新生鼠暴露亚中毒阈剂量毒死蜱诱导黑质多巴胺能神经元损伤和神经行为改变

发布时间：2018-06-22 09:04

本文选题：毒死蜱 + 亚中毒阈剂量　；参考：《中南大学》2006年博士论文

【摘要】： 目的：“安全剂量”的有机磷农药对人体真无害吗?这是涉及疾病控制和公共卫生的一个重要的问题。本研究探讨大鼠新生期暴露亚中毒阈剂量(“安全剂量”)有机磷农药毒死蜱(Chlorpyrifos，CPF)，对成年期中脑黑质多巴胺能神经元和神经行为的影响。方法：出生11天SD大鼠138只，随机分为CPF组(n=46)、对照组(n=92)。对照组又分为溶剂二甲亚砜(dimethysulfoxide，DMSO)对照组(n=46)和生理盐水(normal sodiu，NS)对照组(n=46)。大鼠出生11-14天经腹部皮下注射亚中毒阈剂量(以下简称“亚剂量”，5mg／kg．d)CPF，制作新生SD大鼠暴露CPF动物模型。脂溶性的CPF溶于溶剂DMSO中给药。对照组分别以DMSO、NS替代CPF。各组给药体积均为1ml／kg．d。给次给药后严密观察12小时有无急性有机磷农药中毒症状。设出生后15天、20天、30天、60天共四个观察时点。透射电镜观察CPF对小胶质细胞超微形态结构影响；免疫组织化学方法检测中脑黑质多巴胺能神经元酪氨酸羟化酶(TH)及海马星形胶质细胞胶质纤维酸性蛋白(GFAP)表达；RT-PCR检测中脑黑质小胶质细胞CD11bmRNA及胶质细胞系源性神经营养因子GDNFmRNA的表达；酶联免疫吸附方法检测脑TNF-α、IL-1β水平；高架十字迷[笛槠拦澜孤撬健Ｋ凶柿喜捎肧PSS l 2．O统计软件包进行数据处理。所有数据用均值±标准差((?)±S)表示，首先采用单因素方差分析，，方差齐时采用最小显著差法t检验，方差不齐时用Dunnett’s t检验。P＜0.05为差异有显著性。结果：新生鼠暴露亚剂量CPF未见急性有机磷农药中毒症状。观察至成年期有如下重要发现： 1．CPF在中脑黑质急速诱导小胶质细胞活化，在超微结构小胶质细胞由静息状态转变为具有吞噬作用的激活状态，上调CD11b mRNA表达在暴露CPF早期(出生15天)比晚期(出生20天)更明显(P＜0.05)，增加炎症细胞因子TNF-α表达(P＜0.01)，但对IL-1β无影响(P＞0.05)。 2．暴露CPF下调中脑黑质GDNFmRNA表达并使中脑黑质TH阳性细胞表达(出生30天、60天)下降(P＜0.05)。 3．新生鼠暴露CPF诱导海马星形胶质细胞活化(P＜0.05)并增加成年期(出生60天)焦虑水平(P＜0.01)。 4．DMSO与NS比较，抑制小胶质细胞活性，减少CD11bmRNA表达并增加GDNFmRNA的表达(P＜0.05)。结论：本研究首次证实，新生期动物暴露亚剂量有机磷农药CPF后，通过中脑黑质小胶质细胞激活，增加TNF-α表达，同时减少GDNFmRNA表达，诱导成年期中脑黑质多巴胺能神经元损害。这个发现可以为神经退行性疾病，特别是对帕金森氏病(Parkinson’s disease，PD)的病因提供重要的线索。同时还发现，新生期动物暴露亚剂量CPF后，导致成年期焦虑水平增加，同时伴随海马星形胶质细胞激活。这个发现主要证明在新生儿期暴露环境化学污染物，可以在成年期引起神经精神紊乱，从而部分的阐述神经精神疾病的发病机理。此外，本研究还有一个出乎预料的发现，DMSO通过在中脑抑制小胶质细胞活性，增加GDMFmRNA表达，可能对多巴胺能神经元具有保护作用。这个重要的发现，对神经退行性疾病，特别是对帕金森氏病有潜在的治疗意义。
[Abstract]:Objective: is the "safe dose" of organophosphorus pesticides really harmless to the human body? This is an important problem involved in disease control and public health. This study explores the threshold dose ("safe dose") of Chlorpyrifos (CPF) of the neonatal exposure subthreshold dose ("safe dose"), and to the dopaminergic neurons and deity of the midbrain substantia nigra. The effect of behavior.
Methods: 138 SD rats born 11 days were randomly divided into group CPF (n=46) and control group (n=92). The control group was divided into two methyl sulfoxide (dimethysulfoxide, DMSO) control group (n=46) and normal saline (normal sodiu, NS) control group (n=46). The subcutaneous injection of subcutaneous intoxication threshold dose (hereinafter referred to as "subdose") in the abdominal subcutaneous injection (hereinafter referred to as "sub dose") in the rats was born. CPF, the animal model of neonatal SD rats exposed to CPF was made. The fat soluble CPF was dissolved in the solvent DMSO. The control group was DMSO, NS instead of CPF., the volume of each group was 1ml / kg.d.. After 12 hours, there were no acute organophosphorus pesticide poisoning symptoms. Four observation time points were set up after birth, 20 days, 30 days, and 60 days. The ultrastructural effects of CPF on the ultrastructure of microglia were observed by electron microscopy. Immunohistochemical method was used to detect the expression of dopaminergic tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) in