双酚A对中国林蛙（Rana chensinensis）生精细胞凋亡及Bax，Bcl-2表达的影响

【作者】 林胜男；

【导师】 张育辉；

【作者基本信息】 陕西师范大学 ， 发育生物学， 2008， 硕士

【摘要】 近年来,随着工农业的快速发展,大量危害环境和人类健康的化学品被不断地释放到自然环境中。越来越多的证据证实,环境中许多化学污染物可以干扰野生动物和人类的正常生理活动。研究表明,双酚A（bisphenol-A,BPA）作为环境雌激素可影响两栖动物的生长发育及繁殖,同时,酚类诱导其他动物细胞凋亡的研究也不乏报道,但有关BPA对两栖类生殖细胞凋亡的研究尚未见报道。本实验以中国林蛙（Rana chensinensis）为研究对象,探讨BPA对中国林蛙生精细胞凋亡指数（Apoptotic index,AI）及Bax、Bcl-2表达的影响,旨在揭示酚类环境化学污染物危及两栖类生殖活动的机制,为防治由于BPA污染所造成的两栖类繁殖能力下降提供理论依据。本实验以精原细胞增殖期和精母细胞成熟分裂期雄性中国林蛙为实验动物,将精原细胞增殖期雄性中国林蛙暴露于10^-7、10^-6、10^-5mol/L BPA水体中持续3d、5d、7d:将精母细胞成熟分裂期雄性中国林蛙暴露于10^-7、10^-6、10^-5mol/L BPA水体中持续4d、14d、28d。取其精巢组织,用原位末端转移酶法（TUNEL）和甲基绿-派诺宁法（Methyl Green-Pyronine）检测生精细胞凋亡,用免疫组织化学方法检测生精细胞的Bax和Bcl-2表达。实验结果及结论如下:1.TUNEL和Methyl Green-Pyronine检测结果显示,经过BPA处理后,在中国林蛙的精巢中,无论是在精原细胞增殖期还是在精母细胞成熟分裂期,均可见生精细胞染色质固缩,部分细胞散落于管腔中部,一些受损细胞在接近管腔部位聚集成团,处于即将脱落状态。表明BPA可以导致中国林蛙生精细胞发生凋亡。在精母细胞成熟分裂期,各BPA组处N14d后,生精小管中可见不规则坏死灶。表明BPA可使精母细胞呈现坏死效应。2.统计中国林蛙生精细胞AI结果显示,在精原细胞增殖期,用10^-7mol/LBPA处理3d到7d,生精细胞AI变化不显著,提示用10^-7mol/LBPA从3d处理到7d,由BPA所诱发的生精细胞凋亡程度相似:而在10^-6mol/L,10^-5mol/L处理组中,生精细胞AI随着BPA处理时间的延长呈现增高趋势;在处理时间相同时,生精细胞AI随着BPA处理浓度增高呈现增大趋势。提示BPA在中国林蛙精巢内存有累积效应,生精细胞AI与BPA累积浓度相关,生精细胞AI与BPA之间存在显著剂量-效应及时间-效应关系。在精母细胞成熟分裂期,用10^-7mol/L处理4d后,生精细胞AI与对照组无显著性差异。表明在较低BPA浓度的水体中,由于BPA的降解或机体的自身调节可使BPA效应减轻,受损细胞被及时清除导致AI未显著增大。在10^-5mol/L组,处理28d后,生精细胞AI有所下降,坏死细胞增多。表明机体在启动细胞凋亡途径清除受损细胞的同时,细胞坏死的发生使生精细胞AI下降。3.比较分析两个不同时期生精细胞AI及其相对应的BPA剂量,结果显示,在精原细胞增殖期,用浓度为10^-6mol/L BPA处理7d后,生精细胞AI为8.24±1.03%,而在精母细胞成熟分裂期,用10^-6mol/L BPA处理4d后,生精细胞AI可高达12.96±1.66%。表明在相对较低累积剂量BPA的作用下,精母细胞成熟分裂期时的生精细胞AI比精原细胞增殖期的生精细胞AI值高。提示不同时期生精细胞对BPA诱导凋亡作用的敏感性不同,精母细胞成熟分裂期的生精细胞较敏感。4.用免疫组织化学法检测中国林蛙生精细胞中凋亡蛋白Bax和Bcl-2的表达,结果显示,在精原细胞增殖期和精母细胞成熟分裂期的10^-7mol/L,10^-6mol/LBPA处理组中,在相同的处理时间下,随着BPA处理浓度的增高,Bax表达逐渐上调,Bcl-2表达逐渐下调:在同一BPA处理组中,随着BPA处理时间的延长,Bax表达逐渐上调,Bcl-2表达逐渐下调。表明生精细胞中Bax和Bcl-2表达量与BPA的累积剂量有关,蛋白表达量与BPA之间具有显著的剂量-效应和时间-效应关系。5.在精原细胞增殖期,用10^-5mol/L BPA处理7d后,生精细胞中Bax表达量最大;在精母细胞成熟分裂期,用10^-5mol/L BPA处理4d后,生精细胞中Bax表达量达到最大,以后维持恒定水平。表明如在相同剂量的BPA作用下,精母细胞更易调节Bax的表达。在精原细胞增殖期,用10^-5mol/LBPA处理5d后,Bcl-2表达量最低,以后维持恒定水平;而在精母细胞成熟分裂期,用10^-5mol/L BPA处理14d后,Bcl-2表达量最低。表明精原细胞更易调节Bcl-2的表达。6.通过分析各BPA处理组中生精细胞AI与其相对应的Bax/Bcl-2表达量比值的线性回归关系,可知无论在精原细胞成熟期还是在精母细胞成熟分裂期的中国林蛙精巢中,生精细胞AI与Bax/Bcl-2表达量比值之间具有显著的线性正相关关系。提示在BPA作用后,机体通过上调Bax和下调Bcl-2来调节二者的相对含量,从而促进细胞凋亡的发生。表明Bax与Bcl-2相对含量是决定生精细胞是否发生凋亡的关键因素,BPA诱导的生精细胞的凋亡与Bax/Bcl-2通路有关。更多还原

