【作者】 陈雯雯；

【导师】 邵华；

【作者基本信息】 山东省医学科学院 ， 劳动卫生与环境卫生学， 2009， 硕士

【摘要】 锰(manganese,Mn)是人体正常代谢过程中不可缺少的一种微量元素,又是工作场所和环境中常见的易在体内蓄积的毒物。职业性长期接触锰最明显的是对神经系统的毒作用,表现为中枢神经系统锥体外系受损和震颤麻痹症状,其临床表现酷似神经退行性疾病-帕金森氏病(Parkinson’s disease,PD)。锰所致严重的、不可逆的神经系统损伤一旦发生,临床上无特效控制措施,病程迁延,终生不愈,给患者、家庭和社会造成巨大的经济负担。为预防锰中毒,应在锰对靶器官系统造成不可逆的损害之前识别,并减少锰的接触。因此,长期职业性锰接触所引起的神经系统损伤的防治问题是我国职业医学领域迫切需要解决的重要问题。目前锰及其化合物对人体神经系统的毒作用机制尚不完全清楚,锰主要侵害基底神经节,选择性损伤多巴胺能神经元,其机制可能与干扰中枢神经的能量代谢,改变神经递质,激活氧化系统导致神经细胞退行性变性等有关。本文选择神经系统中易受锰影响的生化指标、与神经递质代谢、神经细胞的脂质过氧化有关的合成酶及降解酶,如血清丙二醛(MDA)、总超氧化物岐化酶(SOD)、谷胱甘肽过氧化物岐化酶(GSH-Px)活力及胆碱酯酶(AChE)活性等指标,研究锰的接触与效应生物标志物。在同样作业环境、同样接触水平的工人中,仅有少部分人发生中毒,提示个体对锰易感性存在着差异,表明引起神经系统损伤的锰接触者可能存在某些敏感基因,遗传因素可能对锰所致神经毒性作用的发生起重要作用。有研究表明多巴胺-β-羟化酶(DBH)、热休克蛋白70(HSP70)、锰超氧化物歧化酶(MnSOD)及单胺氧化酶(MAO)与脂质过氧化,损伤多巴胺能神经元等机制有关,提示这四种酶的遗传多态性可能影响锰对神经损伤的敏感性。DBH、HSP70、MnSOD及MAO基因多态性与锰所致神经损伤的关系研究国内外报道较少,且不同国家、地区和种族间结果不一,有待确定它们是否为锰所致神经损伤的遗传敏感基因。本文同时研究了DBH、HSP70、MnSOD及MAO四种代谢酶基因多态性及mRNA表达,综合分析它们是否为锰职业危害的敏感基因。热休克蛋白(HSPs)在正常细胞中介导其它蛋白质的跨膜转运和正确装配,起着分子伴侣的重要作用,在应激因素的作用下,可被诱导产生,通过应激基因的激活,从而提高细胞对损伤的耐受和应激能力,HSP70是含量最丰富的热休克蛋白,有研究表明HSP70是反映机体应激反应的有价值的生物标志物。本文研究了HSP70表达与锰的神经毒作用关系。【研究目的】1.探讨尿锰、血锰、血清丙二醛(MDA)、总超氧化物岐化酶(SOD)、谷胱甘肽过氧化物岐化酶(GSH-Px)活力及胆碱酯酶(AChE)活性等指标与空气锰个体接触剂量的相关关系,研究锰职业危害的接触与效应生物标志物。2.以微流控芯片为检测平台,应用单核苷酸多态性的方法,探讨四种代谢酶DBH、HSP70、MnSOD与MAO基因多态性与锰所致神经损伤的关系。3.以实时荧光定量为检测平台,探讨空气锰个体接触不同暴露水平对mRNA表达及mRNA指导下的蛋白质含量的影响。探寻锰职业危害的生物标志物,为职业暴露人群的筛选、高危人群的健康监护提供理论依据,从而达到进一步加强病因预防的目的。【研究方法】1.采用个体采样器对锰职业接触工人进行八小时时间加权平均浓度的监测,获得准确的工人工作班内锰个体接触剂量。测定血清MDA含量、SOD、GSH-Px活力及AChE活性等指标。应用石墨原子吸收法测定锰职业接触工人的血锰、尿锰含量及空气锰个体接触剂量。2.选择锰职业接触工人为研究对象,以微流控芯片为检测平台,采用限制性片段长度多态性(RFLP-PCR)方法检测与锰体内代谢过程关系密切的四种生物代谢酶DBH,MAO,HSP70和MnSOD的基因位点多态性,统计分析四种生物代谢酶不同基因型及等位基因对锰所致神经损伤的影响,筛选出锰职业接触遗传易感基因。3.以SYBR Green I嵌合荧光实时定量聚合酶链式反应法检测四种代谢酶mRNA含量表达;应用蛋白印迹法检测HSP70基因的蛋白含量,探讨空气锰个体接触不同暴露水平对四种酶mRNA表达及mRNA指导下的蛋白质含量的影响。【研究结果】1.根据8小时时间加权平均浓度(8h-TWA)分组,高锰暴露组工人血锰及血清MDA含量、SOD、GSH-Px活力及AChE活性等指标与低暴露照组比较均有显著性差异(P<0.05),但与8h-TWA无明显相关关系;尿锰含量在两组间比较没有显著性差异,与8h-TWA也无明显相关关系(P>0.05)。2.以402名锰接触者外周血为样本,测定四种生物代谢酶基因型,结果发现:在神经功能异常组与正常组间,DBH基因A2A2型与A2等位基因分布明显不同,在异常组的分布多于正常组有显著性差异(P<0.05);MnSOD基因的VV基因型在高暴露水平下,异常组的分布明显多于正常组(P<0.05);而MAO与HSP70多态位点基因型及等位基因在高、低暴露水平下两组间分布均没有显著性差异(P>0.05)。3.根据8h-TWA所分为高、低暴露组,锰高暴露组的HSP70的mRNA及HSP70蛋白表达水平明显高于低暴露组(P<0.05)。DBH、MAO与MnSOD的mRNA表达在两组间没有显著性差异(P>0.05)。【结论】1.血锰、MDA含量、SOD、GSH-Px活力及AChE活性虽在高低暴露组间有显著性差异,有助于确定当前锰暴露的程度,但对空气锰个体接触浓度的变化不敏感,不能很好的作为锰职业危害的接触与效应标志物,可以作为参考指标与其他指标联合分析锰的内暴露剂量及产生的神经毒性作用。2. DBH基因TaqI多态性,MnSOD基因Val-16-Ala多态性可能在锰职业危害的遗传易感性中起作用。MAO与HSP70基因多态性可能对锰职业危害影响不大。3.锰接触者外周血淋巴细胞HSP70水平及HSP70mRNA表达的增高与保护神经细胞免受锰刺激引起的脂质过氧化等损伤有关,这可能与锰的神经毒作用机制有潜在联系。更多还原

【Abstract】 Manganese (Mn) is a critical microelement for human normal metabolism, but it also is a poison accumulated in vivo easily which could be found in work site and surrounding. Exposure to excessive level of manganese for a long time would result in a toxic effect on nervous system, which is characterized by the damage of extractor- ticospinal tract and symptom of paralysis agitans. The clinical symptoms of neurological disorder caused by manganese were similar to Parkinson’s disease (PD). The severe and inconvertible nervous system damage caused by manganese