农药毒死蜱对大鼠皮层神经细胞的毒性及儿童监护人农药使用行为调查

发布时间：2018-06-11 14:54

本文选题：有机磷农药 + 毒死蜱　；参考：《中南大学》2014年博士论文

【摘要】：目的探讨低剂量有机磷农药毒死蜱(Chlorpyrifos, CPF)及其代谢产物氧化毒死蜱(Chlorpyrifos oxon, CPO)对原代培养大鼠脑皮层细胞的神经毒性；了解农村儿童监护人的农药安全使用行为,为探索儿童农药暴露防控途径提供新的思路。方法1.原代培养SD胎鼠脑皮层神经元和新生鼠皮层星形胶质细胞,胰酶消化法进行分离纯化星形胶质细胞,采用细胞免疫荧光化学法分别对两种细胞纯度进行鉴定,比色法描绘细胞生长曲线。得到高纯度神经元和星形胶质细胞后选取处于活力上升期的细胞,首先探索两者对毒死蜱及其代谢产物的溶媒二甲基亚砜(Dimethyl sulfoxide, DM SO)的敏感性。分别用0.5%、1.0%、5.0%、10.0%浓度的二甲基亚砜干预原代神经元,0.25%、0.50%、1.00%、5.00%、10.00%浓度的二甲基亚砜干预原代星形胶质细胞,对照组以等量培养基替代二甲基亚砜,于12h、24h、48h时通过动态观察细胞形态、计算存活细胞数并测定O.D值间接判断细胞活力,确定对两种细胞无损伤作用的二甲基亚砜浓度与作用时间。 2.同样选取处于活力上升期的神经元和星形胶质细胞,分别暴露于5μM、20μM、80μM、100μiM浓度的毒死蜱及其代谢产物氧化毒死蜱,对照组仅加入等量二甲基亚砜,分别在12h、24h、48h于倒置光学显微镜下观察细胞形态；采用细胞免疫荧光化学法显示细胞核形态并计算细胞数目；采用比色法(CCK-8)测定WST-8(water-soluble tetrazolium-8)在细胞线粒体中代谢产物甲佨(Formazan)的吸光度值(O.D值),间接反映细胞的增殖率与活力。 3.在湖南省新化县农村采用整群随机抽样的方法选择526名儿童监护人为研究对象。采用自行设计的包括家庭一般情况、农药安全使用行为的调查表进行问卷调查,以了解不同家庭结构农村儿童的监护人安全使用农药的行为。使用EpiData3.1软件整理收集到的资料并建立数据库,SPSS19.0软件进行统计描述与分析。结果1.成功培养胎鼠皮层神经元及新生鼠皮层星形胶质细胞。纯度分别为92.7%±3.1%和95.1%±1.2%,选择活力上升期细胞进行后续实验。 2.与对照组相比,0.5%的二甲基亚砜干预原代神经元各时间点均未见细胞数量与形态发生明显改变；神经元暴露于1.0%的二甲基亚砜12h时,细胞数量减少,轴突皱缩,轴突网络形成减少,此变化在24h和48h时加剧,并出现变形的细胞核；当二甲基亚砜浓度在1.0%以上时,随着浓度的增加,神经元数量减少,差异有统计学意义(P0.05),形态改变加剧,且胞体内出现空泡,细胞核浓缩变形,染色不均匀,呈颗粒样,经比色法测定的神经元O.D值下降,差异有统计学意义(P0.05)；同一浓度下,随着时间的延长,也可见神经元数量减少,形态改变明显,O.D值显著下降,差异有统计学意义(P0.05)；至10.0%二甲基亚砜暴露48h时已较难在视野中寻见神经元。 3.0.25%的二甲基亚砜干预至48h未见星形胶质细胞数量与形态明显改变。浓度为0.50%、1.00%的二甲基亚砜作用于原代星形胶质细胞24h-48h时,细胞数量增多,更加密集,上层附着颗粒样物质,细胞形态无明显变化,细胞活力增强,差异有统计学意义(P0.01)。5.00%二甲基亚砜干预24h后,星形胶质细胞数量减少,胞体内出现空泡,多数细胞皱缩,视野内有较多细胞残渣,随着时间的延长及浓度的增加上述改变加剧,活力降低,差异有统计学意义(P0.01)。 4.