發(fā)布時間：2018-08-23 19:16

【摘要】： 為探討低濃度辛硫磷農藥對肝臟的慢性毒性作用及其機理,本研究通過觀察大鼠肝臟病理組織與超微結構改變及檢測血漿、肝臟ChE、抗氧化物酶和脂質過氧化指標,研究了辛硫磷農藥慢性灌胃染毒條件下對大鼠肝臟的氧化損傷作用;并進行了原代培養(yǎng)大鼠肝細胞的染毒試驗,通過掃描電鏡、熒光顯微鏡下觀察,Annexin-V-FITC和PI雙標記流式細胞儀測定凋亡率,雙氫羅丹明標記細胞、流式細胞儀測定細胞內活性氧(ROS)等方法,探討了辛硫磷對大鼠肝臟的毒性作用、致細胞凋亡作用以及ROS在辛硫磷誘導細胞凋亡中的作用。 結果表明,辛硫磷慢性染毒,大鼠血漿和肝臟中ChE活性均顯著降低,但沒有出現明顯的臨床中毒癥狀,推測中毒癥狀的出現不僅與血漿ChE活性降低程度有關,更主要與ChE降低速度有關。大鼠肝臟的病變程度隨著染毒劑量的增加和染毒時間的延長有加重趨勢。30μmol/kg劑量染毒15d,血漿和肝臟SOD、GSH-Px活性及MDA含量均無明顯變化,這也進一步說明辛硫磷是一種短效型低毒殺蟲劑對人畜的毒性較低。但是隨著染毒時間的延長,染毒30d后,肝臟抗氧化酶活性升高和MDA濃度升高,血漿抗氧化酶活性降低,是否表明肝臟的抗氧化能力代償性增強,有待進一步探討。300μmol/kg劑量的辛硫磷染毒15d,血漿SOD、GSH-Px活性降低,MDA含量明顯上升,但肝臟SOD、GSH-Px活性升高。持續(xù)染毒30d時,血漿和肝臟SOD、GSH-Px活性均明顯下降,MDA含量極顯著升高(P㩳0.01),表明辛硫磷農藥持續(xù)染毒后,不僅可引起大鼠機體氧化應激,而且可以通過誘導肝細胞發(fā)生脂質過氧化造成肝臟結構的損傷。 本試驗中30μmol/L辛硫磷對體外培養(yǎng)的肝細胞持續(xù)染毒24h,掃描電鏡下以及吖啶橙染色后熒光顯微鏡下都觀察到凋亡細胞,證明了低濃度的辛硫磷可以誘導肝細胞凋亡。不同劑量的辛硫磷對原代培養(yǎng)的肝細胞染毒,肝細胞染毒12h,100μmol/L劑量組死亡率極顯著增加(P0.01)、300μmol/L劑量組死亡率達到3.01%。染毒24h,隨著染毒劑量的增大,細胞死亡率呈明顯的上升趨勢,差異均極顯著(P0.01),300μmol/L劑量組死亡率已高達14.28%。說明辛硫磷農藥對肝細胞具有直接的毒性作用,而且細胞死亡率隨染毒劑量增大和染毒時間延長呈明顯的劑量-效應關系,表明辛硫磷作用劑量的增加或作用時間的延長,均可增加對細胞的毒性損傷。本試驗測得各染毒組大鼠肝細胞隨染毒劑量的增大和染毒時間延長,細胞凋亡率增大,與對照組比較,30μmol/L染毒12h凋亡率顯著增加(P0.05),100μmol/L劑量組凋亡率極顯著增加(P0.01),并且300μmol/L劑量組細胞凋亡率達到最大3.93%;染毒24h后,與對照組相比,各染毒組細胞凋亡率極顯著增加(300μmol/L劑量組除外),100μmol/L劑量組細胞凋亡率達到最大7.53%。說明低濃度的辛硫磷農藥可以誘導肝實質細胞凋亡。本研究發(fā)現隨著辛硫磷濃度的升高,肝細胞內ROS水平逐漸升高,細胞凋亡率與細胞內ROS水平呈正相關。提示在辛硫磷農藥致大鼠肝細胞凋亡過程中ROS發(fā)揮重要作用。
[Abstract]:To investigate the chronic toxicity of phoxim pesticide at low concentration on liver and its mechanism, the oxidative damage of rat liver was studied by observing the pathological and ultrastructural changes of liver and detecting the indexes of plasma, liver ChE, antioxidant enzyme and lipid peroxidation. The toxicity of Phoxim on primary cultured rat hepatocytes was studied by scanning electron microscopy, fluorescence microscopy, Annexin-V-FITC and PI double-labeled flow cytometry, dihydrorhodamine-labeled cells and reactive oxygen species (ROS) assay. The toxicity of Phoxim on rat liver and its effect on cell apoptosis were investigated. Apoptosis and the role of ROS in phoxim induced apoptosis.
The results showed that the ChE activity in plasma and liver of rats was significantly decreased after chronic exposure to phoxim, but no obvious clinical toxic symptoms were observed. It was speculated that the occurrence of toxic symptoms was not only related to the degree of decrease of plasma ChE activity, but also to the rate of decrease of plasma ChE activity. There was no significant change in SOD, GSH-Px activity and MDA content in plasma and liver after 15 days of exposure at a dose of 30 micromol/kg, which further indicated that phoxim was a short-acting and low-toxic insecticide with low toxicity to human and livestock. However, with the prolongation of exposure time, liver antioxidant enzyme activity and MDA concentration increased 30 days after exposure. The activity of SOD, GSH-Px and MDA in plasma and liver decreased significantly after 15 days of Phoxim exposure, but the activity of SOD and GSH-Px in liver increased significantly. The activity of SOD, GSH-Px in plasma and liver decreased significantly after 30 days of continuous exposure. The content of phoxim increased significantly (P?0.01), indicating that phoxim could not only induce oxidative stress in rats, but also damage the liver structure by inducing lipid peroxidation in hepatocytes.
Apoptotic cells were observed under scanning electron microscopy and fluorescence microscopy after staining with acridine orange. It was proved that low concentration of phoxim could induce apoptosis of hepatocytes. Different doses of phoxim were given to primary cultured hepatocytes and the hepatocytes were exposed to phoxim for 12 hours and 100 micromol/L for 12 hours. The mortality of L-dose group was significantly increased (P 0.01), and that of 300 micromol/L group was 3.01%. The cell mortality increased significantly with the increase of dose in 24 hours (P 0.01). The mortality of 300 micromol/L group was up to 14.28%. The cell death rate showed a dose-effect relationship with the increase of dose and duration of exposure, indicating that the increase of dose or duration of phoxim could increase the toxic damage to cells. Compared with the control group, the apoptosis rate increased significantly at 12 h (P 0.05), and significantly at 100 micromol/L (P 0.01), and the apoptosis rate reached the maximum of 3.93% in the 300 micromol/L dose group; after 24 h, the apoptosis rate of each exposure group was significantly increased (except 300 micromol/L dose group), and fine in the 100 micromol/L dose group. The apoptosis rate was 7.53%. It indicated that low concentration phoxim pesticide could induce apoptosis of hepatic parenchyma cells. With the increase of Phoxim concentration, the level of ROS in hepatocytes increased gradually, and the apoptosis rate was positively correlated with the level of ROS in hepatocytes. Effect.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R363

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