职业性慢性锰中毒人群及染锰PC12细胞中MnSOD基因表达水平变化研究

发布时间：2018-09-03 08:39

【摘要】：【研究背景】锰是人体必需的微量元素，也是一种重要的环境和工业污染物，由于职业性、医源性和环境接触的原因，锰主要通过呼吸道吸收，体内含量增高，可以通过血脑屏障进入脑组织，损坏中枢神经系统。慢性接触时锰在大脑中蓄积，尤其在纹状体中含量最高，主要影响纹状体苍白球和黑质网状带，引发基底节多巴胺神经元退行性病变。由于发病率的升高，职业性慢性锰中毒受到越来越多的关注。锰中毒的典型症状—震颤和肌张力增高，类似帕金森氏综合征。还可出现学习记忆功能、认知功能和空间方向感的明显障碍及易哭泣、烦躁、胆怯怕人、孤独自闭等明显情绪改变的症状。锰的神经毒性作用机制目前尚不完全清楚，，主要涉及氧化应激、线粒体损伤、神经递质代谢及细胞凋亡等方面。在氧化应激及线粒体损伤机制中，主要的抗氧化剂锰超氧化物歧化酶（MnSOD）具有十分重要的作用，本课题组前期研究发现，MnSOD基因多态性与职业性慢性锰中毒的易感性有关，可能为易感基因。在锰暴露过程中，研究其表达的变化与疾病的发生具的关系具有十分重要的意义。【研究目的】分别以锰暴露病例对照人群和PC12细胞体外染锰模型为研究对象，观察职业性慢性锰中毒病例与对照中MnSOD mRNA表达水平的差异，体外细胞模型在锰不同的时间剂量作用下，细胞增殖抑制及MnSOD mRNA表达水平的变化，探讨MnSOD在锰神经毒作用中的机制。【研究方法】 1.筛选临床上确诊的职业性慢性锰中毒病人31例作为病例组，病例诊断依据国家职业性慢性锰中毒诊断标准GBZ3-2006。选择与病人同性别、同工种、同车间、同期作业，工龄（相差≤3年）、年龄（相差≤5岁）相近，个人生活史和家族遗传史相似等而未发病的人群作为对照组。对每位确定的研究对象采血5ml，提取RNA。采用半定量RT-PCR方法检测MnSOD基因RNA表达水平，统计分析病例组与对照组人群基因表达水平的差异，分析MnSOD基因表达与职业性慢性锰中毒的关系。 2.以大鼠肾上腺嗜铬细胞瘤细胞(PC12)为模型，采用0、100、200、400、600、800、1000μmol/LMnCl2的培养液，分别培养对数生长期PC12细胞1、2、3、4天后，光学显微镜观察细胞增殖及形态变化，用MTT法测定锰的细胞毒性作用，提取细胞RNA，RT-PCR方法检测RNA表达水平变化。【研究结果】 1对照组和病例组在性别构成、年龄和工龄上差异无统计学意义；MnSOD基因mRNA表达水平在职业性慢性锰中毒病例组与对照组间有显著性差异(t=-4.589,p＜0.05)，二者差异的95%可信区间为[-0.277，-0.107]，且病例组较对照组表达减少。 2MTT实验结果显示，在一定浓度的条件下，锰对PCl2细胞的增殖抑制呈时间、浓度依赖效应。随着浓度的增大以及作用时间的延长，锰对PC12细胞增殖的抑制程度逐渐增强；400μmol/L MnCl2作用下3天，细胞生长抑制率超过50%；相关分析发现MnCl2浓度与细胞抑制率的相关系数为0.975，p＜0.05，而MnCl2作用时间与细胞抑制率的相关系数为0.116，p＞0.05。MnSOD mRNA表达水平实验结果显示，相同时间下，各浓度MnCl2所致PC12细胞MnSOD基因RNA表达水平存在显著差异（p＜0.05），随MnCl2浓度增加，RNA表达水平逐渐下降；MnCl2浓度与RNA相对表达量的相关系数为-0.958，p＜0.05，MnCl2作用时间与RNA相对表达量的相关系数为-0.14，p＞0.05。【研究结论】 1MnSOD基因mRNA表达水平可能与职业性慢性锰中毒的发生有关，MnSOD的表达下降可能有助于锰中毒的发生； 2MnCl2对PC12细胞具有毒性作用，浓度越高，毒性作用越强，MnSOD基因mRNA的表达越少。说明MnSOD mRNA的表达，可能参与了锰致神经细胞损伤的过程，推测其高表达可能对锰的神经毒性起到抑制作用。
[Abstract]:[background]
Manganese is an essential trace element of human body and an important environmental and industrial pollutant. Because of occupational, iatrogenic and environmental contact, manganese is absorbed mainly through the respiratory tract, and the content of manganese in the body increases. Manganese enters the brain through the blood-brain barrier and damages the central nervous system. Occupational chronic manganese poisoning has attracted more and more attention due to the increased incidence. The typical symptoms of manganese poisoning are tremor and increased muscle tone, similar to Parkinson's syndrome. Learning and memory can also occur. Significant impairments in function, cognitive function and spatial orientation, and symptoms of obvious emotional changes such as crying, irritability, timidity, fear of others, loneliness and autism.
The mechanism of neurotoxicity of manganese is still unclear, mainly involved in oxidative stress, mitochondrial damage, neurotransmitter metabolism and apoptosis. In oxidative stress and mitochondrial damage mechanism, the main antioxidant manganese superoxide dismutase (MnSOD) plays an important role. Gene polymorphism is associated with the susceptibility to occupational chronic manganese poisoning and may be a susceptible gene.
[research purposes]
The differences of MnSOD mRNA expression between the occupational chronic manganese poisoning patients and the control group were observed. The changes of cell proliferation inhibition and MnSOD mRNA expression in the in vitro cell model under the effect of different time and dose of manganese were investigated. The mechanism of action.
