活化的CTL高表达PF和GrB蛋白，介导皮肤角质细胞的死亡，引起TCE药疹样皮炎患者皮肤的损伤。而健康的TCE接触对照工人体内也存在活化的细胞毒性T细胞（CD4+CTL与CD8+CTL），但并没有发病，其原因仍有待进一步研究。
病例组外周血Fas mRNA表达高于接触对照组（1.2622倍）与非接触对照组（1.1383倍）,接触对照组低于非接触对照组（0.9019倍）；病例组外周血FASL mRNA表达高于接触对照组（1.4239倍）与非接触对照组（1.7507倍），接触对照组低于非接触对照组（0.8133倍）。病例组（0.8166倍）与接触对照组（0.6009倍）PF mRNA表达低于非接触对照组，而病例组高于接触对照组（1.3590倍）。病例组GrB mRNA表达低于接触对照组（0.4477倍）与非接触对照组（0.4835倍），接触对照组高于非接触对照组（1.0800倍）。此研究结果与FAS、FASL、PF、GrB蛋白的表达情况不完全一致，是由于mRNA的表达在转录和翻译水平均受到多种因素的调节。
二、8例TCE药疹样皮炎病例报告分析
对8例TCE药疹样皮炎的病人入院治疗时及肝功能恢复正常时（转归时）各指标进行比较分析，进一步探索TCE药疹样皮炎病人体内各免疫学指标的变化以及可用于评价临床治疗效果的指标，并结合前面病例与对照实验研究结果对TCE药疹样皮炎与各细胞免疫学指标的关系进行综合分析。
1.各群淋巴细胞检测分析
获得6例病人入院治疗时及转归时淋巴细胞检测结果资料。与临床正常参考值相比较，6例（100%）病人入院时NEUT均升高，1例病人肝功能恢复正常时NEUT仍高于正常参考值范围；入院时EO升高者有4例（66.67%）；BASO和LYMPH升高者分别有3例（50%）；MONO升高者有2例(33.33%)，出院时仍有1例病人MONO升高。
对6例病人入院和转归时血常规结果进行比较，其中6例（100%）病人入院时NEUT与BASO均升高，5例（83.33%）病人LYMPH、MONO、EO均升高。提示TCE药疹样皮炎患者在发病的初期机体正在进行着免疫反应，与第一部分研究结果相一致。
对6例病人入院和转归时淋巴细胞亚群分析结果进行比较，6例病人中有4例（66.67%）在入院时T淋巴细胞升高，B淋巴细胞有3例（50%）升高，4例（66.67%）NK细胞降低。入院时4例（66.67%）CD4+T淋巴细胞明显增多，4例（66.67%）CD8+T淋巴细胞明显增多。CD8+CD28+T有3例（50%）入院时降低，CD8+CD28-T有5例（83.33%）入院时升高。6例病人中有4例在入院时CD4+/CD8+比值降低，4例CD28+/CD28-细胞的比值降低。提示TCE药疹样皮炎患者在发病初期机体内各群免疫细胞的数量与比例发生变化。
2.外周血Fas/FasL、PF/GrB的测定
与疾病转归时相比较，入院时6例病人中有4例（66.67%）Fas表达阳性 CD4+T细胞百分比升高；3例(50%)病人Fas阳性 CD8+T细胞百分比升高。6例病人中有3例(50%) FasL阳性 CD4+T细胞升高；3例(50%)病人FasL阳性 CD8+T细胞升高。而两例入院检测前未经激素治疗的病例外周血入院时Fas表达阳性 CD4+T细胞百分比降低、Fas阳性 CD8+T细胞百分比升高、FasL阳性 CD8+T细胞升高，提示病例在发病的早期CD4+CTL未发挥负调节免疫应答的作用，未及时清除活化的T细胞。CD8+CTL通过Fas/FasL途径介导了TCE药疹样皮炎患者的病例损伤。3例(100%)病人在外周血中PF表达阳性淋巴细胞百分率均高于出院时。1例(33.33%)病人外周血中GrB表达阳性细胞百分率高于出院时。提示在TCE药疹样皮炎的发病早期，PF通过杀伤皮肤角质细胞介导TCE药疹样皮炎患者的皮肤损害。此与第一部分研究结果相一致。

总之，本研究通过病例与对照实验研究以及病例入院和转归时细胞免疫学指标的比较分析，揭示TCE药疹样皮炎的发生可能是由其代谢产物而非TCE本身引起，TCE药疹样皮炎的发生与体内免疫细胞的活化增殖有关，TCE暴露能诱导人外周血淋巴细胞亚群的数量发生变化，其中总淋巴细胞、T细胞和CD4+T细胞计数升高可能作为TCE接触工人发生药疹样皮炎的的临床筛检指标，CD3-CD56+细胞计数降低、CD8+T细胞计数升高以及CD8+CD28-T计数升高可作为反映TCE接触的效应指标。CD4+CTL细胞通过表达Fas介导活化的T细胞凋亡，维持机体的免疫自稳。CD8+CTL通过表达FasL并随血液循环与皮肤、肝脏、肾脏等组织细胞表面Fas结合介导靶细胞凋亡，引起组织器官的免疫性损伤。CTL通过PF/GrB途径介导TCE药疹样皮炎患者的皮肤损害。因此本研究为TCE接触工人的健康监护具有重要指导意义和参考价值，并对TCE药疹样皮炎病人采取针对性的免疫治疗措施提供理论依据。

