DEHP致隐睾症跨代遗传的基因组印记修饰机制

发布时间：2018-08-03 14:01

【摘要】：研究背景：儿童时期泌尿生殖畸形中最常见的疾病便是隐睾(Cryptorchidism),足月生产男婴中隐睾的发病率高达3%-4%,近些年研究提示在欧洲、美洲和我国部分工业发达的地区,泌尿生殖畸形包括隐睾的发生率明显增加。越来越多证据表明隐睾症的发生、发展与环境污染、机体内分泌、家族遗传等因素密切相关,环境因素的刺激和变化可能在其中起到主要的作用。隐睾症能引起男性生殖功能障碍导致不育,睾丸癌变几率也比正常睾丸增加了20-60倍。但是环境内分泌因子等究竟是通过什么途径对机体造成的改变和影响目前还不能明确的阐述说明,因此,研究隐睾症的发病机制具有十分重要的现实意义。人类很早就认识到很多的人工合成化学污染物、添加剂通过干扰生物的内分泌系统从而影响泌尿生殖系统的发育和功能,这些因子广泛存在于环境中,人类很容易接触到它们,现已经将这类物质统称为环境内分泌干扰因子(Environmental Endocrine Disruptors, EED)。邻苯二甲酸二乙基己酯(Di(2-Ethylhexyl) Phthalate, DEHP)是邻苯二甲酸酯类化合物(phthalate esters, PAEs)中重要的一员,它已经被证实为环境内分泌干扰物。DEHP通称塑化剂,在我国广泛应用于各行各业,产耗量极大,大量用于塑料产品制造,最常见如食品包装材料、医用材料、玩具制造等。DEHP也是很多化工产品的重要组成成分,不易降解,长期存在于自然界,可以通过多种途径对环境造成严重的“白色”污染,无形之中危害到人类身体健康。研究证实：使亲代(F0)孕期暴露于DEHP,能诱导出子代(F1)隐睾症发生,隐睾症的发病率高达57%,病变睾丸体积明显缩小,睾丸组织曲细精管发育不良,附睾组织管腔内精子数量减少,生育能力下降明显。亲代(FO)孕期暴露DEHP虽然没有造成F1代基因突变或者DNA序列改变,但是能导致F1代DNA甲基化修饰状态的改变,具体表现：(1)F1代基因组DNA甲基化水平较正常对照组增高约10%；(2)DNA甲基化转移酶的表达均有不同程度的增强,分别是Dnmt1、Dnmt3a、Dnmt3b。研究结果提示表观遗传学研究范畴的DNA甲基化修饰模式改变是DEHP导致F1代隐睾发生的机制之一。同时,在前期研究中观察到：染毒孕鼠F0代所产F1代雄性仔鼠与正常雌鼠交配所生F2代雄性小鼠隐睾的发生率较正常对照组明显升高,F2代雄性小鼠隐睾的发生率约为12.5%。环境因素虽然没有造成基因突变或者DNA序列改变,但是所引起的DNA甲基化修饰模式等表观遗传学改变却能促使亲代疾病的发生,更为重要的是理论上这种“获得性”表观遗传修饰改变可以通过生殖细胞(基因印记)遗传给后代,导致子代疾病的发生。研究提示：环境污染源DEHP所致的“获得性”隐睾能够从F1代遗传给F2代。然而,DEHP是如何通过DNA甲基化转移酶影响基因组印记甲基化修饰模式的改变,从而抑制雄性生殖系统发育关键基因的表达,诱导隐睾症发生跨代遗传?目前机制尚不明确,国内外资料也未见报道。本研究拟通过将DEHP于性腺发育的关键时期作用于孕鼠(FO),研究Fl-F4代隐睾跨代遗传的演变情况,筛选雄性生殖系统发育过程中的关键性印记基因,以期探明基因组印记修饰在隐睾症跨代遗传发生、发展过程中的作用和机制,为科学有效干预阻断隐睾症的发生和未来生物制药作用靶位点的选择,提高国民人口素质,提供科学的实验依据。本文的主要研究内容及结果如下：第一部分“获得性”隐睾症跨代遗传模型的建立目的：亲代孕期暴露DEHP诱导子代隐睾症的发生,“获得性”隐睾症跨代遗传模型建立。方法：1.动物分组与给药：妊娠SD大鼠随机分为2组：正常对照组和DEHP实验组,实验组自妊娠第7d到19d持续经口予以DEHP 750 mg/kg·d灌胃,观察子代隐睾发生情况。2.隐睾的发生情况和生殖系统的组织学检查：观察子代隐睾发生情况,雌鼠受孕率；记录大鼠体重和睾丸、附睾重量以及AGD值,观察精子数量和质量；观察连续4代大鼠睾丸组织形态的演变情况。3.胰岛素样因子3、睾酮(T)表达的检测。结果：1.成功建立隐睾症跨代遗传模型,F1代隐睾发生率为30%,F2代隐睾发生率为12.5%,F3、F4代未见隐睾发生。2.交配实验显示F1代使雌鼠受孕率为50%,F2代为75%,F3、F4代100%；HE染色发现F1代睾丸生精上皮明显萎缩,生精细胞少,F2代有所改善,F3、F4代形态趋于正常。3.胰岛素样因子3、睾酮表达在F1代明显减少。第二部分“获得性”隐睾症基因组印记机制研究目的：探讨基因组甲基化状态改变和基因组印记甲基化修饰差异是否为“获得性”隐睾症发生跨代遗传的机制?方法：1. Real-Time PCR 和 Western Blot检测DNA甲基化转移酶mRNA和蛋白质的表达2.甲基化测序检测基因组甲基化水平和CpG甲基化差异检测3.印记基因筛选和鉴定结果：1.Real Time-PCR、免疫组化和Western Blot表明DNA甲基化转移酶的表达是一个动态变化的过程,在F1代表达水平明显增加,随着传代数增加,表达水平逐渐降低,至F4代与正常对照组无明显差异。2.全基因组甲基化测序提示Fl代与F4代及正常对照组的甲基化水平有着明显的差别。3.FGF9、RN5-8S基因启动子区甲基化差异明显与隐睾症的发生相关。结论：DEHP导致大鼠雄性生殖功能受损,且损伤程度随着遗传代数的增加逐渐减弱。这可能是由于甲基化转移酶表达受到调控继而导致DNA甲基化修饰模式产生改变,产生基因组印记并遗传给下一代,从而使子代雄性生殖系统发育的关键性印记基因作用失衡,并最终导致子代产生隐睾,可能是导致生殖系统损害的重要毒理机制之一。隐睾症跨代遗传有自我修复的趋势,是否与DNA去甲基化有关尚需要进一步研究。
[Abstract]:Background: the most common disease in children's genitourinary malformation is cryptorchidism (Cryptorchidism). The incidence of cryptorchidism is up to 3%-4% in full term production of male infants. Recent studies suggest that the incidence of genitourinary malformation including cryptorchidism has increased significantly in Europe, America and some of the developed areas in China. More and more evidence shows that the incidence of