壬基酚对中国林蛙肝细胞中雌激素受体和卵黄蛋白原的影响

【作者】 巨荣菊；

【导师】 张育辉；

【作者基本信息】 陕西师范大学 ， 发育生物学， 2010， 硕士

【摘要】 随着现代工业的快速发展,大量化学物质的使用对野生动物的生存环境已经造成严重污染。其中,酚类环境污染物作为环境内分泌干扰物严重影响着动物的生长发育以及导致动物种群数量下降。壬基酚(Nonylphenol, NP)就是其中一种环境内分泌干扰物,它有着与动物雌激素相似的结构,具有弱雌激素效应。本实验以雄性中国林蛙(Rana chensinensis)作为实验动物,探讨NP对中国林蛙肝细胞中雌激素受体(ER)和卵黄蛋白原(Vtg)表达的影响,从而深入理解NP的雌激素效应,以及揭示酚类环境化学污染物影响两栖类动物内分泌的机制,并为由NP污染所造成的两栖类动物繁殖能力下降提供一定的理论依据。本实验将雄性中国林蛙暴露于10-5、10-6、10-7M NP水体中分别持续10d、20d、30d,以10-8、10-9M 17β-雌二醇(17p-estradiol, E2)作为阳性对照；取其肝脏组织,用免疫组织化学方法检测ER和Vtg蛋白的表达；用原位杂交方法检测ER mRNA的表达；以阳性反应产物的光密度作为表达相对值。实验结果及结论如下：1、用免疫组织化学方法检测NP处理后中国林蛙肝细胞中Vtg的表达,Vtg阳性反应主要在细胞质中呈棕黄或黄色。Vtg表达相对值检测结果表明,除10d时10-7M NP处理组的Vtg表达差异不显著(P>0.05),其余各NP处理组中Vtg表达相对值均高于对照组,且差异显著(P<0.05)。在10-5、10-6M NP处理组中,10d时差异显著(P<0.05),在20d和30d时,Vtg的表达量上升,差异极显著(P<0.01),即表达量随NP处理时间的延长而上调。在相同处理时间,随各NP处理组浓度的升高Vtg表达相对值升高(P<0.05),即Vtg的表达量随NP处理浓度的升高而上调。同时,NP处理组的Vtg表达量低于E2对照组,表明Vtg的表达与NP间有剂量-时间效应关系,这与E2处理组的效应相似。2、用免疫组织化学方法检测肝细胞的ER,在各NP处理组和E2处理组中均有ER表达,ER阳性反应在胞质和胞核中呈棕黄或黄色。ER表达相对值检测结果表明,各NP处理组中肝细胞中ER表达相对值均高于空白对照组,即NP处理组的ER表达量上调；同时NP处理组的ER表达量低于E2对照组。在10-5、10-6和10-7 M NP处理组中,随着处理时间的延长,ER表达相对值显著升高(P<0.05),在30d时极显著(P<0.01),即ER表达量随NP处理时间的延长而上调。在相同处理时间,随各NP处理组浓度的升高ER表达相对值升高(P<0.05),即ER的表达量随NP处理浓度的升高而上调；这与E2处理组中,ER表达的变化趋势相似。3、原位杂交检测肝细胞中ER mRNA的表达,在NP和E2处理组的肝细胞中均存在ER mRNA阳性反应,阳性反应在胞质呈蓝色。ER mRNA表达相对值检测结果表明,各NP处理组中肝细胞中ER mRNA表达量上调,差异显著；同时ERmRNA表达相对值低于E2对照组。在10-5、10-6和10-7MNP处理组中,随着处理时间的延长,ER mRNA表达相对值显著升高(P<0.05),在20d和30d时极显著(P<0.01),即ER mRNA表达量随NP处理时间的延长而上调。在相同处理时间,随各NP处理组浓度的升高ER表达相对值升高(P<0.05),即ER mRNA的表达量随NP处理浓度的升高而上调。上述可见,在NP和E2处理后,肝细胞中ER表达量的变化与ER mRNA基本一致,均呈高调趋势,说明ER mRNA的表达过程伴随着ER的合成。4、从NP处理后肝细胞中ER和Vtg之间的变化趋势分析,NP能诱导ER高调,Vtg的表达量增加,二者的变化趋势也基本一致。说明ER的合成过程伴随着Vtg的生成,Vtg的表达依赖于ER的水平,NP通过高调ER诱导Vtg的合成。5、本实验中E2作为阳性对照,在相同处理时间,NP与E2处理导致ER、Vtg的变化趋势基本一致,说明NP产生的效应与E2相似。但是,NP的最低处理浓度比E2的最高处理浓度高一个数量级,表明NP的雌激素效应低于E2,有较弱的雌激素效应。上述结果表明,NP可通过高调肝细胞中ER使Vtg的合成增加,从而发挥较弱的雌激素效应。更多还原