与对照组相比,5μM、20μM毒死蜱暴露各时间点均未见神经元数量、形态及O.D值的明显改变；80μM毒死蜱暴露至48h虽未见原代培养神经元数量与形态的改变,但细胞O.D值较12h组下降,差异有统计学意义(P0.05)；当毒死蜱浓度大于80μM时,随着浓度的增加,神经元数量无明显改变,但变形的细胞核增多,O.D值下降明显,差异有统计学意义(P0.05)；同一浓度下,随着时间的延长,神经元形态改变加剧,O.D值显著下降,差异有统计学意义(P0.01)。 5.5μM、20μM毒死蜱处理各时间点均未见星形胶质细胞数量与形态的明显改变。80μM毒死蜱处理48h后星形胶质细胞数量减少(P0.01),偶见胞浆内空泡形成和变形细胞核。100μM毒死蜱处理48h时,星形胶质细胞数量减少,较多细胞胞浆内形成空泡且有细胞发生皱缩,随着浓度的增加和时间的延长,星形胶质细胞数量继续减少,多数细胞发生皱缩,核变形,活力下降,差异有统计学意义(P0.01)。 6.与对照组相比,5μM、20μM氧化毒死蜱暴露各时间点均未见神经元数量、形态及O.D值的明显改变；80gM氧化毒死蜱暴露至48h虽未见原代培养神经元数量与形态的改变,但细胞O.D值较12h组下降,差异有统计学意义(P0.05)；当氧化毒死蜱浓度在80gM以上时,随着浓度的增加与时间的延长,O.D值明显下降,差异有统计学意义(P0.05)；400μM氧化毒死蜱暴露至48h时才观察到神经元数量的减少。 7.80μM以下浓度的氧化毒死蜱处理后三个时间点均未见星形胶质细胞数量、形态及活力的明显改变。大于100μM氧化毒死蜱处理24h时,随着浓度的增加和时间的延长,星形胶质细胞数量显著减少,差异有统计学意义(P0.01),细胞聚集明显,胞体内有空泡增多,变形细胞核增多,活力下降明显,差异有统计学意义(P0.01)。 8.毒死蜱浓度在100μM以上暴露48h时,神经元的O.D值显著低于同浓度同时间的氧化毒死蜱暴露组,差异有统计学意义(P0.01)。 9.毒死蜱浓度在80μM以上暴露各时间点,星形胶质细胞的O.D值均低于同浓度同时间的氧化毒死蜱暴露组,差异有统计学意义(P0.05)。 10.留守儿童监护人农药安全使用行为计分为(12.1±2.6)分,低于非留守儿童监护人农药安全使用行为计分(17.3士1.5)分,差异有统计学意义(P0.01),与非留守儿童监护人相比,留守儿童监护人缺乏正确的农药安全使用行为。结论1.毒死蜱及氧化毒死蜱均对原代培养的胎鼠皮层神经元和新生鼠皮层星形胶质细胞有毒性作用,表现在对细胞形态、数量及活力的影响,且该毒性作用在一定浓度范围(≥80μM)存在时间与剂量依赖性,随着浓度的增加与作用时间的延长,细胞数量减少,发生形态改变的细胞比例增加,细胞活力下降。 2.相同条件下,毒死蜱及氧化毒死蜱对原代星形胶质细胞形态、数量与活力的影响较神经元更为明显,提示星形胶质细胞可能是其毒性作用的靶细胞。 3.毒死蜱代谢为氧化毒死蜱后对原代神经元及星形胶质细胞的毒性作用减弱。 4.室温下,使用二甲基亚砜作为有机磷农药的溶媒时,作用于原代胎鼠皮层神经元的浓度不超过0.5%,原代新生鼠皮层星形胶质细胞的作用浓度不超过0.25%。 5.基于不同家庭结构的儿童监护人视野的调查表明,留守儿童监护人农药使用行为不规范,使得留守儿童的安全与健康存在隐患。
[Abstract]:Objective to explore the neurotoxicity of Chlorpyrifos (CPF) and its metabolite Chlorpyrifos Oxon (CPO) on the primary cultured rat cortical cells, and to understand the safe use of pesticides in rural children's guardians, and to provide a new way for exploring the ways to prevent and control pesticide exposure in children.