would be a worrisome burden for patients, family and society, for once it happened, there is no effective approach to control or cure it. So it is significant to achievement the prevention and cure of nervous system damage caused by occupational manganese poisoning asearly as possible.Mechanism of manganese neurotoxicity is not elucidate clearly. It was found the basal nucleus was damaged and dopaminergic neuron was injuried by manganese. The mechanism could be related with interfering the energy metabolism of central nerve, changing neurotransmitters, activating oxidation system and so on. Some biochemical indicators in tissue of nervous system influnced by manganese and some synthetases or catabolic enzymes such as malondialdehyde(MDA), superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), acetylcholinesterase(AChE) could be chosed as biomarkers of effect for the study of manganese.There were only a part of people to be poisoned by manganese in aequales condition which suggested that there was individual variation to the susceptibility of manganese. There would be some sensitive genes in people exposed to manganese with damaged nervous system. Genetic factors could play a significant role in the neurotoxic effect cause by manganese. Some researches had showed that dopamine-β-hydroxylase(DBH), Heat shock proteins 70 (HSP70), manganese superoxide dismutase (MnSOD), monoamine oxidase (MAO) were related with lipid peroxidation and dopaminergic neuron damage, which indicated that the genetic polymorphism of these four enzymes could influence the sensitivity of nerve injury caused by manganese. There were few reports about the relationship between gene polymorphism of DBH, HSP70, MnSOD, MAO and nerve injury cause by manganese. Furtheremore, The results were different among differert countries and races. So it was essential to determine whether they were sensitive genes or not. At the same time the expressions of mRNA and SNP of DBH, HSP70, MnSOD, MAO were investigated for sggregate analysis.Heat shock proteins (HSPs) comprised of a group of highly conserved and stress related proteins with many important physiological and pathological roles such as cellular homeostasis, molecular chaperone tumor markers, molecular adjuvant and so on. HSP70 could strengthen the cytoendurance to damage, maintain the normal metabolism and enhance the viability, it also could protect the damage of central nerve system, cardiacmuscle, liver and lung. HSP70 was proved to be a valuable biomarker to reflect physical stress. So the relationship between HSP70 and stress effect of manganese was investigated.Objective1 The indicators including urine manganese, blood manganese, MDA, SOD, GSH-Px, AChE were investigated to find the relationship with the air concentration of manganese at the work-place. The objective was to find biomarkers of effect for the study of manganese neurotoxicity.2 To detect single nucleotide polymorphism by microfluidic and investigate the relationship between genetic polymorphism of DBH, HSP70, MnSOD, MAO and manganese-induced nerve injury.3 To analysis the expression levels of