[research methods]
1. Thirty-one cases of occupational chronic manganese poisoning were selected as the case group. The case diagnosis was based on the national diagnostic standard of occupational chronic manganese poisoning GBZ3-2006. The patients were selected as the same sex, the same type of work, the same workshop, the same period of work, the working age (difference < 3 years), the age (difference < 5 years), the personal life history and the family genetic history were similar. The expression of MnSOD gene RNA was detected by semi-quantitative RT-PCR. The difference of gene expression between the case group and the control group was statistically analyzed. The relationship between MnSOD gene expression and occupational chronic manganese poisoning was analyzed.
2. Rat adrenal pheochromocytoma cells (PC12) were cultured in 0,100,200,400,600,800,1000 micromol/LMnCl 2 medium for 1,2,3,4 days respectively. The proliferation and morphological changes of PC12 cells were observed under light microscope. The cytotoxicity of manganese was determined by MTT. RNA was extracted and RNA expression was detected by RT-PCR. Horizontal variation.
[results]
There was no significant difference in sex composition, age and length of service between the control group and the case group; the expression level of MnSOD gene mRNA was significantly different between the occupational chronic manganese poisoning group and the control group (t=-4.589, P < 0.05), the 95% confidence interval between the two groups was [-0.277, -0.107], and the expression of MnSOD gene in the case group was lower than that in the control group.
The results of 2MTT assay showed that the inhibition of proliferation of PCl2 cells by manganese was time-dependent and concentration-dependent under certain concentration. With the increase of concentration and prolongation of action time, the inhibition of proliferation of PC12 cells by manganese gradually increased; the inhibition rate of growth of PCl2 cells was more than 50% after 3 days of 400 micromol/L MnCl2 treatment. The correlation coefficient between MnCl2 concentration and cell inhibition rate was 0.975, P < 0.05, while the correlation coefficient between MnCl2 action time and cell inhibition rate was 0.116, P > 0.05. MnSOD mRNA expression level in PC12 cells showed that at the same time, the expression level of MnSOD gene RNA in PC12 cells induced by MnCl2 concentration was significantly different (p < 0.05). The correlation coefficient between MnCl2 concentration and RNA relative expression was - 0.958, P < 0.05, and between MnCl2 action time and RNA relative expression was - 0.14, P > 0.05.
[Conclusion]
The expression level of 1MnSOD gene mRNA may be related to the occurrence of occupational chronic manganese poisoning, and the decrease of MnSOD expression may contribute to the occurrence of manganese poisoning.
The higher the concentration of 2MnCl2, the stronger the toxicity, and the less the expression of MnSOD gene mRNA. It is suggested that the expression of MnSOD mRNA may be involved in the process of manganese-induced neurotoxicity. It is speculated that the high expression of 2MnCl2 may inhibit the neurotoxicity of manganese.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R135.1

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