【作者】 谢惠芳；

【导师】 郑玉建；

【作者基本信息】 新疆医科大学 ， 劳动卫生与环境卫生学， 2010， 博士

【摘要】 目的：了解新疆饮水型地方性砷中毒在高砷区以及非高砷区的流行特征,筛选血浆效应生物标志,MT基因、GST基因多态性与砷中毒的关联,以及对比分析蛋白表达差异,为开展系统性的地方性砷中毒生物标志物的研究,达到揭示慢性砷中毒发病以及致癌的分子机制,以及为地方性砷中毒的早期诊断、防治以及预后危险度评价提供有预测意义和经济快速、特异、敏感的地方性砷中毒生物标志进行探索性研究。方法：在采用采用队列研究方法,以1985年在当地调查的慢性砷中毒患者名单为基础,随访108例为病例组(AP),用病例对照的方法(AP+SI),IC95例,EC 91例。各组性别、年龄基本匹配。根据目前所获得的基础流行病学资料,对地方性砷中毒的影响因素进行探讨。采用硝酸还原酶法测试血浆样品中的一氧化氮(NO)含量,应用放射免疫分析法(Radioimmunoassay, RIA)测试血浆样品中的β2-MG含量。应用聚合酶链反应及限制性片段长度多态性技术(PCR-RELP, PCR-CTPP)检测MT2A、GSTT1以及GSTO1基因多态性,采用病例-对照的关联分析方法对地方性砷中毒患者与内对照、外对照的基因型和等位基因频率进行分析。利用2-DE分辨,并用电泳后的酶切及基质辅助的激光解吸附质谱以定位和分析地方性砷中毒患者与正常人血浆中蛋白质质谱变化,寻找地方性砷中毒患者血浆中的特异差异蛋白质。结果：1)基线流行病学资料分析,IC组与AP组、EC组的年龄、饮水年限、水砷含量三组差异均有统计学意义(P<0.05)。砷暴露组与内对照组、外对照组之间在血压、心电图结果上的差异均无统计学意义(P>0.017);而皮肤色素沉着、皮肤色素缺失上差异有统计学意义(P<0.05)。砷暴露组与内对照组之间皮肤角化差异有统计学意义(P<0.05),与外对照组之间差异无统计学意义(P>0.05)。经对数变换后协方差分析,血浆NO含量的修正均数在三组间比较差异有统计学意义(P<0.05),固定其他因素后,组别与饮水年限的交互作用对NO含量有影响。AP组、IC组与EC组比较,血浆中β2-MG含量差异有统计学意义(P<0.05)。固定其它因素作用下,年龄、组别与年龄的交互作用均对β2-MG的含量有影响；2)MT2A基因rs10636位点基因型分布在病例组和内外对照组之间的分布,不符合Hardy-Weinberg遗传平衡定律(P<0.05)。MT2A rs10636 CC、CG、GG各基因型频率在三组之间,差异有统计学意义(P<0.05)；CG型频率高于CC型高于GG型。CG基因型分布外对照组68.6%多于病例组43.6%(P<0.05)。GG基因型分布频率病例组25.5%高于内对照组9.9%、外对照组10.5%(P<0.05)。MT2A基因rs10636的等位基因C、G频率三组比较,差异无统计学意义(P>0.05)。经非条件Logistic回归分析,三组经过亚元变量的Wald卡方检验,显示在其他因素固定的情况下,性别会影响MT基因的多态性(P<0.05),女性突变是男性的1.941倍。GSTTl基因型、GSTO1基因rs4925位点、rs11509437位点、rsl 1509438位点基因型分布在三组均符合Hardy-Weinberg遗传平衡定律(P>0.05)。GST01基因rs4925位点的等位基因频率C、A在三组间分布,差异无统计学意义(P>0.05)。经非条件Logistic回归分析,在其他因素固定的情况下,饮水年限每增多1年,相应的rs4925位点基因发生突变优势改变0.971倍(P<0.05)。GSTO1基因rs11509438位点基因型A、G等位基因频率三组比较差异无统计学意义(P>0.05)。GSTO1基因rs11509437位点等位基因频率AGG、-AGG,三组比较差异无统计学意义(χ2=3.524,P=0.172)。经非条件Logistic回归分析,在其他因素固定的情况下,男性比女性,相应的rs1 1509437位点基因发生突变优势改变0.439(P<0.05)；3)血浆样品双向电泳,地方性砷中毒组血浆样本检出蛋白点165个,健康对照组血浆样本检出蛋白点113个。选定血浆蛋白质双向凝胶电泳图谱2组血浆蛋白表达量差异2倍以上的蛋白点进行分析,经MALDI-TOF-MSMS质谱分析与Mascot Peptide Mass Fingerprint数据库匹配,成功鉴定出12个差异蛋白质,与对照组比较,地方性砷中毒组表达上调的蛋白点有5个,表达下调的有7个。结论：1)高砷区水砷浓度高于非病区,砷对心脏的影响存在着一定的持续性,砷暴露区居民皮肤病变还没有完全康复,这些损伤的恢复需时较长。皮肤色素沉着、色素缺失和皮肤角化这三种作为诊断用的皮肤病变指征,在地方性砷中毒防治干预后,仍是有较好价值的监测指标；2)在生物标志研究中,血浆中NO尚不能明确其作为饮水型砷中毒的生物标志物,而血浆p2-MG可被视为地方性砷中毒的效应生物标志;3)MT2A基因rs10636位点基因型分布在病例组和内外对照组之间不同,可作为地方性砷中毒的生物标志,并且该基因位点男性发生突变是女性的1.941倍；4)GSTT1、GSTO1基因rs4925位点、GSTO1基因rs11509438位点以及GSTO1基因rs11509437位点,基因型分布在病例组和内外对照组之间相同。GSTT1基因、GSTO1基因rs11509438位点可能与地方性砷中毒无关联,目前尚不能将这4个基因位点作为地方性砷中毒的生物标志；5)对血浆样品的2-DE电泳以及N(?)ALDI-TOF-MSMS质谱分析,成功鉴定出12个差异蛋白质,地方性砷中毒组表达量上调显著的蛋白点有5个,表达量下调的有7个。这些蛋白质可能与地方性砷中毒发病有关,分别在地方性砷中毒的炎症免疫应答、神经功能紊乱、男性生殖功能抑制、补体调节以及DNA修复等方面发挥作用,这些蛋白质可以作为地方性砷中毒的生物标志。更多还原

