【作者】 李春燕；

【导师】 逯晓波；

【作者基本信息】 中国医科大学 ， 卫生毒理学（专业学位）， 2021， 硕士

【摘要】 目的:近年来,在女性相关癌症死亡原因中,卵巢肿瘤已成为主要原因之一,其发病率处于不断上升状态。卵巢肿瘤的危险因素很多,包括家族及遗传史、生育及年龄和环境影响等。随着科技的发展和化工产品的大量应用,近年来,内分泌干扰物引起了人们的关注,它们对身体和环境的稳态具有一定的破坏作用和影响。目前,邻苯二甲酸酯类(PAEs)和双酚类(BPs)这两大类物质是备受关注的内分泌干扰物,其被广泛应用于塑料制品的增塑剂和主要制造原料,在环境及生物体内也都可以被检测到,成为危害较严重的环境污染物。由于环境污染物在实际生活中多以混合暴露形式存在,同时在人体接触途径形式多样,而且与各污染物单独暴露作用相比,此种混合暴露模式会呈现不同毒性作用,因此,对个别污染物的单独毒性作用的研究已不再能足够满足人类社会的需求。邻苯二甲酸二(2-乙基)己酯(DEHP)与双酚A(BPA)分别作为PAEs与BPs的代表性物质,普遍存在于自然和社会环境中。它们的交互作用较复杂,人群研究也存在有一定的局限性,会使得调查结论出现较大偏差,同时实验证据尚不充分,缺乏准确可靠的安全性评价数据。其中,卵巢是DEHP和BPA有害作用的靶器官之一,二者单独或联合暴露是否会影响卵巢肿瘤的发生及其作用机理仍有待探讨。因此,评价邻苯二甲酸酯类和双酚类化合物单独或联合暴露所致卵巢肿瘤发生及其相关机制,为环境内分泌干扰物的安全性评价提供理论依据和科学线索是本文的研究目的。研究方法:1.选用雌性健康SD大鼠(4周龄)160只,构建大鼠卵巢肿瘤模型。采用DMD(二乙基亚硝胺DEN,N-甲基亚硝基脲MNU和二异丙醇亚硝胺DHPN)致癌模型,将大鼠随机分为DA(给予致癌物DMD预处理)组与DN(未给予致癌物DMD预处理)组。随后将DN组分为四组,分别为对照(Control)组,DEHP150mg/kg组,BPA 20 mg/kg组及DEHP 150 mg/kg+BPA 20 mg/kg组,每组20只大鼠,DA组则分为DMD组,DMD+DEHP 150 mg/kg组,DMD+BPA 20 mg/kg组,DMD+DEHP 150 mg/kg+BPA 20 mg/kg组,同样每组20只大鼠。首先DA组造模动物在第1天按照100 mg/kg给予大鼠腹腔注射DEN,在第5、8、11、14天按照20 mg/kg给予大鼠腹腔注射MNU,第1周和第3周按照0.1%DHPN饮水染毒,DN组动物给予生理盐水注射和日常饮水,第4周进行休息调整后,所有动物开始给予DEHP和BPA单独及联合灌胃染毒至第30周后处死。造模期间观察并记录各组大鼠生长状态、体重变化、死亡情况以及各组大鼠卵巢的脏器系数变化;2.染毒结束后留取所有大鼠的卵巢组织,行HE染色后镜下扫片,由病理科医师阅片,观察各组大鼠卵巢组织的病理改变情况并统计其肿瘤发生率;3.采用IHC法测定各组大鼠卵巢组织中肿瘤标记物增殖细胞核抗原(PCNA)的表达情况,通过阳性细胞计数对卵巢组织IHC结果进行统计分析;4.采用RT-PCR方法检测大鼠卵巢组织中ERα、PTEN m RNA表达水平;5.采用RT-PCR方法检测大鼠卵巢组织中候选micro RNA(mi R-let7b、mi R-21、mi R-200c、mi R-200a-3p、mi R-205、mi R-212-3p、mi R-141-3p、mi R-222、mi R-203-3p)的表达水平;6.采用Western blot方法检测大鼠卵巢组织中ERα、PTEN、AKT、P-AKT的蛋白表达水平;7.Kaplan-Meier plotter网站进行在卵巢组织中部分有差异表达的micro RNA生存分析并确定最终筛选出相关micro RNA。结果:1.大鼠在30周的饲养过程中,随时记录各组大鼠的生存及死亡情况。与对照组比较,DA组(给予致癌物DMD预处理组)中大鼠生存曲线趋势均呈降低趋势,并在BPA与DEHP单独及联合暴露之后,大鼠生存曲线出现显著性差异(P<0.05);2.DEHP与BPA单独及联合染毒期间,DN组(无致癌物DMD预处理组)大鼠生长稳定,摄食正常,体毛光滑,同时体重随着时间稳步增长,但组间未见显著性差异。DA组(给予致癌物DMD预处理组)大鼠生长缓慢,摄食量较少,精神萎靡,染毒期间各处理组大鼠均生长缓慢,从第10周开始至第30周染毒结束,各处理组大鼠体重与对照组相比均呈下降趋势(P<0.05)。染毒结束后留取各组大鼠卵巢组织并对其称重,进行大鼠卵巢脏器系数的计算与分析,与对照组相比,各处理组大鼠卵巢脏器系数改变未见显著性差异;3.通过HE染色检测大鼠卵巢病理改变情况,结果显示,DN组大鼠卵巢结构基本正常,颗粒细胞排列规则且紧密,而DA组大鼠卵巢正常结构基本消失,且DEHP与BPA单独及联合暴露后大鼠颗粒细胞排列疏松紊乱,闭锁卵泡增多,部分肿瘤细胞呈乳头状和菜花状排列,分化程度低,与对照组相比,DA组中的DEHP与BPA单独及联合暴露显著提高了大鼠卵巢肿瘤的发生率(P<0.05);4.通过IHC检测大鼠卵巢组织中PCNA的表达,对照组PCNA阳性细胞数较少,而DN组中DEHP和BPA的联合暴露使得大鼠卵巢组织PCNA阳性细胞数升高。DA组PCNA的阳性细胞数显著升高,且在DEHP与BPA单独及联合暴露之后,PCNA的阳性细胞数远高于对照组,差异具有显著性(P<0.05);5.通过RT-PCR和Western blot检测各处理组大鼠卵巢组织ERα的表达,结果显示,与对照组相比,DN组中DEHP和BPA的联合暴露导致大鼠卵巢组织ERα的蛋白表达水平升高,m RNA表达水平未呈现明显升高或降低趋势,而在DA组中,DEHP与BPA单独及联合暴露导致大鼠卵巢组织ERα的蛋白表达水平升高且具有显著性差异(P<0.05),但其m RNA表达水平同样未呈现明显升高或降低趋势;6.通过RT-PCR和Western blot检测各处理组大鼠卵巢组织PTEN表达情况,结果显示,与对照组相比,DN组中,BPA与DEHP单独及联合暴露组大鼠卵巢组织PTEN的m RNA和蛋白表达均低于对照组,且DEHP和BPA联合暴露后PTEN蛋白表达有显著差异(P<0.05)。在DA组中,BPA单独暴露及二者联合暴露诱导大鼠卵巢组织PTEN的m RNA和蛋白表达均降低且差异具有显著性(P<0.05);7.