mRNA and proteins in different exposure to manganese by quantitative real-time reverse transcription-PCR. To find suitable biomarkers for manganese-induced occupational hazard.Methods1 To monitor the 8-hour time weighted average concentration of vocational contact manganese by personal sampler to obtain precise exposure dose. The concentration of MDA, SOD, GSH-Px, AChE in serum were determined, and concentration of blood manganese, urine manganese and air manganese were determined by GFAAS.2 Occupational contact workers of styrene were included in the study. PCR-RFLP was applied to determine the SNPs of DBH, MAO, HSP70, MnSOD and statistically analysed the influence of gene polymorphisms on the metabolism of manganese.3 The mRNA expressions of four different metabolic enzyme were detected by SYBR Green I quantitative real-time polymerase chain reaction The protein level of HSP70 was detected by western-blotting.Results1 The groups were divided into high exposure group and low exposure group by 8-hour time weighted average concentration(8h-TWA). There was significant difference between two groups of MDA concentration and the activities of SOD, GSH-Px, AchE(P<0.05). But there was no obvious correlation with 8h-TWA. There was no significant difference between two groups of urine manganese and no obvious correlation with 8h-TWA.2 Detecting the genotype of DBH, MnSOD, MAO and HSP70 of 402 workers exposed to manganese through collecting peripheral blood. The distribution of A2 A2 genotype and A2 allele of DBH was obviously different. The distribution was wiser in abnormal group than normal group(P<0.05), the VV genotype of MnSOD was obviously different. The distribution was wiser in abnormal group than normal group(P<0.05), the genotype and allele of MAO or HSP70 was with no significant difference(P>0.05).3 The groups were divided by 8h-TWA, and there was significant difference between the two groups on the expressions of HSP70 mRNA and protein, and the HSP70 mRNA level and HSP70 protein level of high exposure group were higher than low exposure group(P<0.05). The expressions of DBH mRNA, MAO mRNA and MnSOD mRNA was found no significant difference between the two groups (P>0.05).Conclusions1 There was significant difference of blood manganese, MDA concentration and the activities of SOD, GSH-Px, AchE between two groups, but these indicators were insensitive to the change of air manganese concentration. So these indicators could not be chosed as biomarkers of effect for the study of manganese. They could be used as reference indicators coupled with others to analyze the exposure dosage and indeced neurotoxicity.2 TaqI polymorphism of DBH and Val-16-Ala polymorphism of MnSOD could play great roles in hereditary susceptibility of occupational hazard cause by manganese. The polymorphism of MAO and HSP70 gene may not related to the occupational hazard cause by manganese.3 The increase of HSP70 protein and mRNA could be concerned with lipid peroxidation which was induced to preotcet nerve cell. The mechanism of manganese-induced neurotoxicity could be related with HSP70 and HSP70 mRNA.更多还原