【作者】 江涛；

【导师】 朱启星； 杨永坚； 沈彤；

【作者基本信息】 安徽医科大学 ， 劳动卫生与环境卫生学， 2012， 硕士

【摘要】 研究背景三氯乙烯（Trichloroethylene，TCE）是工业上广泛使用的一种有机溶剂，在五金、电子、玩具等行业作为金属部件和电子元件的清洗剂、脱脂剂。在我国职业性三氯乙烯药疹样皮炎（ODMLT）的发病人数不断上升，已成为TCE接触工人健康危害的突出问题。ODMLT的发病机制，大多观点认为是T淋巴细胞介导的IV型变态反应。对于ODMLT中炎症性反应的发生机制方面，多数学者进行了大量研究，但有关TCE免疫性损伤的调节机制研究还刚起步。调节性T细胞是一种不同于Th1和Th2的具有免疫抑制功能的T细胞群，在免疫抑制或免疫耐受过程中发挥重要作用，主要通过分泌IL-10、TGF-β发挥抑制作用。目的通过检测TCE致敏小鼠脾脏Treg细胞比例，以及外周血和皮肤组织中抗炎性细胞因子白细胞介素（IL-10）、转化生长因子-β（TGF-β）的表达水平，研究CD4+CD25+Foxp3+Treg细胞在TCE引起的免疫应答中动态变化情况，探讨CD4+CD25+Foxp3+Treg细胞在TCE致敏中的作用，为最终阐明TCE的危害及其机理并制定防治对策提供理论依据。方法选用6~8周健康雌性BALB/c小鼠80只，随机分成空白对照组、TCE处理组。建立BALB/c小鼠致敏模型，观察记录小鼠背部受试部位的皮肤致敏反应情况。根据末次激发24h后小鼠的致敏结果以及取材时点的不同(分别于末次激发后1d、2d、3d、7d、14d采血，无菌取皮肤组织和脾脏)，将TCE处理组分为致敏1d组、致敏2d组、致敏3d组、致敏7d组、致敏14d组和未致敏1d组、未致敏2d组、未致敏3d组、未致敏7d组、未致敏14d组。脾脏单个核细胞悬液用相关荧光标记的抗体染色后采用流式细胞术分析。采用酶联免疫吸附法检测各组血清中IL-10、TGF-β水平。免疫组织化学法检测皮肤组织中IL-10、TGF-β的表达。结果1.致敏率皮肤致敏反应试验中，空白对照组背部皮肤均没有发现红斑和水肿；TCE处理组75只小鼠中有27只出现背部皮肤的红斑或水肿，致敏率为36%。2.脾脏Treg细胞的比例末次激发后，各组小鼠脾脏组织CD4~+CD25~+Foxp3~+Treg细胞比例：致敏1d组CD4~+CD25~+Foxp3~+Treg细胞比例较空白对照组升高不明显；与空白组对照组、致敏1d组相比，致敏2d组和3d组均显著升高，差异均有统计学意义；致敏3d组比例最高，但与致敏2d组比较，差异无统计学意义；与致敏2d组和3d组比较，致敏7d组和14d组又明显下降。同时点的比较，致敏2d、3d组分别高于未致敏2d、3d组，差异均有统计学意义。3.血清IL-10和TGF-β水平末次激发后，各组小鼠血清中IL-10的水平：所有致敏组显著高于空白对照组； IL-10的水平在致敏组有“先升后降”的趋势，在末次激发后3天达到最高，且致敏2d、3d、7d、14d组均高于致敏1d组，差异有统计学意义。同时点的比较，致敏组IL-10的水平均高于未致敏组，但差异无统计学意义，除了未致敏14d组。末次激发后，各组小鼠血清中TGF-β的水平：致敏1d、2d、3d、14d组显著高于空白对照组，差异有统计学意义；致敏3d、7d、14d组均低于致敏1d组，差异有统计学意义；致敏3d、7d组低于致敏2d组，差异有统计学意义。同时点的比较，致敏2d组TGF-β的水平均高于未致敏2d组（P<0.05），其他致敏组均高于未致敏组，但是差异无统计学意义。4.皮肤组织IL-10和TGF-β表达末次激发后，各组小鼠皮肤组织中IL-10的水平：致敏1d、2d、3d组较空白对照组显著升高，差异有统计学意义；致敏7d、14d组显著降低，与致敏1d、2d、3d组比较，差异均有统计学意义。同时点的比较，致敏1d、2d、3d组分别高于未致敏1d、2d、3d组，差异均有统计学意义。末次激发后，各组小鼠皮肤组织中TGF-β的水平：致敏1d、2d、3d、7d组较空白对照组显著升高，差异有统计学意义；致敏2d、3d组较致敏1d组升高，差异均有统计学意义；与致敏3d组比较，致敏7d、14d组下降，差异均有统计学意义；致敏14d组降至最低，与致敏7d组比较，差异有统计学意义。同时点的比较，致敏1d、2d、7d组分别高于未致敏1d、2d、7d组，差异均有统计学意义。结论1. TCE在一定剂量下能对小鼠致敏，致敏率为36%，TCE对BALB/c小鼠皮肤具有中度致敏作用。2. Treg细胞及其分泌的IL-10、TGF-β在TCE接触过敏性皮炎发生过程中可能发挥重要的免疫下调作用。更多还原