通过RT-PCR检测各处理组大鼠卵巢组织micro RNA的表达情况,结果显示,与对照组相比,mi R-200c和mi R-222的表达水平呈现升高趋势,在DA组中,单独给予致癌物和DEHP与BPA单独及联合暴露后使其表达水平与对照组相比有显著差异(P<0.05),同时与DMD组相比,DEHP和BPA联合暴露组大鼠卵巢组织的mi R-200c和mi R-222的表达有显著差异(P<0.05);与对照组相比,mi R-let7b和mi R-21的表达水平上调,在DA组中DEHP与BPA单独及联合暴露后,其表达水平有显著差异(P<0.05),同时与DMD组相比,DEHP和BPA联合暴露组大鼠卵巢组织mi R-let7b和mi R-21表达有显著差异(P<0.05);mi R-200a-3p的表达水平呈现降低趋势,与对照组相比,在DA组中给予DEHP与BPA单独及联合暴露后,其表达水平有显著差异(P<0.05);mi R-203-3p的表达水平呈现不规律变化趋势;mi R-141-3p和mi R-205的表达水平与对照组相比均呈现降低趋势,但整体表达水平呈现不规律变化趋势;mi R-212-3p的表达水平与对照组相比均呈现降低趋势;8.Kaplan-Meier plotter网站对在卵巢组织中具有显著升高趋势的mi R-let7b、mi R-200c,mi R-222,mi R-21进行人群生存分析验证。结果显示,与低表达组相比,hasmir-222高表达组预后差,差异具有显著性(P=0.028);has-let-7b(P=0.092)、hasmir-200c(P=0.21)高表达组预后较差,但差异不具显著性;has-mir-21高表达组预后较好,同样差异不具显著性(P=0.25);9.大鼠卵巢肿瘤组织中,ERα表达水平与mi R-222表达水平呈正相关(P<0.05),即ERα高表达,则mi R-222高表达;mi R-222表达水平与PTEN表达水平呈负相关(P<0.05),即mi R-222高表达,则PTEN低表达。结论:1.双酚A和邻苯二甲酸二(2-乙基)己酯长期联合暴露会影响大鼠卵巢卵泡和颗粒细胞等的正常生长发育,但尚未观察到两者诱发卵巢肿瘤的直接致癌作用。2.双酚A和邻苯二甲酸二(2-乙基)己酯长期联合暴露可协同促进致癌剂所致大鼠卵巢肿瘤的发生,两者作为促长剂增加卵巢肿瘤的发生率。3.双酚A和邻苯二甲酸二(2-乙基)己酯长期联合暴露后可能会经ERα促进mi R-222表达水平的升高,进而抑制PTEN并激活下游PI3K/AKT信号通路,增加大鼠卵巢肿瘤发生的易感性。更多还原

【Abstract】 Objective:In recent years,ovarian cancer has become one of the major causes of womenrelated cancer deaths,and its incidence is on the rise.There are many risk factors for ovarian cancer,for example,family and genetic history,fertility,age,and environmental influence.Recently,with the development of science and technology and the extensive application of chemical products,endocrine disruptors have attracted people’s attention,which can affect the homeostasis of the body’s inside environment and can produce a certain damaging effect on the human body.Nowadays,PAEs and bisphenols are major types of endocrine disruptors that have drawed much more attention.As the main raw materials of plasticizers and plastic products,they are diffusely used in production and our life,and can be disclosed in the environment or human body,and it has become a more dangerous and harmful chemicals.Due to the fact that environmental pollutants exist in multiple mixed states in real life,and the human body is exposed to complex and diverse ways,and the single action mode of each substance may present different toxic effects in contact with this mixed,so the study on the toxic effect of a single pollutant has been unable to meet the practical needs.DEHP and BPA respectively as representative material of PAEs and BPs,is common in the environment,and the interaction is more complex,because of the limitations on population study,cause investigation conclusion differences,and experimental evidence is not sufficient,it is the lack of accurate and reliable data for safety evaluation.The ovary is one of the target organs of the harmful effects of DEHP and BPA.Whether single or combined exposure of DEHP and BPA can influence the occurrence of ovarian tumors and the mechanism of action are still unclear.As a result,this article want to evaluate the affect of phthalate esters and bisphenol compounds on ovarian tumorigenesis and their related mechanisms caused by singly or combined exposure,and to provide theoretical basis and scientific clues for the safety evaluation of EDCs.Methods:1.160 healthy