cryptorchidism in urogenital deformities is increasing. The occurrence of cryptorchidism is closely related to environmental pollution, endocrine, family heredity and other factors. The stimuli and changes of environmental factors may play a major role in it. Cryptorchidism can cause male reproductive dysfunction to lead to infertility, and the probability of testicular carcinogenesis is also increased by 20-60 times than that of normal testicles. It is very important to study the pathogenesis of cryptorchidism. It is very important for us to study the pathogenesis of cryptorchidism. Human beings have known many synthetic chemical pollutants early, and the additives can affect the urinary reproduction by interfering with the endocrine system of the organism. The development and function of the system are widely existed in the environment. Human beings are easily exposed to them. They are now known as Environmental Endocrine Disruptors (EED). Two ethylhexyl (Di (2-Ethylhexyl) Phthalate, DEHP) is o-phthalic acid ester (phthal) (phthal) (phthal). As an important member of ate esters, PAEs, it has been proved to be an environmental endocrine disruptor.DEHP generic plasticizer. It is widely used in all walks of life in our country. It is widely used in the manufacture of plastic products. The most common.DEHP, such as food packaging materials, medical materials, toy manufacture, and so on, is also an important component of many chemical products. It is easy to degrade and exist in the natural world for a long time. It can cause serious "white" pollution to the environment through a variety of ways, and it can jeopardize human health. It has been confirmed that the exposure of F0 during pregnancy to DEHP can induce the occurrence of F1 cryptorchidism, the incidence of cryptorchidism is as high as 57%, the volume of the testicular disease is obviously narrowed, and the testicular group is reduced. The dysplasia of the fine seminiferous tubules, the decrease in the quantity of sperm in the epididymal tissue and the decrease of fertility. Although the exposure of DEHP in FO during pregnancy did not cause the mutation of F1 generation gene or the change of the DNA sequence, the changes in the DNA methylation status of the F1 generation could be changed, and the specific performance was as follows: (1) the level of DNA methylation in the F1 generation was more than that of the normal control group. Increased by about 10%; (2) the expression of DNA methyltransferase was enhanced in varying degrees. The results of Dnmt1, Dnmt3a, and Dnmt3b. suggest that the DNA methylation modification of epigenetics is one of the mechanisms of DEHP leading to the occurrence of the F1 generation of cryptorchidism. Meanwhile, it was observed in the previous study that the F1 generation male produced by the F0 generation of pregnant mice was produced. The incidence of cryptorchidism in F2 