the hippocampal astrocytes. RT-PCR was used to detect microglia microglia CD11bmRNA and glial cell derived neurotrophic factors in the middle brain The expression of subGDNFmRNA; enzyme-linked immunosorbent assay for the detection of brain TNF- alpha, IL-1 beta, and the data processing of the viaduct cross fan [? Seylis Castanopsis, K? Kenzo persimmon with PSS L 2.O statistical package. All data were expressed with mean mean standard deviation ((?) + S), and the single factor variance analysis was used first and the least significant difference method was used in the variance. T The test showed that when the variance was not homogeneous, Dunnett 's t test was used to test.P < 0.05, the difference was significant.
Results: no acute organophosphorus pesticide poisoning symptoms were found in neonatal rats exposed to sub dose CPF.
1.CPF induced the activation of microglia rapidly in the mesencephalic substantia nigra, and the ultrastructural microglia changed from resting state to phagocytic activation state, and up regulation of CD11b mRNA expression was more obvious (P < 0.05) at the early stage of exposure to CPF (P < 0.05), and increased the expression of inflammatory cytokine TNF- alpha (P < 0.01), but IL-1 beta (P < 0.01). No effect (P > 0.05).
2. exposure to CPF reduced the expression of GDNFmRNA in substantia nigra and decreased the expression of TH positive cells in the substantia nigra (30 days, 60 days) (P < 0.05).
3. neonatal rats exposed to CPF induced hippocampal astrocyte activation (P < 0.05) and increased anxiety level in adulthood (60 days) (P < 0.01).
Compared with NS, 4.DMSO inhibited the activity of microglia, reduced the expression of CD11bmRNA and increased the expression of GDNFmRNA (P < 0.05).
Conclusion: This study is the first to confirm that after exposure to subdose organophosphorus pesticide CPF in newborn animals, the activation of mesencephalic nigral microglia increases the expression of TNF- A and reduces the expression of GDNFmRNA, and induces damage to dopaminergic neurons in the midbrain of the midbrain. This discovery may be a neurodegenerative disease, especially for Parkinson's disease (Park The etiology of inson 's disease (PD) provides important clues. It is also found that neonatal exposure to a sub dose of CPF leads to increased anxiety in adulthood and the activation of astrocytes in the hippocampus. This discovery is mainly a discovery that exposes environmental chemical contaminants in the newborn period and can cause neuropsychiatric disorders in adulthood. The pathogenesis of neuropsychiatric disorders is described in part.
In addition, there is an unexpected discovery that DMSO may have protective effects on dopaminergic neurons by inhibiting the activity of microglia in the middle brain and increasing the expression of GDMFmRNA. This important discovery is of potential therapeutic significance for neurodegenerative diseases, especially for Parkinson's disease.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

【引证文献】