【Abstract】 With the rapid development of modern industry, a large number of chemical substances used in the environment have caused serious pollution to the survival of wild animals. Among them, phenolic environmental pollutants as endocrine disruptors have seriously affected the growth of animals and caused animal populations decline. Nonylphenol(Nonylphenol,NP) is one of endocrine disrupting chemicals, which has similar structure to animal estrogen, with weak estrogenic effects. In this experiment, male Rana chensinensisas as the experimental animals were used to investigate effects of NP on hepatic cells of estrogen receptor (ER) and vitellogenin (Vtg) expression, which can deeply reveal the estrogenic effects of NP and the impact of phenolic environmental chemical pollutants on amphibian endocrine system, so as to provide a theoretical basis.at amphibian decline in reproductive capacity caused by NP pollution.In this study, male Rana chensinensisas were treated in 10-5,10-6,10-7 M NP water for 10d,20d,30d,10-8,10-9 M 17(3-estradiol (17β-estradiol, E2) as a positive control; then liver tissue was got from R.chensinensis at certain days. Hepatic cells are detected the expression of protein of ER and Vtg by immunohistochemical technique, and the expression of ER mRNA by in situ hybridization technique.The results and conclusions of experiment as follows:1. The results of immunohistochemical technique of Vtg show that the positive effect of Vtg is brown or yellow in the cytoplasm. The results of the relative intensity of Vtg show that in addition to the 10-(?)M NP treatment group in lOd was not significant (P>0.05), the remaining NP treatment groups were significantly higher(P<0.05). the 10-5、10-6 M NP treatment groups were significant different (P＜0.05), in the 20d and 30d, Vtg expression increase was significantly different (P＜0.01). The expression of Vtg in NP treated group is less than E2 treated group. The expression of Vtg and NP is the dose-time-response relationship, which is similar to the effects of E2 treatment groups.2. The results of immunohistochemical technique of ER show that the positive effect of ER is brown or yellow in the nuclear and cytoplasm. The results of the relative intensity of ER show that the expression of ER increased significantly in hepatic cells of R.chensinensis in NP treated groups compared with the control group. And the expression of ER in NP treated group is significantly lower than the E2 treated group. The 10-5、10-6 and 10-7M NP treatment groups were significant different(P<0.05). in the 30d, ER expression increase was significantly different(P<0.01).In NP treated groups, the expression of ER is increased with the increasing concentration of NP or with the increasing treated time. In E2 treated group, the expression of ER is changed in a similar trend.3.The results of in situ hybridization detection of ER mRNA show that the positive effect of ER mRNA is blue in the cytoplasm in NP treated groups and E2 treated group. The 10-5、10-6 and 10-7M NP treatment groups were significant different(P<0.05), in the 20d and 30d, ER mRNA expression increase was significantly different(P<0.01).In each NP group, the expression of ER mRNA is significant increased with the increasing treated time and the increasing treated concentration. These can be seen the change of ER mRNA expression in hepatic cells is consistent with ER mRNA with a high-profile trend in the NP and E2 treatment, which was indicated that the synthesis process of ER was along with ER mRNA expression.4. The relationship between ER and Vtg in hepatic cells of R. chensinensis treated by NP shown that NP can induce the increasing expression of ER and Vtg and the change trend is the same. It’s illuminated that the expression of ER along with the synthesis of Vtg, and NP induces the synthesis of Vtg protein through with the effect way of ER.5. This experiment use E2 as positive control. NP and E2 have the similar effect. But the lowest concentration of NP is higher than the highest concentration of E2, which indicated that the estrogenic effect of NP is lower than the effects of E2, the results showed that NP can play weak estrogenic effects.The results showed that NP can affect the expression of ER and Vtg in hepatic cells of R. chensinensis, NP can induce the synthesis of Vtg though ER and plays weak estrogenic effects.更多还原