【Abstract】 Objective:The goal in this study was to explore the distribution characteristics of water-born endemic arsenism in Kuitun, Xinjiang, chose biomarker in plasma of endemic arsenism patients. To analysis any probable relationship between MT gene, GST gene polymorphisms and endemic arsenism. To contrast the difference expression of proteins from endemic arsenism to normal control.So to discover the mocular mechanism of arsenism and genetic factors on the metabolism of arsenic, to offer specific, sensitive, fastern and economic biomarker for early diagnosis, prevension and risk assessment of Xinjiang endemic arsenism. Methods:This study using cohort study to followed-up 108 individuals who were diagnosed endemic arsenism in Kuitun, Xinjiang in 1985, and matched 95 individuals as intra-control (IC),91 individuals as extra-control (EC) by sex and age. NO and (32-MG of plasma were compared by biochemical tests and radioimmunoassay. We performed genotyping of Metallothionein 2A (MT2A) gene, Glutathione-S-transferase Theta 1 (GSTT1), Glutathione-S-transferase Omega 1 (GSTO1) gene by Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) to investigate the allele frequencies of these polymorphisms in arsenism patients (AP) and IC, EC.Two-dimensional electrophoresis (2-DE) was applied to segregate the total proteins in the plasma form 25 cases arsenism patients and 25 cases health control. then, the differential expression proteins were identified by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Results:1) AP had no significant difference with IC and EC in hypertension, ECG results abnormal (P> 0.05); in the skin pigmentation, skin hypopigmentation were statistically significant (P<0.05). AP and IC had statistically difference in skin hyperkeratosis (P<0.05), had no statistically difference with EC. (P> 0.05). After logarithmic transformation and covariance analysis, corrected mean of NO content of blood plasma was statistically significant (P<0.05) for AP vs. IC, EC controls. Fixed other factors, interaction of groups and drinkingwater time could effect NO content. AP, IC compared with EC,β2-MG of blood plasma was statistically significant (P<0.05), Fixed other factors, interaction of groups and age could effectβ2-MG content, age could also effect; 2) After genotyping GSTT1 and GSTOI (rs4925, rs11509438, rs11509437) polymorphisms, firstly there was no statistically significant difference in AP vs.IC and EC, concerning both, genotype and allele frequencies, respectively (P>0.05). secondly binary logistic regression showed:fixed other factors, the mutation superiority of GSTO1 (rs4925) gene will be increased 0.971, corresponding to drinking-water time increased 1 year (P<0.05); fixed other factors, fale compared to female, the mutation superiority of GSTO1 (rs11509437) gene will be increased 0.439 (P<0.05); 3) The frequencies of MT2A (rs10636) genotypes CC, CG, GG was statistically significant between AP and IC, EC (P<0.05). The frequency of CG>CC>GG, The frequency of CG of EC (68.6%) higher than its of AP (43.6%) (P<0.05). The frequency of GG of AP (2.5%) higher than its of IC (9.9%) and EC (10.5%) (P<0.05).The frequencies of MT2A (rs10636) allele C, G was not statistically significant between AP and IC, EC (P>0.05). Binary logistic regression showed:fixed other factors, gender could effect MT2A genetic polymorphism (P<0.05), the mutation superiority of female higher than male 1.941; 4) To Analysis different expression proteins of 25 cases of blood plasma in Xinjiang endemic arsenism 1:1 matched with normal control. Performed two-dimensional gel electrophoresis and MALDI-TOF-MS proteomic analyses of blood plasma, By the analyses of match differentially displayed map,12 prominent (5up-regulated and 7down-regulated) proteins. Conclusion:Water arsenic content was excessive in Xinjiang Kuitun arsenic exposure area. the health state of inhabitants, as skin lesion.can be used to diagonsis of endemic arsenism. NO of blood plasma was not a biomarker of endemic arsenism. Butβ2-MG of blood plasma was confirmed to be a biomarker of endemic arsenism in this study. The frequencies of MT2A (rs10636) genotypes was statistically significant between AP and IC, EC, so MT2A (rs10636) gene was a biomarker of endemic arsenism.In this study, genotype of GSTT1 and GSTO1 (rs4925, rs 11509438, rs11509437) polymorphisms had no relationship with endemic arsenism, maybe those four gene were not biomarker of endemic arsenism.Performed two-dimensional gel electrophoresis and MALDI-TOF-MS proteomic analyses of blood plasma, By the analyses of match differentially displayed map,12 prominent (5up-regulated and 7down-regulated) proteins, each being unique in endemic arsenism were identified with mass spectrometry and bioinformatic database queries. These identified proteins were immune response protein, neural degeneration protein, male reproduction protein, antioxidant related proteins, those protein could be biomaker of endemic arsenism and might be for the diagnosis, treatment and prevention of endemic arsenism in the future.更多还原