【作者】 越飞；

【导师】 陈学敏； 黄汉林；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2006， 博士

【摘要】 本文由5个部分组成:第一部分发现病例场所所用三氯乙烯成份分析;第二部分三氯乙烯药疹样皮炎患者部分免疫功能变化观察;第三部分三氯乙烯染毒小鼠免疫功能变化研究;第四部分三氯乙烯药疹样皮炎与免疫相关基因易感性关系的研究;第五部分三氯乙烯药疹样皮炎患者基因表达变化的初步研究。第一部分发现病例场所所用三氯乙烯成份分析目的:选取部分三氯乙烯药疹样皮炎患者使用的三氯乙烯进行全成份气相色谱-质谱联机检测,检测三氯乙烯及各种杂质的含量,了解不同工厂使用的导致三氯乙烯药疹样皮炎的三氯乙烯是否存在共同杂质,为确定三氯乙烯致病机制提供依据。方法:选取9个发生三氯乙烯药疹样皮炎工厂病人发病时使用的三氯乙烯,采用HP 6890/ 5973气相色谱-质谱联用仪,抽取0.4ml三氯乙烯直接进样进行全成份分析。在经气相色谱分离后,对离子流中每一峰的质谱图与谱库中谱图比对,与某物质谱图匹配率达90%以上时确定为该物质。然后将所有样品进行汇总比较,观察是否存在共同杂质。如存共同杂质则判断其是否存在致敏可能。结果:9个工作场所三氯乙烯样品所含杂质不尽相同,其中9个样品共同的成份为三氯乙烯、1-1-2-三氯乙烷和四氯乙烯。另外,三氯甲烷、二甲苯和四氯化碳分别在约2/3样品中检出。结论:综合本次研究结果,所有样品中除三氯乙烯外存在共同杂质:1-1-2-三氯乙烷和四氯乙烯,但尚未见二者引起致敏的报道。初步认为三氯乙烯药疹样皮炎的过敏原是三氯乙烯可能性较大。第二部分三氯乙烯致药疹样皮炎患者部分免疫功能变化目的:通过对三氯乙烯致药疹样皮炎患者部分免疫功能指标的观察,了解三氯乙烯药疹样皮炎患者发病时机体部分变化情况,为探索发病机制提供线索。方法:采集病人外周静脉血进行体外实验。采用MTT法观察患者淋巴细胞在加入三氯乙烯前后增殖情况差异;观察患者白细胞在体外加入三氯乙烯后其移动范围的变化以及将三氯乙烯药疹样皮炎患者CD4+/CD8+比值与正常对照进行比较。结果:三氯乙烯(40 mg/L及80mg/L)可显著促进三氯乙烯致药疹样皮炎患者淋巴细胞增殖(P<0.05);该类患者CD4+/CD8+与正常对照组相比差异无显著性意义;三氯乙烯对该类患者白细胞移动的抑制指数与对照组相比无显著性差异。结论:提示TCE可能是该类患者免疫反应的抗原(或半抗原)。第三部分三氯乙烯染毒小鼠免疫功能变化研究目的:本部分研究应用NIH小鼠染毒后,进行小鼠足跖肿胀实验和脾淋巴细胞增殖实验等,观察了三氯乙烯染毒小鼠免疫功能的变化,为探索三氯乙烯致敏作用提供依据。方法:给予NIH小鼠皮下染毒三氯乙烯后,研究胸腺、脾、肝脏等脏器系数变化情况、进行足跖肿胀实验、脾淋巴细胞增殖实验等。结果:三氯乙烯染毒小鼠胸腺、脾脏器系数等未见明显变化。24h、48h时小鼠足跖肿胀系数明显高于对照组。80mg/L三氯乙烯可抑制对照组脾淋巴细胞的增殖,但可促进TCE染毒小鼠脾淋巴细胞增殖。结论:三氯乙烯可能诱导NIH小鼠产生迟发性变态反应。第四部分三氯乙烯药疹样皮炎与免疫相关基因易感性关系的研究目的:根据HLA-DM、HLA-DQA和Lag-3基因多态性在三氯乙烯药疹样皮炎和正常对照分布情况,探索三氯乙烯药疹样皮炎的易感性基因,为其危害防治提供依据。方法:提取病例组(61例)及对照组(60例)淋巴细胞DNA,应用降落式PCR扩增,分别采用限制性片断长度多态性检测方法、直接测序或二者结合方法,确定其HLA-DM、HLA-DQA和Lag-3基因多态性,比较两组之间三个基因的等位基因型和基因型出现频率。结果:病例组HLA-DMA*0101等位基因频率明显高于对照组(P<0.05),病例组HLA-DMA*0102等位基因频率明显低于对照组。病例组HLA-DMB*0103等位基因频率明显高于对照组(P<0.05)。其他等位基因型频率在两组之间均无显著性差异。对照组HLA-DMA*0102-*0102纯合子比例明显高于病例组(P<0.05),OR为3.86,CI为2.78-4.94。而对于HLA-DMB,病例组*0101-*0102杂合子比例明显低于对照组(P<0.05),OR为3.59,CI为2.71-4.47。其他HLA-DMA、DMB基因型频率在两组之间均无显著性差异。HLA-DQA等位基因型中,*0201频率在病例组明显高于对照组(P<0.05)。*05频率在病例组明显低于对照组,但P值经校正后大于0.05。所有受检的病例及对照Lag-3基因的exon1-6及exon-8序列与Genebank中序列一致,未发现缺失、突变及多态性情况。exon-7存在多态性,但其在两组间分布情况差异无显著性(P>0.05)。结论: HLA-DM基因、HLA-DQA基因多态性可能与三氯乙烯药疹样皮炎的易感性有关。Lag-3基因多态性可能与三氯乙烯药疹样皮炎的易感性无关。第五部分三氯乙烯药疹样皮炎患者基因表达变化的初步研究目的:了解三氯乙烯药疹样皮炎患者治疗前后(治疗后看作近似正常)基因表达谱是否存在差异,涉及哪些基因。进而选取病人共同改变的部分基因,应用荧光定量PCR方法在其他病人加以验证。通过观察三氯乙烯药疹样皮炎治疗前后外周血淋巴细胞中基因表达改变,探索三氯乙烯药疹样皮炎涉及哪些基因及其病理过程。为研究三氯乙烯药疹样皮炎发病机制提供基础资料。方法:收集12例病人入院及激素治疗一个月后的外周血淋巴细胞,分别提取总RNA。其中2例应用基因芯片检测治疗前后基因表达谱改变。然后比较两例结果,找出其共有的改变。在基因芯片筛选出的共有差异表达基因中选取部分表达差异较大的10个基因在其他10个病人中加以验证。结果:将治疗前后两样品信号强度间的比值(Ratio)大于2或小于0.5的数据作为有表达变化的基因数据。在506号病例中有基因表达改变317个,在治疗前样品中表达上调的基因有262个,下调表达的基因有55个;在507号病例中这类基因有591个,在治疗前样品中表达上调的基因有467个,下调表达的基因有124个。两例同为上调的基因147个,同为下调的基因9个。从初步的结果分析来看,与细胞表面抗原、细胞因子以及免疫系统相关的基因有比较明显的表达变化,其他具有明显的功能类型特征的基因包括与蛋白质合成及降解相关的基因表达的变化。结论:初步研究显示三氯乙烯药疹样皮炎病人病理过程中可能存在相似的基因表达改变,从而引发相同的病理过程。更多还原

【Abstract】 Part One Analysis of the components of Trichloroethylene used by patientsObjective To investigate the concentration of the impurity and find out whether or not the coexistent impurity presence in the Trichloroethylene which cause to the Occupational medicamentosa-alike dermatitis due to trichloroethylene in the factory, and to give the basis data for the role of trichloroethylene in the mechanisms of the Occupational medicamentosa-alike dermatitis due to trichloroethylene.Methods Using HP 6890/5973 Gas chromatogram-mass spectrum instrument, 0.4ml of trichloroethylene of 9 samples was taken out to detect, which is used in the factories by the patients of Occupational medicamentosa-alike dermatitis due to