关键词：三氯乙烯，细胞免疫，药疹，接触性过敏性皮炎，半抗原，细胞毒性T细胞，淋巴细胞亚群，职业接触

Study on Index of Cell-mediated immunity and Pathogenesis
of Dermatitis medicamentosa-like of trichloroethylene
英文摘要
Abstract
Dermatitis medicamentosa-like of trichloroethylene(DMLT) in workers appears to be an occupational health issue and has aroused general concern in China in recent years. However, little is known about its pathogenesis to date, there are also lack of specific indexes for health surveillance as well as specific measures for clinical treatment.
DMLT may involve in type Ⅳ hypersensitivity according to its characteristics. Formation of T memory lymphocytes and proliferation are the two steps of cell-mediated immunity. Activated T cells regulate the proliferation of NK and B cells through secrete a series of cytokines. Cytotoxic T lymphocytes(CTL) play an important part in cell-mediated immune system. Its cytotoxicity can be induced by two ways: a) granuledependent exocytosis pathway, which participate immune defenses. b) Fas-FasL intercellular linkage-mediated pathway, which down-regulate immune response through clean activated peripheral CTL at the end of the immune response. The two ways play an important role in maintaining the immune homeostasis. The process of DMLT may related to the proliferation of lymphocytes and the function of CTL. Those two approaches play an important role in cleaning foreign antigens to maintain immune homeostasis. In normal condition, specific immune lymphocytes which stimulated by antigen proliferate in peripheral immune organs. In the end of the immune response, the activated cells apoptosis through Fas/FasL pathway, thus to control the immune process and maintain immune homeostasis. Therefore, DMLT might be induced by activated cells which were not cleared in time, and strong immune response breakout.
In order to test the above hypothesis, we conducted a three groups design analysis and cases analysis to explore the relationship between abnormal index of cell-mediated immunity and DMLT. The results were as follows:
1. Study on Index of Cell-mediated immunity and Pathogenesis of DMLT
There are there groups in this study: case group, cases are 16 patients of DMLT, all examinations executed on admission; TCE-exposure group, 30 workers came form the workshops the cases occurred but no skin disorders examined by the occupational physicians after the first 3 months of TCE exposure. Healthy worker group, 28 workers were never exposed to TCE and no history of previous skin disease. The main results are as follows:
Ⅰ.Test for functions of antigen-specific lymphocyte proliferation
16 patients with severe rash, accompanied by different degree of liver damage, 15 cases (93.33%) in 16 with higher ALT and 12 cases (73.33%)in 16 with higher AST. Peripheral blood lymphocytes(2×106/ml) of three groups cultured with different concentrations TCE (0.2mmol/L,1mmol/L, 5mmol/L). In any group, peripheral blood lymphocyte survival rates gradually reduced with the increase of TCE concentration. No significant difference was found about survival rates of lymphocytes under the same concentrations in three groups. The result suggest that TCE performed great cell toxicity on peripheral blood lymphocytes, and there was no TCE-specific memory lymphocyte.
Ⅱ. Analysis of lymphocyte subsets in peripheral blood
From the routine blood test of 16 patients, 6 cases(37.5%) peripheral blood lymphocyte(LYMP) higher than the normal clinical range, 1(6.25%) below the normal clinical range; 6 cases(37.5%) mononuclear cells (MONO) higher than normal reference range of clinical; 14 cases(87.6%) neutrophils NEUT) higher than normal clinical range; 8 cases(50%) eosinophils(EO) higher than normal clinical range, 2 cases (12.5%) below the normal clinical range; 8 cases (50%) basophilic granulocyte (BASO) higher than the normal range of clinical range. In both healthy TCE-exposed workers and unexposed workers, the indexes are all in clinical normal reference ranges. Those indicate immune response on going in patients of DMLT. The change of peripheral immune cells are closely related to the organization of immune cell infiltrates, human eosinophils mainly distributed in the respiratory tract, enteron and genitourinary tract mucosa tissue, the increase of EO in peripheral blood might relate to the damage of mucous membrane in eye, mouth and genital of patients.
The absolute counts of lymphocyte, T cell, CD4+ T cell were significantly increased in patients with hypersensitivity dermatitis compared with healthy TCE-exposed workers and unexposed workers(P<0.05); meanwhile, no significant differences in counts of lymphocyte, T cell, CD4+ T cell were demonstrated between exposed and unexposed groups(P>0.05), so those three indexes could be disease markers of DMLT. CD8+ T cell, CD8+CD28- T cell counts among the three groups showed case group>exposed group>unexposed control group, and the difference was significant(P<0.05). NK cell counts among the 3 groups showed the case group<exposed group<unexposed control group, and the difference was significant(P<0.05), so those three indexes could be immunology effect makers for TCE exposure.
Ⅲ.Examination of Fas/FasL、PF/GrB expression of protein and mRNA in peripheral blood
The percentage of CD8+CD95+T cell in CD8+T cell in case is higher than exposed group and unexposed group(P<0.05), no significant difference between exposed group and unexposed group(P>0.05). The percentage of CD4+CD178+T cell in CD4+T cell in case and exposed group are higher than unexposed group(P>0.05), no significant difference between case group and unexposed group(P>0.05).The percentage of CD8+CD178+T cell in CD8+T cell among the three groups showed case group>exposed group>unexposed control group, and the difference was significant (P<0.05). In case group, PF protein in peripheral lymphocyte is higher than exposed group and unexposed group(P<0.05), no significant difference between exposed group and unexposed group(P>0.05). It indicate CTL (CD4+CTL and CD8+CTL) induce apoptosis of effector cells in healthy TCE-exposed workers. However, in cases, some CTLs are removed through Fas/FasL pathway, others migrate to skin, liver, kidney, etc, and mediated the target cell apoptosis. Meanwhile, the high expression of PF and GrB cause skin damage in cases.
In case group the expression of FAS mRNA is higher than exposed(1.2622 fold) and unexposed group(1.1383 fold), exposed group is lower than unexposed group(0.9019 fold); In case group the expression of FasL mRNA is higher than exposed(1.4239 fold) and unexposed group(1.7507 fold), exposed group is lower than unexposed group(0.8133 fold); In case (0.8133 fold)and exposed group (0.6009 fold)the expression of PF mRNA are lower than unexposed group, in case group (1.3590 fold)is higher than unexposed group. In case group the expression of GrB mRNA is lower than exposed(0.4477 fold) and unexposed group(0.4835 fold), exposed group(1.0800 fold) is higher than unexposed group. The expression of FAS, FASL, PF, GrB in protein and mRNA level are not identical, that is due to the expression of mRNA in transcription and translation are regulated by various factors.

