【作者】 李俊；

【导师】 翟金霞；

【作者基本信息】 安徽医科大学 ， 劳动卫生与环境卫生学， 2017， 硕士

【摘要】 目的:十溴联苯醚(decabromodiphenyl ether,BDE-209)作为一种重要的环境内分泌干扰物(environmental endocrine disruptors,EEDs)被广泛用做阻燃剂添加于电器和生活用品中,是一种能够干扰体内激素的合成、分泌、运输、结合、作用、代谢的物质,具有神经、生殖、甲状腺和免疫毒性。因有研究表明多溴联苯醚的浓度与新生儿的体重呈负相关,减轻附睾重量,其表现出生命早期毒性作用,同时雌激素受体(estrogen receptor,ER)有调控睾丸支持细胞增殖的作用,故为研究睾丸雌激素受体α(estrogen receptorα,ERα)在围生期BDE-209暴露对F1雄性小鼠血睾屏障的破坏作用,使用ICR小鼠探讨BDE-209对睾丸ERα、Formin1、F-actin,血睾屏障(blood-testis barrier,BTB)相关蛋白(claudin-11,occludin,ZO-1)表达以及BTB结构破坏的影响;此外,使用BDE-209以及ERα的特异性抑制剂MPP(Methyl-Piperidino-Pyrazole)共同作用于SerW3细胞,检测ERα,Formin1,F-actin和BTB相关蛋白表达水平影响,阐明BDE-209通过上调睾丸ERα致BTB破坏及其分子机制。方法:动物实验中,取6周龄ICR雄鼠24只,雌鼠48只适应性饲养5天后,合笼取孕鼠,当天记为孕0天(GD0),按随机数字表法将小鼠分到对照组(玉米油100mg/kg BW组),BDE-209 100 mg/kg BW组,BDE-209 300mg/kg BW组,BDE-209 500 mg/kg BW组。对妊娠(GD)7天后的F0母鼠进行经口灌胃BDE-209处理,每天清晨灌胃处理一次,连续灌胃至产后(PND)21天。每天清晨对F0孕鼠称重,纪录F0孕鼠致F1小鼠出生后21天F0孕鼠体重;记录PND1 F1小鼠窝仔数,以及PND1至PND35 F1小鼠体重。于PND35和PND105选取F1雄鼠,摘取小鼠眼球保留血样,待取血结束后选择颈椎脱臼处死,于冰浴上分离各脏器,将需制作组织形态学切片脏器按照相应条件储存,其余脏器立即放于液氮罐中速冻储存,并及时转移致-80℃冰箱储存,当需要对生物样品进行检测时取出,且禁止反复冻融。采用实时定量聚合酶链反应实验法,免疫蛋白印迹法,免疫荧光化学发光法以及免疫组织化学法检测小鼠睾丸erαmrna和蛋白表达改变,血睾屏障相关蛋白如claudin-11,occludin,zo-1mrna与蛋白表达改变,以及formin1和f-actin蛋白改变,。选择serw3细胞进行细胞实验,采用mtt方法检测24h不同浓度bde-209(0μg/ml、10μg/ml、50μg/ml、100μg/ml和150μg/ml)和mpp作用后的细胞生存率,确定bde-209染毒剂量。当复苏或传代细胞生长状态稳定后,待细胞覆盖培养皿80%面积后加入bde-209与mpp,此时细胞密度约为1×108,在bde-209与mpp作用细胞24h后观察细胞形态学变化,免疫蛋白印迹法和免疫荧光方法检测erα,formin1,f-actin,血睾屏障相关蛋白(claudin-11,occludin,zo-1)表达变化。结果:(1)围生期bde-209暴露致f1雄性小鼠体重与脏器系数改变:各bde-209剂量组与对照组相比,不同时期体重均出现了不同程度下降(p<0.05);f1雄性小鼠生殖系统的各脏器系数表现为:pnd35时期,附睾的100mg/kgbw组与对照组相比,脏器系数上升,且差异有统计学意义(p<0.05),pnd105时期睾丸:各bde-209组与对照组相比脏器系数下降,且差异有统计学意义(p<0.05);(2)围生期bde-209暴露上调f1雄性小鼠睾丸erαmrna,erα蛋白且mpp抑制bde-209上调serw3细胞erα蛋白表达:与对照组相比,pnd35时期的500mg/kgbw组的erαmrna表达上调(p<0.05);而pnd105时期f1小鼠的300mg/kgbw组,500mg/kgbw组的erα表达上调(p<0.05);通过免疫组织化学法观察雄性f1小鼠睾丸组织中erα的表达水平,pnd35时期300mg/kgbw与500mg/kgbw组睾丸中erα表达明显增加;pnd105时期500mg/kgbw组睾丸中erα表达明显增加;细胞实验中serw3细胞的bde-209组的erα蛋白的表达量明显高于对照组(p<0.05);(3)围生期bde-209暴露下调f1雄性小鼠睾丸formin1且mpp抑制bde-209下调serw3细胞formin1表达:pnd35时期300mg/kgbw组,500mg/kgbw组与对照组相比,formin1表达下调(p<0.05);而pnd105时期小鼠的所有bde-209组的formin1的表达明显低于对照组(p<0.05);细胞实验中serw3细胞的bde-209组的Formin1的表达明显低于对照组(P<0.05);(4)围生期BDE-209暴露下调F1雄性小鼠睾丸F-actin且MPP抑制BDE-209下调SerW3细胞F-actin表达:PND35时期的F-actin的表达变化为300mg/kg BW组,500mg/kg BW组与对照组相比,表达下调(P<0.05),细胞实验中SerW3细胞的各个BDE-209组的F-actin的表达明显低于对照组(P<0.05);(5)围生期BDE-209暴露下调F1雄性小鼠睾丸BTB相关蛋白的mRNA与蛋白表达且MPP抑制BDE-209下调SerW3细胞BTB相关蛋白表达:PND35与PND105两个阶段的claudin-11 mRNA:300mg/kg BW组,500mg/kg BW组表达明显低于对照组(P<0.05),而ZO-1与occludin分子的mRNA的表达为只有PND35时期各BDE-209组的表达明显低于对照组(P<0.05);PND35时期的claudin-11:500mg/kg组相对于对照组表达下降(P<0.05),而在PND105时期,300mg/kg BW组,500mg/kg BW剂量组表达低于对照组(P<0.05);PND35与PND105时期ZO-1 300mg/kg BW组,500mg/kg BW组表达量均显著低于对照组(P<0.05);occludinPND105时期BDE-209的300mg/kg BW组,500mg/kg BW组表达明显低于对照组(P<0.05);细胞实验中BDE-209组的claudin-11,occludin的表达量与对照组相比,明显较低(P<0.05),其余BDE-209组claudin-11,occludin的表达量相对于对照组差异无统计学意义,而ZO-1表达量在BDE-209组与对照组间无差异。(6)围生期BDE-209暴露致F1雄性小鼠组织形态学指标:HE切片发现两个时期小鼠的睾丸组织的曲系精管出现不同程度损伤以及精子数目的改变,超微电镜中各剂量组中紧密连接结构出现了不同程度的破坏,Sertoli细胞内细胞器也出现不同程度改变。结论:十溴联苯醚可以引起睾丸组织以及睾丸支持细胞SerW3细胞系中ERα表达上升,Formin1,F-actin以及血睾屏障紧密连接相关蛋白表达下调,并且引起睾丸组织中支持细胞紧密连接破坏,提示BDE-209通过上调ERα表达破坏小鼠血睾屏障。更多还原