【Abstract】 At present,alkylphenol polyethoxylates（APEs） as industrial material are used broadly in the world.Therefore it is very easy for the APEs to enter organism,disturb the normal hormone effects and have profound and all-pervading influence on the creatural reproduction.Recently,some reports shows that Bisphenol A has caused serious effects on the growth development and propagation of amphibian;it is the one of the main reasons which induces the number of amphibian.Most reports about the effects of BPA on amphibian focus on its estrogenic effects,the report about whether it can induce the apoptosis in spermatogenetic cells of R.chensinensis hasn’t been studied.So we take R.chensinensis as experimental animal to investigate the effects of BPA on the apoptosis index and the expression of Bax,Bcl-2 in spermatogenetic cells of R.chensinensis.The aim of this experiment is to reveal the mechanism of environmental chemical pollutants endanger the propagation activity of animal,whether this mechanism is implemented though Bax/bcl-2 pathway. Provide theoretical basis for protecting decreasing propagation ability caused by BPA.We take the R.chensinensis in spermatogonia proliferation stages and in spermatocyte maturation division stage as experiment animals.R.chensinensis in spermatogonia proliferation stages had been treated in 10^-7,10^-6,10^-5mol/L BPA and control group for 3d,5d,7d,R.chensinensis in spermatocyte maturation division stage had been treated in 10^-7,10^-6,10^-5mol/L BPA and control group for 4d,14d,28d.Then testis was got from R.chensinensis at certain days.Apoptosis cells are tested by TUNEL and Methyl Green-Pyronine technique.The relative intensity of Bax and Bcl-2 are tested by immunohistochemical technique.The results and the conclusions are listed as follows:1.Detecting apoptosis cells in spermatogenetic cells of R.chensinensis.by TUNEL and Methyl Green-Pyronine technique.In both spermatogonia proliferation stages and spermatocyte maturation division stage,Spermatogenetic cells have failed into the middle of lumen with cell chromatin pyknosis.Some damaged cells which gather into cluster in soon abscisic status close to the middle of lumen.It shows that BPA induce the Spermatogenetic cells apoptosis.In spermatocyte maturation division stage,some irregular necrosis focus in the lumen can be seen in every BPA treatment group which has been treated for 14d.It shows that BPA induce the cell necrosis of spermatogenetic cells.2.Detecting the expression of Bax,Bcl-2 in spermatogenetic cells of R.chensinensis by immunohistochemical technique,in spermatogonia proliferation stages,apoptotic index in 10^-7mol/LBPA treatment group from 3d to 7d change little,but apoptotic index is increased with concentration and time in 10^-6mol/LBPA and 10^-5mol/L BPA groups.It shows that BPA has accumulation effect in the R.chensinensis testis,apoptotic index of spermatogenetic cells have relation to the BPA accumulate concentration.There exist significant dose-effect relationship between BPA and AI.In spermatocyte maturation division stage,AI of 10^-7mol/L BPA treated for 4d is similar with control group.It shows that because the low concentration of BPA and organism’s regulating ability,the effects of BPA has been reduced,so the AI of damage cells hasn’t increased obviously.In 10^-5mol/L treated for 28d,although the AI is higher than it in control group,but lower in 14d BPA treatment group.It shows that organism not only start the apopotosis pathway but also the cell necrosis pathway to exclude the damage cells.So the AI is decreasing.3.Comparing the AI and its relative BPA concentration in these two stages,in spermatogonia proliferation stages,AI of spermatogenetic cells in 10^-6mol/L treated for 7d is 8.24±1.03%,while in spermatocyte maturation division stage,AI reach to 12.96±1.66%when treated with 10^-6 mol/L BPA for 4d.It shows that compared to spermatogonia,spermatocyte’s AI is higher in relative low BPA accumulate concentration.It suggested that sensitivity of the two kinds of cells is different. Spermatocyte is much more sensitive.4.Detecting the expression of Bax,Bcl-2 by immunohistochemical technique,In spermatogonia proliferation stages and in spermatocyte maturation division stage,the results shows that,the expression of Bax is increasing with concentration and time while Bcl-2 expression is decreasing with the concentration and time in 10^-7 mol/L,10^-6 mol/L BPA.It shows that the expression of Bax has relationship with BPA concentration;the number of protein expression has significant relation with time and concentration.5.In spermatogonia proliferation stages,the results suggested that the expression of Bax is the biggest in 10^-5 mol/LBPA treated for 7d,while Bax is the highest in10^-5 mol/LBPA after treated for 4d in spermatocyte maturation division stage.In spermatogonia proliferation stages the expression of Bcl-2 is the lowest in 10^-5 mol/L treated for 5d while the lowest expression of Bcl-2 after treated for 14d in spermatocyte maturation division stage.It suggests that the spermatocyte is easy to regulate the expression of Bax in relative low BPA concentration,while the spermatogonia are easy to regulate the expression of Bcl-2.6.Analyzing the linear regression relation of AI and the ratio of Bax/Bcl-2 expression,it suggests that in these two stages,significant linear correlation exist between AI and the ratio of Bax/Bcl-2 expression.It suggests that the relative expression of Bax and Bcl-2 is the key factor which determinates whether cell apoptosis happens.After treated with BPA,organism can increase the expression and decrease the expression of Bcl-2 to regulate the cell apoptosis.It shows that cell apoptosis induced by BPA is though Bax/Bcl-2 pathway.更多还原