female SD rats at the age of 4 weeks were used to construct the rat ovarian tumor model,which was randomly divided into the carcinogen DMD(diethylnitrosamine DEN,n-methyl nitrosamine MNU and diisopropanol nitrosamine DHPN)administration group(DA group)and sham administration group(DN group).The DA group was divided into four groups: control group,DEHP 150 mg/kg group,BPA 20mg/kg group and DEHP 150 mg/kg +BPA 20 mg/kg group,with 20 rats in each group,and the DN group was also divided into the above four groups.Namely DA group animals according to 100 mg/kg intraperitoneal injection of DEN on the first day,and on the 5,8,11,14 days according to 20 mg intraperitoneal injection of MNU,the first week and the third week according to the 0.1% DHPN by water,animals in DN group given saline injections and daily drinking water.After the forth week adjustment rest,all animals were given DEHP and BPA alone and combination for 30 weeks,and the growth status,body weight of rats in each group were observed and recorded during the experiments.At the end of experiments,all tissues of the rats were retained,the changes of organ coefficients of the rats in each group were analyzed;2.After HE staining,the ovary tissues were scanned under the microscope,and the films were read by the pathologists;3.IHC method was used to determine the expression of tumor marker proliferating nuclear antigen(PCNA)in ovarian tissue of rats in each group,and immunohistochemical results were analyzed by positive cell count;4.RT-PCR method to detect the expression levels of ERα and PTEN m RNA in rat ovary tissue;5.RT-PCR method to detect candidate micro RNAs of rat ovary tissue(mi R-let7 b,mi R-21,mi R-200 c,mi R-200a-3p,mi R-205,mi R-212-3p,mi R-141-3p,mi R-222,mi R-203-3p)expression levels;6.Western blot method was used to detect the protein expression levels of ERα,PTEN,AKT and P-AKT in rat ovary tissue;7.KaplanMeier plotter website conducts survival analysis of some differentially expressed micro RNAs and confirms that the relevant micro RNAs will be find out.Results: 1.During the 30-week feeding process,the survival and death of rats in each group were recorded at any time.the survival curve of rats in the DA group(pretreated with carcinogen DMD)showed a decreasing trend,and the survival curve of rats was significantly different after BPA and DEHP were exposed individually and in combination(P <0.05);2.During the long-term exposure of DEHP and BPA alone and in combination,the growth of the rats in DN group(without DMD pretreatment)was stable,the food intake and body weight increased gradually,the reaction was rapid,the body hair was thick and smooth,the vulva was dry,and the stool was granular.The rats in group DA(pretreated with DMD)grow slowly,eat less,showed low spirits,and wet hair around the vulva,with more loose stool.The death time of the rats in the DMD group was earlier,but there was no statistically significant difference between the survival