【作者】 苗颂；

【导师】 徐贵发； 高志贤；

【作者基本信息】 山东大学 ， 流行病与卫生统计学， 2008， 博士

【摘要】 目的1.通过对F₀代大鼠的围生期双酚A染毒,观察围生期双酚A暴露对F₀、F₁两代大鼠血清IgG（immune globulin G）、IgM（immune globulin M）的影响。2.通过对F₀代大鼠的围生期双酚A染毒,观察围生期双酚A暴露对F₀、F₁两代大鼠雌激素受体-α（estrogen receptor-α,ER-α）相对含量的影响。3.通过对F₀代大鼠的围生期双酚A染毒,观察围生期双酚A暴露对F₀、F₁两代大鼠脾脏白介素-2（interleukin-2,IL-2）、白介素-12（interleukin-12,IL-12）、肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、γ-干扰素（interferon-γ,IFN-γ）和胸腺Fas、FasL这6种细胞因子mRNA表达的改变。为进一步探讨环境内分泌干扰物（environmental endocrine disruptors,EEDs）对机体免疫功能的影响提供依据。方法1.动物染毒健康F344大鼠,雌雄各20只,4周龄,适应喂养一周后,按体重随机分为4组,每组10只,雌雄各半。将双酚A溶于玉米油中,配成不同浓度的溶液,按低剂量组（4 mg/kg·d）,中剂量组（40mg/kg·d）,高剂量组（400 mg/kg·d）,从第五周开始,每早8～9点对动物进行灌胃,对照组给予同体积的玉米油。大鼠进入青春期后（9周龄）,将同剂量组大鼠按雌雄1:1比例合笼,雌鼠受孕后,单独饲养,直至子鼠出生后30天,子鼠由母鼠哺乳喂养。2.体重和一般情况观察每周称2次体重,密切观察各组动物的一般状况变化,如被毛、粪便、行为、精神状态等。3.组织病理学观察取胸腺、脾脏、肝脏和肾脏,称重后各剪取一部分,固定于10%的中性福尔马林中。苏木精、伊红染色（HE染色）,石蜡包埋,制成5μm厚的片子,显微镜下观察并拍照,比较各组间的差别。4.血清抗体测定取全血,离心分离血清,采用酶联免疫间接法测定大鼠血清IgG、IgM的相对含量。5.Western杂交分别取出各处理组大鼠的脾脏和胸腺组织,匀浆,离心,用SDS-PAGE和Western Blot方法分别分析脾脏和胸腺组织中雌激素受体α（estrogen receptorα,ER-α）的相对含量变化。6.实时定量PCR取大鼠脾脏和胸腺组织,提取RNA后,检验RNA的浓度和完整性,以GAPDH为内参基因,进行逆转录和PCR反应后,得到IL-2,IL-12,IFN-γ,TNF-α以及Fas,Fasl基因的相对表达量。结果1.F₀代体重、脏器指数变化情况大鼠体重持续增长,但与对照组相比,F₀代低剂量组的体重略有增加但无显著统计学意义;中剂量组的体重无明显改变;高剂量组大鼠的体重却显著降低（P＜0.05）,其它指标无明显改变（P＞0.05）。2.F₁代体重、脏器指数变化情况与对照组相比,低剂量组子鼠的体重虽无显著变化,脾脏重量和脾脏指数（脾脏与体重之比）却显著增加（P＜0.05）;中剂量组子鼠的体重和脾脏重量均无显著变化,脾脏指数显著增加（P＜0.05）;高剂量组子鼠的体重和脾脏重量显著降低（P＜0.05）,脾脏指数无显著变化（P＞0.05）。3.F₁代性别比例的变化情况各组雌性仔鼠的比例较大,但与对照组相比,其他各剂量组仔鼠数及性别比例无显著变化（P＞0.05）。4.组织病理学改变（1）F₀代组织病理学改变肝脏各给药组与对照组的部分动物肝细胞浊肿变性,肝细胞疏松化,以高剂量组略明显,未见坏死。肾脏与对照组相比,高剂量组部分动物出现肾小球萎缩。脾脏高剂量组部分动物白髓减少,脾小体缩小。胸腺无明显病理改变。（2）F₁代组织病理学改变各给药组与对照组相比,高剂量组部分动物肝细胞浊肿变性,肝细胞疏松化,但未见坏死。肾脏与对照组相比,高剂量组部分动物出现肾小球萎缩。脾脏、胸腺无明显病理改变。5.血清IgG、IgM的改变（1）F₀代血清IgG、IgM的改变与对照组相比,高剂量组雌性大鼠的血清IgG、IgM含量明显减少（P＜0.05）,而低剂量组和中剂量组均无明显变化（P＞0.05）;雄性大鼠也出现了相同的变化。（2）F₁代血清IgG、IgM的改变各剂量组雌性大鼠血清IgG、IgM均无明显改变（P＞0.05）;雄性大鼠高剂量组血清IgG、IgM明显降低（P＜0.05）,低剂量组和中剂量组无显著变化（P＞0.05）。6.雌激素受体α（estrogen receptorα,ER-α）的变化（1）脾脏ER-α的改变F₀代,低、中剂量组雌性F344大鼠的ER-α表达量没有明显改变,但高剂量组ER-α的表达明显提高（P＜0.05）,而雄性大鼠恰恰相反,低、中剂量组无明显变化,高剂量组ER-α表达量降低（P＜0.05）。F₁代,低剂量组雌性F344大鼠的ER-α表达量没有明显改变,但中、高剂量组ER-α的表达明显提高（P＜0.05）,而雄性大鼠各剂量组ER-α均无明显变化。（2）胸腺ER-α的改变F₀代,低剂量组雌性F344大鼠的ER-α表达量没有明显改变,但中、高剂量组ER-α的表达明显提高（P＜0.05）,而雄性大鼠各剂量组ER-α均无明显变化。F₁代,低剂量组雌性F344大鼠的ER-α表达量没有明显改变,但中、高剂量组ER-α的表达明显提高（P＜0.05）,而雄性低剂量组大鼠ER-α无明显变化,中、高剂量组表达量明显降低（P＜0.05）。7.IL-2、IL-12、IFN-γ、TNF-α、Fas、FasL表达的改变（1）F₀代基因表达的变化与对照组相比,低、中、高剂量组脾脏IL-2、IL-12、IFN-γ、TNF-α的mRNA表达均显著降低（P＜0.05）,胸腺Fas表达下降（P＜0.05）,FasL表达在低剂量组出现一个短暂的下降后（P＜0.05）,在中、高剂量组明显上升（P＜0.05）。（2）F₁代基因表达的变化。与对照组相比,低、中、高剂量组脾脏IL-2、IL-12、IFN-γ、TNF-α的mRNA表达均显著降低（P＜0.05）,胸腺Fas表达下降（P＜0.05）,FasL表达在低剂量组明显下降（P＜0.05）,在中、高剂量组却明显上升（P＜0.05）。更多还原

【Abstract】 Object:1.To observe the influence of bisphenol A on the concentration of immune globulin G,immune globulin M,after the exposure of bisphenol A.2.To observe the influence of bisphenol A on the expression of estrogen receptor alpha when the rats of F₀ generation were administered bisphenol A.3 To observe the effect of bisphenol A on the expression of IL-2,IL-12,TNF-α, IFN-γin spleens and the expression of Fas,FasL in thymus,when the rats of F₀ generation were administered bisphenol A..This is