【Abstract】 Objective:As an important environmental endocrine disruptors(EEDs),Decadrominated diphenyl ethers(BDE-209)is widely used in electronic devices,upholstery and carpets.BDE-209 has neurotoxicity and reproductive toxicity that interferes with the the synthesis,secretion,transport,binding,action,metabolism and elimination of hormones in vivo.Studies have shown that PBDEs concentration was negatively correlated with the newborn,reducing the weight of the epididymis,which exhibits early life toxicity.To explore the effect of testis estrogen receptor α(ERα)exposure to BDE-209 on the blood testis of F1 male mice,ICR mice were used to investigate the effect of BDE-209 on the expression of testis ERα,Formin1,F-actin,blood-testis barrier(BTB)-related protein(claudin-11,occludin,ZO-1)and the disruption of BTB structure.In addition,SerW3 cell was co-treated with BDE-209 and Methyl-Piperidino-Pyrazole(MPP),the expression of ERα,Formin1 and other BTB related proteins were detected using Westerblot.This study provides a scientific basis for elucidating that BDE-209 exposure to disrupt blood-testis barrier of mice via upgraduated ERα signal pathway.Methods:In animal experiment,72 six-week-old mice(24 male and 48 female)was selected which feeded for 5 days adaptively.The mice were randomly divided into the control group(corn oil 100 mg / kg BW),BDE-209 100 mg / kg BW group,BDE-209 300 mg / kg BW group,BDE-209 500 mg / kg BW group.Pregnancy(GD)7 days after the F0 rats were orally administered BDE-209,treated once every morning,continuous feeding to postpartum(PND)21 days.Weighed and recorded the weight of rats(F0-PND21)every morning and the number of PND1 F1.In the PND35 and PND105 selected F1 male rats,remove the mouse eye to keep the blood sample,then cervical dislocation,separated organs in the ice bath,then stored organs in the corresponding conditions.The rest of the organs immediately placed in the liquid nitrogen tank frozen storage,and timely transfer to-80℃ refrigerator storage,when the need for biological samples to be detected,and prohibit repeated freezing and thawing.Real-time quantitative polymerase chain reaction(RT-PCR),immunoblotting,immunofluorescence chemiluminescence and immunohistochemistry were used to detected the expression of ERα mRNA and protein,BTB related proteins and Formin1 and F-actin proteins.In vitro,SerW3 cell was o-treated with different concentrations of BDE-209(0μg / ml,10μg / ml,50μg / ml,100μg / ml and 150μg / ml)and MPP.When the cell growth stable,the cell density was about 1 × 108.Cell viability was determined by MTT assay.The expressions of ERα,Formin1,F-actin and claudin-11,occludin and ZO-1 were detected by immunoblot and immunofluorescence.Results:(1)Peritoneal BDE-209 exposuredecreased F1 male mice body weight and changed organ coefficient: Compared with the control group,each BDE-209 group exist difference of the mean body weight of PND1-PND35 F1 mice(P<0.05).In the PND35 mice,the epididymis organs coefficient in the 100mg/kg BW group was lower than that in the other groups(P<0.05),in the PND105 mice,the testis coefficient in control group was higher than other BDE-209 groups(P<0.05).(2)Peritoneal BDE-209 exposure upregulated the expression of ERα mRNA and ERα in F1 male mice testis and MPP inhibited BDE-209 upregulated ERα of Ser W3 cells: In PND35,the expression of ERα mRNA in the 500mg/kg BW group was significantly higher than that in the control group(P<0.05).In PND105,the expression of ERα in the 300mg/kg BW group and the 500mg/kg BW group was significantly higher than that in the control group(P<0.05).The expression of ERα in the testes of F1 mice was observed by immunohistochemical method.The expression of ERα in the testes of PND35 was shown higher in 300mg/kg BW and 500mg/kg BW,in PND105,the expression of ERα was significantly increased in the 500 mg/kg BW group.The expression of ERα in BDE-209 group was significantly higher than that in control group.In cell experiments: the expression of ERα protein in SerW3 cells was significantly higher than that in control group(P<0.05).