curve of the control group.However,after the pretreatment with DMD,the rats in the treatment group in DA group all grow slowly.the weight of the rats in the treatment group in DA group showed a significant decrease(P < 0.05).After the exposure,the ovary tissues of each group of rats were collected and weighed,and the organ coefficients of ovary of rats were calculated and analyzed.the changes in the ovary organ coefficients of rats in each treatment group were not statistically significant;3.HE staining to verify the pathological changes of the rat ovary,the results showed that the ovary structure of the DN group was basically normal,and the granular cells were arranged regularly and tightly,while the normal structure of the ovary of the DA group basically disappeared,and After DEHP and BPA single and combined exposure,the rat granulosa cells are arranged loosely and disordered,atretic follicles increase,some tumor cells are arranged in papillary and cauliflower-like arrangements,and the degree of differentiation of ovarian tumors is low,suggesting that in rat ovarian tumor models,DEHP and BPA are combined Exposure has a synergistic effect on its occurrence.DEHP and BPA in the DA group alone and combined exposure significantly increased the incidence of ovarian tumors in rats(P<0.05);4.Detection of rat ovary tissue The expression of PCNA by IHC,which is a tumor marker,was basically negative in the control group,and the combined exposure of DEHP and BPA in the DN group increased the number of PCNA-positive cells in rat ovarian tissue.The number of PCNA-positive cells in the DA group was significantly increased,and after DEHP and BPA exposure alone and in combination,the number of PCNA-positive cells was much higher than that in the control group,and the difference was statistically significant(P<0.05);5.RT-PCR and western blot showed that the combination of DEHP and BPA in the DN group resulted in an increase in the protein expression level of ERα in the ovary tissue and m RNA expression level in the DN group.There was no significant increase or decrease trend.In the DA group,DEHP and BPA alone and combined exposure caused an increase in the protein expression level of ERα in rat ovary tissue and there was a significant difference(P <0.05),but its m RNA expression level didn’t show a significant increase or decrease trend,similarly;6.The expression of PTEN in the ovary tissue of rats in each treatment group was disclosed by RT-PCR and Western blot.The results showed that in the DN group,BPA and DEHP alone and in the combined exposure group were exposed to PTEN m RNA and the protein expression was lower than the control group,and the protein expression of DEHP and BPA combined exposure group was significantly different(P <0.05).In the DA group,BPA alone and the combined exposure of the two induced the decrease of m RNA and protein expression of PTEN in rat ovarian tissue,and the difference was significant(P<0.05);7.The