分类号Q2学校代码10590 UDC570密级公开
绝对式角度编码器深圳大学硕士学位论文
(CYP1A1,CYP2E1)的单核苷酸多
态性研究
陈冠豪邮政专营
学科门类理学
专业名称细胞生物学
学院（系、所）生命科学学院
指导教师田生礼副教授，刘建军研究员
摘要
三氯乙烯（trichloroeythlene, TCE）是一种常见的工业原料，可用做金属去脂剂、干洗剂、溶剂、萃取剂等，在五金、电子、玩具、印刷等行业应用广泛。职业性TCE中毒国内外已有报道，临床较多表现为慢性神经系统损害，少数可致急性重度药疹样皮肤损害和肝功能损害。深圳市涉外企业TCE中毒病例近几年也居高不下，危害较严重，造成较大经济损失。TCE在体内主要经两种途径进行代谢：细胞素P450（CYP450）氧化途径和谷胱甘肽（GSH）结合途径。 据相关文献报道，TCE能使其代谢酶基因（CYP450）表达上调，另外该基因中的rs1048943以及CYP2E1 -1053 C→T等SNP位点的突变率增加同样可以造成其表达上调，所以我们针对上述两个位点设计并合成了TaqMan-MGB探针及其相关引物，检测TCE易感性与CYP450基因单核苷酸多态性的关系。本研究从深圳地区内采集了三氯乙烯接触者(没有中毒症状)40例及中毒者40例的外周血样。通过抽提外周血DNA作为模板，以接触者作为对照，利用所合成的特定引物进行荧光定量PCR（QPCR）反应。对QPCR的反应结果进行了分型统计和χ2检验，分析了对照组基因型及等位基因与TCE 中毒组基因型及等位基因的差异性。实验结果表明，在对29例接触者及35例TCE中毒病人的rs1048943突变位点的基因型和等位基因频率检测发现，病人组GG纯合突变频率为 5.7％，与对照组比较，经卡方检验分析没有统计学意义（χ2＝0.03，P>0.05）；而对G等位基因频率分析的结果表明，G等位基因突变频率显著高于接触组（χ2＝4.79，0.01<P<0.05）。说明G等位基因的突变可能与TCE的易感性是有关。进一步对34例接触者和29例TCE中毒病人的CYP2E1基因 -1053 C→T位点的基因型和等位基因频 率进行荧光定量PCR，检验其单核核苷酸多态性。发现CYP2E1基因 -1053 C→T位点中病人组TT纯合突变频率为3.45％，与对照组比较没有统计学意义（χ2＝0.37，P>0.05）,T 等位基因频率显著高于接触组（χ2＝15.33，P<0.01），说明CYP2E1基因5′-端调控区-1053 C→T位点的SNP对于TCE的易感性有显著差异。表明CYP2E1基因 -1053 C→T位点的突变可能与TCE的易感性是有关。结果说明，TCE中毒易感可能是由于TCE代谢酶基因（CYP450）的rs1048943突变位点G突变的频率增高和CYP2E1基因 -1053 C突变为T的结果。这为进一步探讨和研究CYP450基因单核苷酸位点多态性与三氯乙烯中毒易感性机理提供了可靠的实验依据。
由于从外周血液提取DNA的方法要抽取一定量的血液样本，患者不愿接受。我们开展了从小量的血液样本，如血浆，血痕中提取DNA用于SNP分型实验的探索。最后为了证实SNP分型结果的可靠性，我们分别对rs1048943位点以及CYP2E1 -1053 C→T 位点设计测序引物并在NCBI上进行BLAST特异性比对，然后分别用两套引物对rs1048943位点以及CYP2E1 -1053 C→T位点的三种不同基因型样本进行PCR扩增，扩增产物进行测序分析。测序结果表明，rs1048943突变位点和CYP2E1 -1053 C→T突变位点与QPCR的实验结果一致。说明利用血浆，血痕进行三氯乙烯代谢酶基因的多态性分析是可行的。
以上实验结果表明，通过研究CYP450基因的相关SNP位点，对于揭示三氯乙烯易感性机理，寻易感基因，探讨其发病机制提供了新的资料。同时血浆，血痕提取DNA 进行QPCR SNP分型实验的结
果为今后的大规模实验提供了一种新的有效方法。
关键词：三氯乙烯; CYP1A1; CYP2E1; 单核苷酸多态性; TaqMan—MGB探针；
荧光定量PCR；突变
Abstract
Trichloroeythlene (TCE) is a normal industry raw material, can be used as metal degreasant, dry clean agent, solvent, extractant and so on, and widely used in hardware, electronic, toy, printing industries. The professional toxicosis of TCE was reported in domestic and overseas, the clinic features are usually chronic nervous system damage, few example could cause severe drug eruptional skinny damage and hypohepatia. The patients of TCE toxicosis in Shenzhen’s foreign capital enterprises maintained a high level in recent years, and caused a big economic loss. The TCE have two metabolic pathways in the body: The CYP450 oxidative pathway and the GSH combine pathway.