2. 8 cases report of DMLT
Compare the differences of DMLT when hospitalized and liver function recovered to normal, further explore the indexes of immunological changes and that could be used to evaluate the clinical treatment effect, and combining with the former group analysis to analyze the relationship between immunological indexes and DMLT.
Ⅰ.Analysis of lymphocyte subsets
There are lymphocyte results of six patients when hospitalized and liver function recovered to normal. NEUT in 6 cases(100%) were higher when checked into hospital; 4 in 6 cases(66.67%)had a higher EO than normal clinical range; 3 in 6(50%) cases had a higher BASO and LYMPH than normal clinical range. 6 cases(100%) had higher NEUT and BASO, 5 cases in 6 had higher LYMPH、MONO and EO when hospitalized than liver function recovered to normal. Those indicate immune function disorder in DMTL, all consistent with the former results.
Lymphocyte subsets analysis results of six patients in hospital and liver back to normal, 4 cases (66.67%)in 6 have significantly higher T lymphocytes in admission; B lymphocytes, 3 cases (50%) was obviously higher than normal liver, 4 cases (66.67%) NK cell below normal liver. On admission, 4 cases (66.67%) CD4+T lymphocyte increased obviously, 4 cases (66.67%) CD8+T lymphocytes increased obviously. In 3(50%)cases CD8+CD28+T below in admission, 5 cases (83.33%) CD8+CD28-T higher than liver back to normal. 4 patients in 6 patients (66.67%) CD4+/CD8+ ratio decreased when hospitalized, 4 cases CD28+/CD28- cell ratio decreased. It indicated there are changes of immune cells number and proportion onset of DMLT.
Case 1, 5, and 6 discharged from hospital when the liver function returning to normal. NK cells elevated、 CD28+CD8-T cells reduced and the ratio of CD4+/CD8+ returned to normal in 3 patients when discharged from hospital. Those three indexes could be the index of rehabilitation. In case 1 and case 6, T lymphocytes、CD8+T lymphocytes declined when discharged from hospital, it might be a reference index of rehabilitation.
Ⅱ.Examination of Fas/FasL、PF/GrB expression of protein and mRNA in peripheral blood
4 patients in 6 (50%) the percentage of Fas+CD4+T cells in CD4+T were higher when hospitalized 、3 patients (50%) patients the percentage of FAS+CD8+Tcells in CD8+T were higher at admission than normal liver function, 3 patients in 6 (50%) the percentage of FasL+CD4+T cells in CD4+T were lower, 3 cases (50%) the percentage of FasL+CD8+T cells in CD8+T were lower than rehabilitation. Those results are consistent with the former results.
PF protein in peripheral lymphocyte was higher in 3 cases(100%) when hospitalized than liver function recovered to normal; 1 in 3 cases(33.33%) GrB protein in peripheral lymphocyte was higher when hospitalized than liver function recovered to normal. It indicated PF damage skin keratinized cell at the early stage of DMLT.

In summary, this study revealed the immune response in DMLT might induc