Methods 1. primary cultured SD fetal rat cortical neurons and neonatal rat cortical astrocytes were cultured. Astrocytes were isolated and purified by trypsin digestion. The purity of two cells was identified by cell immunofluorescence chemical method. The cell growth curve was depicted by colorimetric method. The selection of high purity neurons and astrocytes was obtained. In the cells of the active phase, the sensitivity of the two methyl sulfoxide (Dimethyl sulfoxide, DM SO) to chlorpyrifos and their metabolites was first explored. The 0.5%, 1%, 5%, 10% concentrations of the two methyl sulfoxide, respectively, intervened in the primary neurons, 0.25%, 0.50%, 1%, 5%, 10%, methylsulfoxide, intervened in the primary astrocytes. In the control group, two methyl sulfoxide was replaced by the same medium, and the cell morphology was dynamically observed at 12h, 24h and 48h. The number of surviving cells was calculated and the O.D value was measured to determine the cell viability indirectly. The concentration and action time of two methyl sulfoxide without damage to the two cells was determined.
2. the neurons and astrocytes in the active period were also selected, exposed to 5 M, 20 mu M, 80 mu M, and 100 micron iM, and their metabolites were oxidized to chlorpyrifos, and the control group only added two methyl sulfoxide to observe the cell morphology under the inverted optical microscopy, 12h, 24h, 48H, and the cell immunofluorescence chemistry was used. The cell nucleus morphology was displayed and the number of cells was calculated. The absorbance value (O.D value) of WST-8 (water-soluble tetrazolium-8) metabolism product Formazan (Formazan) in cell mitochondria was measured by colorimetric method (CCK-8), which indirectly reflected the cell proliferation and vitality.
3. in the rural areas of Xinhua County, Hunan Province, 526 children guardians were selected by cluster random sampling method. The questionnaire was used to investigate the behavior of the rural children in rural areas with different family structure and the behavior of the safety use of pesticides. The use of EpiData3 .1 software collects and collects data and builds up a database. SPSS19.0 software is used for statistical description and analysis.
Results 1. the cultured fetal rat cortical neurons and neonatal rat cortical astrocytes were successfully cultured. The purity was 92.7% + 3.1% and 95.1% + 1.2%, respectively.
2. compared with the control group, 0.5% of two methyl sulfoxide did not significantly change the number and morphology of the primary neurons at each time point. When the neurons were exposed to 1% of the two methyl sulfoxide 12h, the number of cells decreased, the axon shrinkage and the formation of the axon network decreased. The changes were aggravated at the time of 24h and 48h, and the nuclei were deformed; when two When the concentration of methyl sulfoxide was above 1%, with the increase of concentration, the number of neurons decreased, the difference was statistically significant (P0.05), the morphological changes were aggravated, and vacuoles appeared in the cell. The nuclei concentrated and deformed, the staining was uneven, and the O.D value of nerve element was decreased by colorimetric method, the difference was statistically significant (P0.05). Under the same concentration, the difference was statistically significant. With the prolongation of time, the number of neurons decreased, the morphological changes were obvious, the O.D value decreased significantly, the difference was statistically significant (P0.05). To 10% two methyl sulfoxide exposure 48h had been more difficult to find neurons in the field of vision.
The number and morphology of astrocytes were not significantly changed by the intervention of 3.0.25% two methyl sulfoxide to 48h. The concentration was 0.50%, and 1% of the two methyl sulfoxide was used in the primary astrocyte 24h-48h, the number of cells increased, the density was more dense, the upper layer attached to granular material, the cell morphology did not change obviously, the cell vitality increased, the difference was statistically significant. After P0.01.5.00% two methyl sulfoxide was interfered with 24h, the number of astrocytes decreased, vacuoles appeared in the cell body, most of the cells crinkled and there were more cell residues in the field of vision. With the prolongation of time and the increase of concentration, the above changes were aggravated and the vitality decreased, the difference was statistically significant (P0.01).