【Abstract】 BackgroundTrichloroethylene（TCE）is an organic solvent in industry, mainly used as cleaningand degreasing agents for metal parts and electronic components in hardware,electronics, toy industry, etc. The rising number of Occupational DermatitisMedicamentosa-like of Trichloroethylene (ODMLT) patients in our country has becomethe outstanding health hazards of workers exposed to TCE. There were many studies onthe pathogenesis of ODMLT, which considered it as delayed-type hypersensitivitymediated by T lymphocyte. Although most researches on inflammatory mechanism hadbeen carried out, the study on the regulatory mechanism of immunologic injury inducedby TCE has just started. Regulatory T cell is an new T cell population different fromTh1and Th2, and play an important role in the immune suppression or immunetolerance, mainly by the secretion of IL-10and(or) TGF-β.ObjectiveTo explore the dynamic changes of CD4+CD25+Foxp3+Treg cells in spleen ofTCE-induced mice by flow cytometrytechnology, and measure the level of TGF-β andIL-10in skin and sera at different time points, providing a theoretical basis forillustrating the hazards and mechanisms of ODMLT and countermeasures of TCE.MethodEighty healthy female BALB/c mice in6to8weeks, were randomly divided intoblank control group and TCE treatment group. Establish a BALB/c mice sensitizedmodel, observe and record the of tested part of the mice skins. At24h after the lastchallenge, the TCE treatment group, according to the sensitization status and thematerial collection points (on the1st day,2nd day,3rd day,7th day,14th day after thelast challenge, the blood samples, skin tissues and spleens were aseptically collected,respectively), was divided into sensitized1d group, sensitized2d group, sensitized3d group, sensitized7d group, sensitized14d group and unsensitized1d group,unsensitized2d group, unsensitized3d group, unsensitized7d group, unsensitized14dgroup. PBMCs of spleen were stained with fluorochrome-conjugated mAbs andanalyzed by flow cytometry. Detection of TGF-β and IL-10in sera was examined byenzyme-linked immunosorbent assays. Expression of TGF-β and IL-10in skin wasexamined by immunohistochemical technique.Result1. Sensitization rateIn the skin sensitization test, the control group showed no erythema or edema.Twenty-seven of seventy-five mice in TCE treatment group showed erythema or edema.Sensitization rate was36%.2. The proportion of spleen Treg cellsThe proportion of CD4~+CD25~+Foxp3~+Treg in sensitized2d or3d group wassignificantly higher than that in control group or sensitized1d group. It had reached thehighest level in sensitized3d group, which no statistically significant differencecompared with sensitized2d group. Sensitized7d or14d group was significantly lowerthan sensitized2d or3d group. At the same material collection point, the proportion ofCD4~+CD25~+Foxp3~+Treg in sensitized2d or3d group was significantly higher thanthat in the corresponding unsensitized group.3. Level of IL-10and TGF-β in seraThe levels of IL-10in all sensitized groups were significantly higher than that ofcontrol group. The level of IL-10in sensitized groups showed an ‘first upward and thendownward’ trend. It had reached the highest level in sensitized3d group, and sensitized2d3d,7d, or14d group was significantly higher than sensitized1d group. At the samematerial collection point, the level of IL-10in sensitized group was higher than that inunsensitized group, and the difference was not statistically significant but theunsensitized14d group.The level of TGF-β in sensitized1d,2d,3d, or14d groups was significantly higherthan that in control group. Sensitized3d,7d, or14d group was lower than sensitized1d group, and the difference was statistically significant. Sensitized3d or7d group waslower than sensitized2d group, and the difference was statistically significant. At thesame material collection point, the level of TGF-β in sensitized group was respectivelyhigher than unsensitized group, but only in sensitized2d group there was an statisticallysignificant difference compared with unsensitized2d group (P <0.05).4. Level of IL-10and TGF-β in skinsThe level of IL-10in sensitized1d,2d, or3d group was significantly higher thanthat of control group. The level of IL-10in sensitized1d,2d, or3d group wassignificantly higher than sensitized7d or14d group. At the same material collectionpoint, the level of IL-10in sensitized1d,2d, or3d group was significantly higher thanthat in the corresponding unsensitized group.The level of TGF-β in sensitized1d,2d,3d, or7d group was significantly higherthan that in control group. Sensitized2d or3d group was higher than sensitized1dgroup, and the difference was statistically significant. Sensitized7d or14d group wassignificantly lower than sensitized3d group. Sensitized7d was significantly higher thansensitized14d group which was to a minimum. At the same material collection point,the level of TGF-β in sensitized1d,2d, or7d group was significantly higher than that inthe corresponding unsensitized group.Conclusion1. TCE can cause sensitive effect on BALB/c mice with proper dose,at this experimentcondition, the sensitive rate is36%.2. The level of CD4+CD25+Foxp3+Treg cells and its related cytokines IL-10、TGF-βshowed statistical significannce between sensitized and non-sensitized groups, they mayparticipate in the formation of TCE-induced cutaneous sensitized reaction and plays animportant role in DMLT.更多还原