【作者】 薛彬；

【导师】 刘宗平；

【作者基本信息】 扬州大学 ， 临床兽医学， 2008， 硕士

【摘要】 为探讨低浓度乐果对大鼠肝脏氧化应激状态和肝细胞凋亡的影响及其机理,本试验通对大鼠肝脏病理组织和超微结构的观察及肝脏AChE、抗氧化酶和脂质过氧化指标的检测,研究了亚慢性染毒条件下乐果对大鼠肝脏氧化应激状态的影响;同时,通过体外原代培养肝细胞,研究了乐果对肝细胞的一般毒性和凋亡的影响。细胞凋亡用Annexin-V/PI双染法检测,细胞内活性氧、线粒体膜电位和细胞内Ca2+浓度变化,分别用DCFH-DA、罗丹名123和Fluo-2-AM标记检测,并探讨了以上因素在乐果诱导肝细胞凋亡中的可能机制。试验结果表明,分别灌服0、1、6和30mg/kg b.w.乐果染毒30d后,血浆和肝脏AChE活性降低,但并无明显的临床症状,推测中毒症状的出现不仅与血浆AChE活性降低程度有关,更主要与AChE降低速度有关。同时,各染毒组大鼠肝脏SOD活性均明显升高,而GSH-Px、CAT活性变化随染毒剂量增加呈先下降后上升趋势。脂质过氧化产物MDA含量呈上升趋势,与对照组相比,MDA含量分别升高29%、69%和129%。组织学和超微结构变化显示肝小叶充血、脂肪变性、肝细胞核皱缩、裂解,形成凋亡小体等。表明低剂量乐果亚慢性暴露不仅可造成大鼠肝脏氧化应激状态升高,且可造成肝脏一定程度的损伤。体外试验结果表明,0、3、30、300μmol/L乐果染毒12、24h均可不同程度抑制肝细胞的增殖,且存在时间-剂量效应。与对照组相比,染毒后48h达到生长高峰时,染毒组细胞存活率分别为对照组的85%、72%和67%,且随染毒时间延长,细胞活率进一步下降。细胞培养液上清中LDH含量升高,与对照组相比差异显著或极显著(P<0.05或P<0.01),300μmol/L组染毒12h和24h培养液上清中LDH含量分别为对照组的7.35倍和8.75倍;染毒组细胞内GSH含量降低,30和300μmol/L组与对照组差异极显著(P<0.01)。本研究表明,不同浓度乐果染毒后,细胞凋亡率明显升高,与对照组相比差异极显著(P<0.01),并且呈时间-剂量效应。细胞内ROS水平在3-100μmol/L范围内随染毒剂量的增大和染毒时间的延长而升高,而在300μmol/L组略有下降。细胞内线粒体膜电位(Δψm)除24h高剂量染毒组(300μmol/L)外均出现持续下降。3μmol/L组细胞内Ca2+浓度极显著高于对照组(P<0.01),但随染毒剂量的进一步增加,细胞内Ca2+浓度逐渐下降。表明乐果诱导的细胞凋亡可能与细胞内ROS生成及细胞膜的损伤有关。更多还原

【Abstract】 The present study was carried out to investigate the effects and mechanisms of dimethoate-induced oxidative stress and hepatocytes apoptosis in rats’liver, the histopathology and ultramicrostructure changes, anti-oxidative enzymes and lipid peroxidation were detected to determine the role of dimethoate in production oxidative stress in rats’liver. At the same time, the general toxicity and apoptosis effects of dimethoate were researched via primary cultured hepatocytes in vitro. Apoptotic rate was detected by Annexin-V/PI double staining, intracellular ROS, mitochondrial membrane potential and concentration were detected with DCFH-DA, Rodamin 123 and Fluo-2-AM respectively. The mechanism of these factors in dimethoate-induced aoptosis in hepatocytes were disccused in this paper too.The results showed that the activity of AChE were both decresed in plasma and liver of experimental rats exposured with 0, 1, 6 and 30mg/kg b.w. dimethoate for 30d respectively, but no obvious clinical symptoms were observed. It could be presumed that the occurrence of clinical symptoms was not only related with the degree of AChE reduction, but mainly with the speed of reduction. The activity of SOD were increased in all treated group accompanied with GSH-Px and CAT decreased in low group but increased in high group. MDA concentration were increased 29%、69% and 129% in treated groups when compared with control group respectivity. Histopathology and ultramicrostructure survey showed congestion of hepatic lobules, fatty degenerationthe, shrinkage, split and apoptotic body of the nucleus. The results showed that subchronic esposure of dimethoate for 30d not only induced oxidative stress, but also cause liver damage in experimental rats.The results of in vitro test showed that exposured with dimethoate of 0, 3, 30, 300μmol/L for 12h and 24h could inhibited hepatocytes proliferation at different degree, and present time-dose effects. The survival rate of hepatocytes were 85, 72 and 67% at 48h in treated groups and aggregate with the prolongation of time. Increase of LDH activity were observed in all treated groups which concentration were 7.35 and 8.75 times in medium supernant when compared with control groups in 300μmol/L group at 12h and 24h respectively (P<0.01). Decrease of GSH concentration were decreased in all treated groups and significant in 30 and 300μmol/L groups. Hepatocytes apoptotic rates were increased significantly from 3.1% in control group to 16.9% in 300μmol/L group by flow cytometry detection use Annexin V/PI staining. Intracellular ROS level were increased in 3μmol/L to 100μmol/L group and slightly subside in 300μmol/L group. Decreased of mitochondria membrane potential owing to permeability transition was observed in our study too except in 300μmol/L group at 24h. Intracellular Ca2+ concentration were increased in all group and reached highest peak in 3μmol/L group, diffirent was significant(P<0.01). All indicated that dimethoate-induced apoptosis was related with generation of ROS and damage of hepatocytes membrane.更多还原