本文選題：有機(jī)磷農(nóng)藥 + 毒死蜱��； 參考：《中南大學(xué)》2014年博士論文

【摘要】：目的探討低劑量有機(jī)磷農(nóng)藥毒死蜱(Chlorpyrifos, CPF)及其代謝產(chǎn)物氧化毒死蜱(Chlorpyrifos oxon, CPO)對(duì)原代培養(yǎng)大鼠腦皮層細(xì)胞的神經(jīng)毒性；了解農(nóng)村兒童監(jiān)護(hù)人的農(nóng)藥安全使用行為,為探索兒童農(nóng)藥暴露防控途徑提供新的思路。 方法1.原代培養(yǎng)SD胎鼠腦皮層神經(jīng)元和新生鼠皮層星形膠質(zhì)細(xì)胞,胰酶消化法進(jìn)行分離純化星形膠質(zhì)細(xì)胞,采用細(xì)胞免疫熒光化學(xué)法分別對(duì)兩種細(xì)胞純度進(jìn)行鑒定,比色法描繪細(xì)胞生長(zhǎng)曲線。得到高純度神經(jīng)元和星形膠質(zhì)細(xì)胞后選取處于活力上升期的細(xì)胞,首先探索兩者對(duì)毒死蜱及其代謝產(chǎn)物的溶媒二甲基亞砜(Dimethyl sulfoxide, DM SO)的敏感性。分別用0.5%、1.0%、5.0%、10.0%濃度的二甲基亞砜干預(yù)原代神經(jīng)元,0.25%、0.50%、1.00%、5.00%、10.00%濃度的二甲基亞砜干預(yù)原代星形膠質(zhì)細(xì)胞,對(duì)照組以等量培養(yǎng)基替代二甲基亞砜,于12h、24h、48h時(shí)通過動(dòng)態(tài)觀察細(xì)胞形態(tài)、計(jì)算存活細(xì)胞數(shù)并測(cè)定O.D值間接判斷細(xì)胞活力,確定對(duì)兩種細(xì)胞無損傷作用的二甲基亞砜濃度與作用時(shí)間。 2.同樣選取處于活力上升期的神經(jīng)元和星形膠質(zhì)細(xì)胞,分別暴露于5μM、20μM、80μM、100μiM濃度的毒死蜱及其代謝產(chǎn)物氧化毒死蜱,對(duì)照組僅加入等量二甲基亞砜,分別在12h、24h、48h于倒置光學(xué)顯微鏡下觀察細(xì)胞形態(tài)；采用細(xì)胞免疫熒光化學(xué)法顯示細(xì)胞核形態(tài)并計(jì)算細(xì)胞數(shù)目；采用比色法(CCK-8)測(cè)定WST-8(water-soluble tetrazolium-8)在細(xì)胞線粒體中代謝產(chǎn)物甲佨(Formazan)的吸光度值(O.D值),間接反映細(xì)胞的增殖率與活力。 3.在湖南省新化縣農(nóng)村采用整群隨機(jī)抽樣的方法選擇526名兒童監(jiān)護(hù)人為研究對(duì)象。采用自行設(shè)計(jì)的包括家庭一般情況、農(nóng)藥安全使用行為的調(diào)查表進(jìn)行問卷調(diào)查,以了解不同家庭結(jié)構(gòu)農(nóng)村兒童的監(jiān)護(hù)人安全使用農(nóng)藥的行為。使用EpiData3.1軟件整理收集到的資料并建立數(shù)據(jù)庫,SPSS19.0軟件進(jìn)行統(tǒng)計(jì)描述與分析。 結(jié)果1.成功培養(yǎng)胎鼠皮層神經(jīng)元及新生鼠皮層星形膠質(zhì)細(xì)胞。純度分別為92.7%±3.1%和95.1%±1.2%,選擇活力上升期細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn)。 2.與對(duì)照組相比,0.5%的二甲基亞砜干預(yù)原代神經(jīng)元各時(shí)間點(diǎn)均未見細(xì)胞數(shù)量與形態(tài)發(fā)生明顯改變；神經(jīng)元暴露于1.0%的二甲基亞砜12h時(shí),細(xì)胞數(shù)量減少,軸突皺縮,軸突網(wǎng)絡(luò)形成減少,此變化在24h和48h時(shí)加劇,并出現(xiàn)變形的細(xì)胞核；當(dāng)二甲基亞砜濃度在1.0%以上時(shí),隨著濃度的增加,神經(jīng)元數(shù)量減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05),形態(tài)改變加劇,且胞體內(nèi)出現(xiàn)空泡,細(xì)胞核濃縮變形,染色不均勻,呈顆粒樣,經(jīng)比色法測(cè)定的神經(jīng)元O.D值下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；同一濃度下,隨著時(shí)間的延長(zhǎng),也可見神經(jīng)元數(shù)量減少,形態(tài)改變明顯,O.D值顯著下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；至10.0%二甲基亞砜暴露48h時(shí)已較難在視野中尋見神經(jīng)元。 3.0.25%的二甲基亞砜干預(yù)至48h未見星形膠質(zhì)細(xì)胞數(shù)量與形態(tài)明顯改變。濃度為0.50%、1.00%的二甲基亞砜作用于原代星形膠質(zhì)細(xì)胞24h-48h時(shí),細(xì)胞數(shù)量增多,更加密集,上層附著顆粒樣物質(zhì),細(xì)胞形態(tài)無明顯變化,細(xì)胞活力增強(qiáng),差異有統(tǒng)計(jì)學(xué)意義(P0.01)。5.00%二甲基亞砜干預(yù)24h后,星形膠質(zhì)細(xì)胞數(shù)量減少,胞體內(nèi)出現(xiàn)空泡,多數(shù)細(xì)胞皺縮,視野內(nèi)有較多細(xì)胞殘?jiān)?隨著時(shí)間的延長(zhǎng)及濃度的增加上述改變加劇,活力降低,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。 4.