trichloroethylene. The graph of mass spectrum of every peak was compared with the graph of mass spectrum bank after separated by the gas chromatogram. The substance was confirmed when the identical rate is over 90%. All the components and all the samples were analyzed. We observed whether or not the coexistent impurity presenting in the samples. And if the coexistent impurity is exist, the possibility of hypersusceptibility was estimated.Results Some of the components are the same in the 9 samples come from 9 different working place. The coexistent impurity are trichloroethylene, 1-1-2-trichloroethane and tetrachloroethylene in all the 9 samples. In addition, trichloromethane, dimethylbenzene and carbon tetrachloride were detected in the 2/3 samples.Conclusion Except of trichloroethylene, all the samples have coexistent impurity: 1-1-2-trichloroethane and tetrachloroethylene. But the hypersusceptibility of the two matters has not been reported. It is suggested that the sensitinogen of the Occupational medicamentosa-alike dermatitis due to trichloroethylene may be trichloroethylene.Part TwoThe main changes of immune function on the patients of Occupational medicamentosa-alike dermatitis due to trichloroethyleneObjective To investigate changes of some immune function indexes on the patients of the Occupational medicamentosa-alike dermatitis due to trichloroethylene, understand the changes of the body and to provide a clue of the mechanisms of the disease.Methods Blood of the patients was used in vitro. The differences of proliferation of the lymphocyte induced by trichloroethylene were compared with control group. The changes of the move region of the leukocytes and the ratio of CD4+/CD8+ of the patients were also compared with the control group.Results Trichloroethylene (40mg/L and 80mg/L) could significantly increase the proliferation of lymphocytes of the patients of the Occupational medicamentosa-alike dermatitis due to trichloroethylene, and no differences of the ratio of CD4+/CD8+ has been found in the patients compared to the control group. Trichloroethylene had no significantly differences to the inhibition exponent of the leukocyte moving compared to the control group.Conclusion It is suggested that the trichloroethylene may be the antigen or hapten of the immune reaction in the Occupational medicamentosa-alike dermatitis due to trichloroethylene. Part ThreeStudy on the changes of immune functions in mice induced by trichloroethyleneObjective The experiments of plantar swelling test and lymphocyte proliferation in NIH mice were used to investigate the changes of the immune functions induced by trichloroethylene, and give evidence for the hypersensetivity of trichloroethylene.Methods The organ coefficients changes of thymus, spleen, and liver, the experiments of plantar swelling test and lymphopoiesis test were carried out by hyodermic to mice.Results The organ coefficients of thymus and spleen had no obviously changes in the mice induced by trichloroethylene. The coefficient of plantar swelling test in mice were obviously higher in 24h and 48h induced by trichloroethylene compared to control group. Trichloroethylene (80mg/L) could inhibit the proliferation of spleen lymphocytes compared to the control group. but could promote the proliferation of spleen lymphocytes of the mice.Conclusion TCE may be related with the induction of the delayed type hypersensitivity in NIH mice.Part FourStudy on the susceptibility