important to supple for the research of environmental endocrine disruptors on the immune function.Methods:1.Treatment of ratsFisher 344 rats were four weeks old and were divided into control and 4,40 and 400 mg·kg^-1BPA groups.Rats were allowed to acclimatize for 1 week prior to the treatment.Bisphenol A was given orally to the three treatment groups daily at 4,40, 400 mg/kg respectively.Controls were treated with the same volume corn oil.When the rats were nine weeks old,vaginal smears were examined daily after female rats were placed with males.Sperm-positive smear was used to determine gestational day （GD0）.After positive detection,pregnant rats were placed individually.Twenty pregnant rats and twenty male rats were divided into four groups in random and the sex ratio of each group was 1:1.Treatments were prolonged for the offspring were thirty days old.Ten offspring were selected randomly from each group.2.General conditionsTo weigh the rats once a week and observe their general conditions such as clothing hair,excrement and urine,action and mental station et al.3.Histological parameters The rats were sacrificed by cervical dislocation after the last treatment.The surplus portions of all organs,were fixed in Bouin’s Solution and buffered formalin（4%）, dehydrated and processed for paraffin wax embedding.The sections were cut at 5um and stained with hematoxiylin and eosin（H&E）.The sections of the paraffin blocks were monitored and compared.4.Determination of antibodyTo observe the concentration of IgG、IgM by enzyme-linked immunosorbent assay（ELISA）,after the exposure of bisphenol A.5.Western BlottingMicrosomes from the spleens and thymus of rats treated with bisphenol A were prepared by differential centrifugation as described previously.The relative concentration of estrogen receptor alpha was evaluated by separated by SDS-polyacrylamide gel electrophoresis（SDS-PAGE,10%gel）and Western Blotting.6.Real- time RT-PCRTotal RNA was prepared from spleens and thymus using Total RNA Kit following the instruction.The relative quantity of IL-2,IL-12,IFN-γ,TNF-α,Fas,and Fasl were gained by Rea-time RT-PCR,and GAPDH was the housekeep gene.Result1.The change of weight and organ index in F₀ generationIn F₀ generation,one female rat died during the exposure at 400mg/kg level.Our results showed that rats exposed to 400mg/kg bisphenol A caused significant decrease in body weights（P＜0.05）.There were no significant changes in spleen weights and spleen index（P＞0.05）.2.The change of weight and organ index in F₁ generationIn F₁ generation,the terminal body weights and the spleen weights were significantly decreased in the high dose group（P＜0.05）.There were no weight changes in the low and middle dose groups,but the spleen index increased significantly.In the middle and high dose groups,not any significantly changes were observed in the liver weights,but the liver index increased greatly（P＜0.05）.Both thymus and thymus index had not any change contrasted to the control（P＞0.05）.3.The change of sex ratio in F₁ generationThere were more female rats than males in F₁ generation.But there were no significant changes in the number of