(3)Peritoneal BDE-209 exposure downregulated the expression of Formin1 in F1 male mice testis and MPP inhibited BDE-209 downregulated Formin1 of Ser W3 cells: In PND35,the expression of Formin1 in 300mg/kg BW and 500mg/kg BW group were significantly lower than that in control group(P<0.05),the expression of Formin1 in all BDE-209 group was significantly lower than that in control group(P<0.05)in PND105;In cell experiments: the expression of Formin1 in BDE-209 group of SerW3 cells was significantly lower than that in the control group(P<0.05).(4)Peritoneal BDE-209 exposure downregulated the expression of F-actin in F1 male mice testis and MPP inhibited BDE-209 downregulated F-actin of SerW3 cells: The expression of F-actin in the PND35,compare with control group,300mg/kg BW group,500 mg / kg BW group decreased(P<0.05),In cell experiments,the expression of F-actin in each BDE-209 group of SerW3 cells was significantly lower than that of the control group(P<0.05).(5)Peritoneal BDE-209 exposure downregulated the expression of BTB-related mRNA and proteins in F1 male mice testis and MPP inhibited BDE-209 downregulated BTB-related proteins of Ser W3 cells: In PND35 and PND105,The expression of claudin-11 mRNA in the 300 mg/kg BW and 500 mg / kg BW group were significantly lower than that in the control group(P<0.05).The BDE-209 groups expressed ZO-1 and occuldin mRNA were significantly lower than that in the control group(P<0.05).In PND35,the expression of claudin-11 in the testes showed different changes between 500mg/kg BW group and control group.In PND105,the 300mg/kg BW and 500mg/kg BW group expression of claudin-11 decreased compare with control group(P<0.05).In PND35 and PND105,the expression of ZO-1 in the 300 mg / kg BW group and the 500mg/kg BW group was lower than that in the control group(P<0.05).The expression of BTB-related protein in testis was observed by immunofluorescence.The expression of claudin-11 protein in PND35 and PND105 was also decreased in the 500mg/kg BW group,and the expression of claudin-11 in PND35 was obvious.The decrease of occludin PND35 and PND105 expression in each dose group showed a decrease in expression.ZO-1 showed only a decrease in the expression of ZO-1 during PND35,while the expression of ZO-1 in PND105 was lower and the expression of ZO-1 was not obvious.In cell experiments,the expression of claudin-11 protein and occludin protein in each BDE-209 group of SerW3 cells was lower than that of control(P<0.05).but the expression of ZO-1 protein in BDE-209 group showed no change compared with the control group.(6)Perinatal BDE-209 exposure induced F1 male mice histomorphological indicators: HE slices found two periods of mouse testicular tissue of the seminiferous tubules appear sperm damage and number of different degree of change,Microscopic electron microscopy in the dose group in the close connection structure of varying degrees of destruction,Sertoli cells within the organelles also varying degrees of change.Conclusion:BDE-209 can increase the expression of ERα in testis and SerW3 cells,and decrease the expression of Formin1,F-actin and BTB-related proteins,and damage the tight connection of the supportingcells in testicular tissue,suggesting that BDE-209 disrupts blood-testis barrier of mice via upgraduated ERα signal pathway.更多还原