4. compared with the control group, the number of neurons and the morphologic and O.D values of 5 M and 20 M chlorpyrifos were not observed at all time points, while 80 M chlorpyrifos exposed to 48h did not change the number and morphology of the primary cultured neurons, but the O.D values of the cells were lower than those in the 12h group, and the difference was statistically significant (P0.05). When the concentration of chlorpyrifos was greater than 80 UX, the number of chlorpyrifos was more than 80 micron. With the increase of concentration, there was no significant change in the number of neurons, but the number of deformed nuclei increased and the O.D value decreased significantly (P0.05). Under the same concentration, the morphological changes of neurons increased with time, and the value of O.D decreased significantly, the difference was statistically significant (P0.01).
The number and morphology of astrocytes were not obviously changed at 5.5 M and 20 M chlorpyrifos at each time point. The number of astrocytes decreased (P0.01) after 48h treatment with chlorpyrifos. Even when the vacuoles were formed in the cytoplasm and the deformed nuclei.100 u M of chlorpyrifos, the number of astrocytes decreased and the vacuoles were formed in more cytoplasm. With the increase of concentration and prolongation of time, the number of astrocytes continued to decrease, and most of the cells shrink, nuclear deformation and vitality decreased, the difference was statistically significant (P0.01).
6. compared with the control group, the number of neurons, the morphology and the O.D value of chlorpyrifos exposed at 5 M and 20 mu M were not seen at all time points. Although 80gM oxidation chlorpyrifos exposed to 48h did not change the number and morphology of the primary cultured neurons, the O.D value of the cells decreased as compared with the 12h group, and the difference was statistically significant (P0.05); when the concentration of chlorpyrifos was oxidized, the concentration of chlorpyrifos was oxidized by chlorpyrifos in the 12h group. At above 80gM, with the increase of concentration and time, the value of O.D decreased significantly, the difference was statistically significant (P0.05), and the number of neurons was decreased when 400 M oxidized chlorpyrifos was exposed to 48h.
The number of astrocytes, morphology and activity of chlorpyrifos were not seen at three time points under the concentration of 7.80 mu M, and the number of astrocytes decreased significantly with the increase of concentration and the prolongation of time, and the difference was statistically significant (P0.01), cell aggregation was obvious, cell aggregation was obvious. The number of vacuoles increased, the number of deformed nuclei increased and the viability decreased significantly (P0.01).
8. when the concentration of chlorpyrifos was above 100 M, the O.D value of the neurons was significantly lower than that of the same time concentration of chlorpyrifos exposure group with the same concentration, and the difference was statistically significant (P0.01).
9. the concentration of chlorpyrifos was above 80 M, and the O.D values of astrocytes were all lower than those of chlorpyrifos exposure group with the same concentration and time, and the difference was statistically significant (P0.05).
The safety use behavior of 10. left guarded children's guardians was scored (12.1 + 2.6), which was lower than that of the non left guarded child guardians (17.3 and 1.5). The difference was statistically significant (P0.01). Compared with the non left guardians, the guardians of the left behind children lacked the correct use of pesticide.
Conclusion 1. chlorpyrifos and chlorpyrifos have toxic effects on the primary cultured fetal rat cortical neurons and neonatal rat cortical astrocytes, which are affected by the cell morphology, quantity and activity, and the toxicity is dependent on the time and dose dependence in a certain concentration range (> 80 M), with the increase of concentration and the action time. The number of cells decreased and morphological changes increased, and cell viability decreased.
2. under the same condition, the effects of chlorpyrifos and chlorpyrifos on the morphology, quantity and activity of astrocytes were more obvious than those of the neurons, suggesting that astrocytes may be the target cells of their toxic effects.
3. the toxicity of chlorpyrifos to chlorpyrifos is weakened after the oxidation of chlorpyrifos.
4. at room temperature, when two methyl sulfoxide was used as a solvent for organophosphorus pesticides, the concentration of the primary cultured rat cortical neurons was not more than 0.5%. The concentration of the original neonatal rat cortical astrocytes was not more than 0.25%..
5. the survey of the eyes of the guardians of children based on different family structures shows that the use behavior of the guardians of the guardians of the left behind children is not standard, which makes the safety and health of the left behind children have hidden dangers.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R725.9

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