offspring and the sex ratios contrast to the control（P＞0.05）.4.The change in Histology（1）The histological change in F₀ generationIn F₀ generation,white pulp of spleens reduced and splenic corpuscle contracted in 400mg/kg group.There was not any histology change in other groups.No abnormalities or treatment related changes were observed in any of the thymus sections of the treatment groups when compared with the control.In F₀ generations, not any histology change was observed in livers at low and middle dose.But in high dose,granular degeneration was visible in hepatocyte.It was observed that depauperation were occurred in acinus renis at 400mg/kg in F₀ generation.（2）The histological change in F₀ generationIn F₁ generations,not any histology change was observed in livers at low and middle dose.But in high dose,granular degeneration was visible in hepatocyte.It was observed that depauperation were occurred in acinus renis at 400mg/kg in F₁ generation.No abnormalities or treatment related changes were observed in any of the thymus and spleens sections of the treatment groups.5.The change of IgG、IgM（1）The change of IgG、IgM in F₀ generationCompare to the control,the concentration of IgG、IgM in high dose group increased significantly in both female and male rats（P＜0.05）,but no significant change was observed in low and middle dose groups（P＞0.05）.（2）The change of IgG、IgM in F₀ generationCompare to the control,there was no significant change in female rats in all treatment groups（P＞0.05）.Whereas,the concentration of IgG、IgM in high dose group of male decreased significantly in（P＜0.05）,but no significant change was observed in low and middle dose groups（P＞0.05）.6.The change of ER-α（1）The change of ER-αin spleenIn F₀ generation,the expression of ER-αreduced in male rats but increased in female rats in the high dose group（P＜0.05）.There were no any changes in the other groups.Similar findings were found in the middle and high dose groups in F₁ generation.（2）The change of ER-αin thymusIn F₀ generation,the expression of ER-αincreased in female rats in the middle and high dose group（P＜0.05）.There were no any changes in the low dose groups and male rats.In F₁ generation,the expression of ER-αincreased in female rats in the middle and high dose group（P＜0.05）,but reduced in male rats in the middle and high dose group.There were no any changes in the low dose groups in both female and male rats.7.The expression of IL-2、IL-12、IFN-γ、TNF-α、Fas、FasL（1）The change of gene in F₀ generationThe expression of IL-2 IL-12 IFN-γand TNF-αmRNA down-regulated greatly in all the treatment groups compared to the control group（P＜0.05）.The expression of Fas decrease in all treatment groups but the expression of FasL up-regulated in middle and high dose groups after it decrease in low dose group（P＜0.05）.（2）The change of gene in F₁ generationThe expression of IL-2 IL-12 IFN-γand TNF-αmRNA down-regulated greatly in all the treatment groups compared to the control group（P＜0.05）.The expression of Fas decrease in all treatment groups but the expression of FasL up-regulated in middle and high dose groups after it decrease in low dose group（P＜0.05）更多还原