micro RNA expression of each treatment group in the rat ovary tissue were detected by RT-PCR,the results showed that the expression levels of mi R-200 c and mi R-222 showed an increasing trend,In the DA group,the group which was given carcinogen alone,and the DEHP and BPA alone and combined exposure group.,and their expression level was significantly different(P<0.05).At the same time,the expression of mi R-200 c and mi R-222 in the ovarian tissue of the DEHP and BPA combined exposure group was significantly different(P<0.05);the expression levels of mi R-let7 b and mi R-21 showed an upward trend.In the DA group,the expression levels of DEHP and BPA were significantly up-regulation(P<0.05).meanwhile,compared with the DMD group,the expression of mi R-let7 b and mi R-21 in the ovary tissue of the DEHP and BPA was significantly different(P<0.05);the expression level of mi R-200a-3p showed a decreasing trend.In the DA group,the expression levels of DEHP and BPA were significantly different from those of the control group after exposure to DEHP and BPA alone and in combination(P<0.05);the expression level of mi R-203-3p showed an irregular trend;the expression level of mi R-205 and mi R-141-3p showed a decreasing trend,but the overall expression level showed an irregular trend;the expression level of mi R-212-3p showed a decreasing trend;8.Survival analysis of mi R-let7 b,mi R-200 c,mi R-222,mi R-21 was confirmed by the Kaplan-Meier plotter website.The results showed that compared with the low expression group,the has-mir-222 high expression group had a worse prognosis,and the difference was statistically significant(P=0.028);The prognosis of the has-let-7b(P=0.092)and has-mir-200c(P =0.21)high expression group was poor,and the difference was not statistically significant;the high expression group of has-mir-21 had a good prognosis,but the difference was not statistically significant(P=0.25);9.In ovarian tumor tissue,The level of mi R-222 expression is positively correlated with the expression level of ERα(P<0.05);the expression level of mi R-222 is negatively correlated with the expression level of PTEN(P<0.05).Conclusions: 1.Long-term combined exposure of bisphenol A and bis(2-ethylhexyl)phthalate will affect the normal growth and development of rat ovarian follicles and granulosa cells,but the direct carcinogenic effects of the two inducing ovarian tumors have not been observed.2.Long-term combined exposure of bisphenol A and bis(2-ethylhexyl)phthalate can synergistically promote the occurrence of ovarian tumors in rats caused by carcinogens,and both act as growth promoters to increase the incidence of ovarian tumors.3.After long-term combined exposure of bisphenol A and bis(2-ethyl)hexyl phthalate,it may promote the increase of mi R-222 expression level via ERα,thereby inhibiting PTEN and activating the downstream PI3K/AKT signaling pathway,thus increasing the susceptibility of ovarian tumors in rats.更多还原