related to immune genes in the Occupational medicamentosa-alike dermatitis due to trichloroethyleneObjective To reveal the susceptibility genes of the Occupational medicamentosa-alike dermatitis due to trichloroethylene according to the frequency of allele genotypes of HLA-DM、HLA-DQA and Lag-3 in the patients and the controls, to provide evidences for the preventive and treatment of the disease.Methods We extracted the DNA of lymphocytes of the Occupational medicamentosa-alike dermatitis due to trichloroethylene patients and controls, amplified the HLA-DM,HLA-DQA and Lag-3 by using touchdown PCR, Then through restriction fragment length polymorphism (RFLP) and sequence base typing, confirmed the alleles and genotypes. Compared the frequency of alleles and genotypes of HLA-DM, HLA-DQA and Lag-3 of the two groups.Results Our study shows that in the Occupational medicamentosa-alike dermatitis due to trichloroethylene patients a significant increase in the HLA-DMA*0101 allele frequency. The allele Frequency of HLA-DMA*0103 increased in patients group than controls. The ratio of HLA-DMA*0102-*0102 in patients is significantly higher than in controls. The ratio of heterozygote HLA-DMB *0101-*0102 in patients group is lower than controls group. And no differences were found in other alleles and genetypes of HLA-DMA and HLA-DMB.The frequency of *0201 in patients was higher than in control groups. Though the frequencies of *05- was lower than controls, but the correct P was higher than 0.05.No differences had been found in exon 1-6 and exon8 of patients and control group. Though there were polymorphism in exon 7 , but the frequencies of it had no differences in two groups.Conclusion The polymorphisms of HLA-DM, HLA-DQA may be related to the susceptibility of the Occupational medicamentosa-alike dermatitis patients, and polymorphisms of Lag-3 may be not.Part FiveResearches on changes of gene expression of the Occupational medicamentosa-alike dermatitis due to trichloroethylene patientsObjective To research the differences of gene expression of the Occupational medicamentosa-alike dermatitis due to trichloroethylene patients between prêt-reatment and convalescence, and confirmed by using real time PCR in other patients. By observed the changes of pre- and post-treatments , understand the mechanism of the disease and provided the data.Methods The total RNA were extracted from the samples of 12 patients in pre- and post-treatment. Micro array had been used to test the gene expression changes in 2 patients. Compared results of the two, the mutual changes had been found. By using real time PCR, the highest 10 different expressed genes had been confirmed in other 10 patients.Results In patient No.506, there were 317 genes differently expressed between pre-treatment and post-treatment. 262 genes of them were up-regulated and 55 of them were down-regulated. In patient No.507, there were 591 genes differently expressed. And the number of up- and down-regulated genes were 467 and 124 separately. In the two patients, 147genes were the same of up-regulated, and 9 genes were the same of down-regulated. The results showed that the functions of the differently expressed genes mabe be involved in cell surface antigen, cytokine and other genes related to immune system.Conclusion: The results showed that there were some similar changes of genes in different patients, and maybe these genes induced similar pathologic processes.更多还原