與對(duì)照組相比,5μM、20μM毒死蜱暴露各時(shí)間點(diǎn)均未見神經(jīng)元數(shù)量、形態(tài)及O.D值的明顯改變；80μM毒死蜱暴露至48h雖未見原代培養(yǎng)神經(jīng)元數(shù)量與形態(tài)的改變,但細(xì)胞O.D值較12h組下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；當(dāng)毒死蜱濃度大于80μM時(shí),隨著濃度的增加,神經(jīng)元數(shù)量無明顯改變,但變形的細(xì)胞核增多,O.D值下降明顯,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；同一濃度下,隨著時(shí)間的延長(zhǎng),神經(jīng)元形態(tài)改變加劇,O.D值顯著下降,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。 5.5μM、20μM毒死蜱處理各時(shí)間點(diǎn)均未見星形膠質(zhì)細(xì)胞數(shù)量與形態(tài)的明顯改變。80μM毒死蜱處理48h后星形膠質(zhì)細(xì)胞數(shù)量減少(P0.01),偶見胞漿內(nèi)空泡形成和變形細(xì)胞核。100μM毒死蜱處理48h時(shí),星形膠質(zhì)細(xì)胞數(shù)量減少,較多細(xì)胞胞漿內(nèi)形成空泡且有細(xì)胞發(fā)生皺縮,隨著濃度的增加和時(shí)間的延長(zhǎng),星形膠質(zhì)細(xì)胞數(shù)量繼續(xù)減少,多數(shù)細(xì)胞發(fā)生皺縮,核變形,活力下降,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。 6.與對(duì)照組相比,5μM、20μM氧化毒死蜱暴露各時(shí)間點(diǎn)均未見神經(jīng)元數(shù)量、形態(tài)及O.D值的明顯改變；80gM氧化毒死蜱暴露至48h雖未見原代培養(yǎng)神經(jīng)元數(shù)量與形態(tài)的改變,但細(xì)胞O.D值較12h組下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；當(dāng)氧化毒死蜱濃度在80gM以上時(shí),隨著濃度的增加與時(shí)間的延長(zhǎng),O.D值明顯下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；400μM氧化毒死蜱暴露至48h時(shí)才觀察到神經(jīng)元數(shù)量的減少。 7.80μM以下濃度的氧化毒死蜱處理后三個(gè)時(shí)間點(diǎn)均未見星形膠質(zhì)細(xì)胞數(shù)量、形態(tài)及活力的明顯改變。大于100μM氧化毒死蜱處理24h時(shí),隨著濃度的增加和時(shí)間的延長(zhǎng),星形膠質(zhì)細(xì)胞數(shù)量顯著減少,差異有統(tǒng)計(jì)學(xué)意義(P0.01),細(xì)胞聚集明顯,胞體內(nèi)有空泡增多,變形細(xì)胞核增多,活力下降明顯,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。 8.毒死蜱濃度在100μM以上暴露48h時(shí),神經(jīng)元的O.D值顯著低于同濃度同時(shí)間的氧化毒死蜱暴露組,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。 9.毒死蜱濃度在80μM以上暴露各時(shí)間點(diǎn),星形膠質(zhì)細(xì)胞的O.D值均低于同濃度同時(shí)間的氧化毒死蜱暴露組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 10.留守兒童監(jiān)護(hù)人農(nóng)藥安全使用行為計(jì)分為(12.1±2.6)分,低于非留守兒童監(jiān)護(hù)人農(nóng)藥安全使用行為計(jì)分(17.3士1.5)分,差異有統(tǒng)計(jì)學(xué)意義(P0.01),與非留守兒童監(jiān)護(hù)人相比,留守兒童監(jiān)護(hù)人缺乏正確的農(nóng)藥安全使用行為。 結(jié)論1.毒死蜱及氧化毒死蜱均對(duì)原代培養(yǎng)的胎鼠皮層神經(jīng)元和新生鼠皮層星形膠質(zhì)細(xì)胞有毒性作用,表現(xiàn)在對(duì)細(xì)胞形態(tài)、數(shù)量及活力的影響,且該毒性作用在一定濃度范圍(≥80μM)存在時(shí)間與劑量依賴性,隨著濃度的增加與作用時(shí)間的延長(zhǎng),細(xì)胞數(shù)量減少,發(fā)生形態(tài)改變的細(xì)胞比例增加,細(xì)胞活力下降。 2.相同條件下,毒死蜱及氧化毒死蜱對(duì)原代星形膠質(zhì)細(xì)胞形態(tài)、數(shù)量與活力的影響較神經(jīng)元更為明顯,提示星形膠質(zhì)細(xì)胞可能是其毒性作用的靶細(xì)胞。 3.毒死蜱代謝為氧化毒死蜱后對(duì)原代神經(jīng)元及星形膠質(zhì)細(xì)胞的毒性作用減弱。 4.室溫下,使用二甲基亞砜作為有機(jī)磷農(nóng)藥的溶媒時(shí),作用于原代胎鼠皮層神經(jīng)元的濃度不超過0.5%,原代新生鼠皮層星形膠質(zhì)細(xì)胞的作用濃度不超過0.25%。 5.基于不同家庭結(jié)構(gòu)的兒童監(jiān)護(hù)人視野的調(diào)查表明,留守兒童監(jiān)護(hù)人農(nóng)藥使用行為不規(guī)范,使得留守兒童的安全與健康存在隱患。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R725.9

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