lsd法The research project is aiming at rs1048943 site and CYP2E1 gene -1053 C→T site which were reported that they could induce the rising of TCE metabolic enzyme (CYP450) gene expression, so 卡车司机的自述
we designed and synthesized TaqMan-MGB probes and primers, firstly detect the relationship between TCE susceptibility and SNPs of the CYP450 gene in China. Peripheral blood samples collected from TCE contactee (not toxicosis) 40 examples and patient 40 examples in Shenzhen district, using the DNA extracted from peripheral blood as template, and the designed primers for QPCR on ABI7500. We statistical typed and χ2 tested the QPCR results, analyzed the genotype and allele differences between of the control group and the patients group. By calculating the rs1048943 allele and genotype frequency in 29 contactees and 35 patients the results shows that the frequency of VV genotype is 5.7%, compare with the control group is not a significant difference. （χ2＝0.03，P>0.05）However, the G allele compare with the control group is a significant difference. （χ2＝4.79，0.01<P<0.05）The results indicate the G allele may have relations with the TCE susceptibility. We further calculate the frequency of the CYP2E1 gene -1053 SNP site allele and genotype in 34 contactees(not toxicosis) and 29 patients, the results shows that the frequency of C2,C2 genotype is 3.45%, compare with the control group is not a significant difference. （χ2＝0.37，P>0.05）The frequency of T allele compare with the control group is a significant difference. （χ2＝15.33，P<0.01）The result indicate the CYP2E1 gene 5′ flank region RsaI polymorphism may have relations with the TCE susceptibility. All the results suggest the TCE susceptibility may be caused by the rs1048943 G allele and the CYP2E1 gene 5′ flank region
RsaI polymorphism. These results provide a reliable experimental basis for further discussing and researching the CYP450 gene SNPs and the mechanism of TCE susceptibility.
Because of colleting the blood sample for traditional method of phenol / chloroform DNA extraction is relatively hard, and the patients are reluctant to accept. That’s why our issue started an exploration in extracting DNA from blood sample of small quantity, like the plasma, blood stain. In order to prove the reliability of SNP typing results, we designed the sequencing primers for rs1048943 site and CYP2E1 gene -1053 C→T site, and BLAST the primers in NCBI. Then the two sets of primers were used to amply the three different genotypes’ samples for the rs1048943 site and CYP2E1 gene -1053 C→T site by PCR respectively. The PCR products were loaded on 2% Agarose M gel and electrophoresis. Purified specific bands were sent to the Shanghai Shenggong Bioengineering company sequencing. The sequencing results shows that the feasibility of the TaqMan-MGB probe and QPCR test the SNP site of P450 gene by using the plasma and blood stain.
All the above results show the issue offers a basis for exploring TCE toxicosis susceptibility, finding susceptible gene, discovering its pathogenesis, preventing and therapy. At the same time, the QPCR SNP typing results using DNA extracted from plasma and blood stain offer an original and effective method for the large scale experiment in the future.
阿克毛事件>李钟瑞Key words: Trichloroethylene; CYP2E1; CYP1A1; Single nucleotide polymorphism; TaqMan-MGB probe; QPCR; Mutation
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