DEHP对雄性中国林蛙肝细胞的雌激素效应和凋亡的影响

【作者】 王娟娟；

【导师】 张育辉；

【作者基本信息】 陕西师范大学 ， 发育生物学， 2013， 硕士

【摘要】 近年来,伴随着工农业的迅速发展,大量人工合成的化合物的被不断释放到自然界,在野生动物的生存环境中,许多化合物可干扰动物的正常生长与发育。邻苯二甲酸酯(phthalatic esters,PAEs)是一类广泛应用于塑料工业的增塑剂,对动物产生雌激素效应,邻苯二甲酸二乙基已酯(di-2-ethylhexyl phthalate,DEHP)是其中的一种,有关DEHP对两栖动物的肝组织的雌激素效应以及对于肝细胞凋亡的影响尚未见报道。本实验以雄性中国林蛙(Rana chensinensis)为对象,探讨DEHP对雄性成体动物的雌激素效应以及对肝细胞的凋亡的影响。揭示DEHP影响雄性的繁殖力的机制以及对肝细胞的毒性。本实验将中国林蛙(Rana chensinensis)雄性成体分别暴露在10-7、10-6、10-5、10-4mol·L-1DEHP水体20d、30d、40d,取其肝组织,用免疫组织化学方法检测肝细胞中雌激素受体(estrogen receptor, ER)、卵黄蛋白原(vitellogenin, VTG)、Bax和Bcl-2蛋白的表达。用末端转移酶法(TUNEL)检测肝细胞的凋亡。结果如下：1.免疫组化检测结果表明,通过DEHP暴露处理后,雄性林蛙肝细胞质中均有VTG阳性反应。对VTG表达相对值的检测统计表明,与对照组相比,各DEHP处理组的VTG表达显著上调,且VTG表达量随着DEHP浓度的升高而增高,但随暴露时间延长的变化不具有规律性。2. DEHP处理后,林蛙肝细胞的胞质和胞核中均有ER阳性反应。ER表达相对值检测表明,各DEHP组的肝细胞ER表达相对值均高于对照组,ER表达在一定暴露时间范围随DEHP浓度的升高而上调,但随暴露时间延长的变化规律不明显。ER与VTG表达趋势的相似性表明,DEHP可通过高调肝细胞ER表达诱导VTG合成。3.对肝组织进行TUNEL检测,DEHP处理后部分肝细胞呈深黑色。对肝细胞凋亡率(AI)统计显示,对照组的AI为3.43%,在DEHP组暴露40d,AI为20.37±2.13%；在10-4mol·L-1DEHP组暴露40d,AI为28.38±±1.03%,达到峰值。总体显示,AI随DEHP浓度的升高和暴露时间的延长均呈现剂量效应和时间效应。但在10-5mol·L-1DEHP组,暴露40d比30d的AI有所下降。4.免疫组化检测肝细胞中Bax和Bcl-2表达显示,DEHP处理后,Bax和Bcl-2表达随着DEHP浓度升高而上调,随着暴露时间的延长Bax表达相对值呈增高趋势,而Bcl-2表达相对值呈下降趋势。对AI与Bax/Bcl-2表达量比值的相关性分析显示,肝细胞的AI与Bax/Bcl-2之间具有显著性的正相关。表明DEHP通过影响肝细胞Bax表达上调和Bcl-2表达下调而导致肝细胞凋亡。更多还原

【Abstract】 In recent years, a large number of synthetic compounds have been released into the nature along with the rapid development of industry and agriculture.Many of compounds can disturb the normal growth and development in wild animal. Phthalic acid esters (PAEs) as industrial products are used universally in our daily life which can induce effect of estrogen. Di2-Ethyl Hexyl Phthalate is one of the PAEs. The report about whether it can induce estrogenic effect or apoptosis in hepatic cells of amphibians hasn’t been studied. So we take R. chensinensis as experimental anmial to understand the estrogenic effect and to investigate the effect of hepatocyte apoptosis of DEHP. The aim of this experiment is to reveal the mechanism of propagation and the hepatotoxicity in males.In this study, adult male frogs Rana chensinensis were exposed to different concentrations of10-7,10-6,10-5and10-4mol·L-1DEHP consecutively for20,30and40d respectively, then liver tissue was got from R.chensinensis at certain days. Hepatic cells are detected the expression of protein of Bax, Bcl-2, ER and Vtg by immunohistochemical technique, and apoptosis cells are tested by TUNEL. The results and the conclusions are listed as follows:1. The results of immunohistochemical technique of VTG show that VTG express in every treated group.The positive effect of VTG is yellow or brown in the cytoplasm.The results of the relative intensity of VTG show that the expression of VTG increased significantly in hepatic cells of R. chensinensis in DEHP treated group compared with the control group.In DEHP treated group, the expression of VTG is increased with the increasing concentration of DEHP, but not with the treated time.2. The positive effect of ER is yellow or brown in the cytoplasm and nuclear.The results of the relative intensity of ER show that the expression of ER increased significantly in hepatic cells of R. chensinensis in DEHP treated group compared with the control group.in DEHP treated group, the expression of ER is increased with the increasing concentration of DEHP. For the same concentration of DEHP treatment, the expression of ER did not significantly fluctuate with prolonged treatment. The change trend of ER is similar to the VTG. These results suggested that DEHP could caused the synthesis of VTG by dint of induce overexpression of ER in hepatocytes.3. The some of hepatic cells present deep black were detected by TUNEL technique. There are apoptotic cells in control group, AI is about3.43%. AI is20.37±2.13%in10-6mol·l-1DEHP treated for40d. AI is28.38±1.03%in10-4mol·L-1DEHP treated for40d, which is the highest. There exist dose-effect and time-effect relationship between DEHP and AI. Whereas, in10-5mol·L-1DEHP treated for40d, the AI is lowerer than treated for30d.4. Detecting the expression of Bax and Bcl-2by immunohistochemical technique in hepatic cells of R.chensinensis. The results show that the expression of Bax and Bcl-2are increasing with concentration along with the time. The expression of Bax is increasing with time, but decreasing with Bcl-2. Analyzing the linear regression relation between AI and the ratio of Bax/Bcl-2expression, the results show that significant positive correlation exist between AI and the ratio of Bax/Bcl-2expression in hepatic cell. It suggests that DEHP can increase the expression of Bax and decrease the expression of Bcl-2to induce the cell apoptosis.更多还原