generation male mice was significantly higher than that in the normal control group. The incidence of cryptorchidism in F2 generation male mice was about 12.5%. environmental factors, although it did not cause genetic mutation or DNA sequence change, but the epigenetic changes caused by DNA methylation modification could promote parental generation. It is more important that this "acquired" epigenetic modification can be inherited from the reproductive cells (gene imprints) to the offspring and lead to offspring disease. The study suggests that the "acquired" cryptorchidism caused by environmental pollution source DEHP can be inherited from the F1 generation to the F2 generation. However, how is DEHP through DNA methyl? The changes in the model of methylation modification of genomic imprinting by transferase can inhibit the expression of the key genes in the male reproductive system and induce the cross generation of cryptorchidism, the mechanism is not yet clear, and the data at home and abroad have not been reported. This study is to study the role of DEHP in the critical period of gonadal development (FO) for the study of Fl-F4 The evolution of the cross generation inheritance of cryptorchidism and the screening of key imprinting genes in the development of male reproductive system, in order to explore the role and mechanism of genomic imprinting in the development of cryptorchidism, in order to effectively intervene to prevent the occurrence of cryptorchidism and the selection of target sites for future biopharmaceutical action, and to improve the country. The main research contents and results of this paper are as follows: the first part of this paper is as follows: the first part is to establish a cross generation genetic model of "acquired" cryptorchidism: the occurrence of DEHP induced cryptorchidism in pregnancy and the establishment of a cross generation genetic model of "acquired" cryptorchidism. Methods: 1. groups of animals and drug delivery: Pregnant SD rats were randomly divided into 2 groups: normal control group and DEHP experimental group. The experimental group was given DEHP 750 mg/kg d from pregnancy 7d to 19d. The occurrence of cryptorchidism and the histological examination of reproductive system were observed. The occurrence of cryptorchidism in the offspring, the pregnancy rate of the female rats, the weight and the testosterone of the rats were recorded. The weight and AGD value of the epididymis and the value of AGD, observe the quantity and quality of spermatozoa, observe the evolution of the testicular formation of the 4 generation rats,.3. insulin-like factor 3, the expression of testosterone (T). Results: 1. the genetic model of cryptorchidism was established successfully, the incidence of cryptorchidism in the F1 generation was 30%, the incidence of cryptorchidism in the F2 generation was 12.5%, F3, and the.2. of the F4 generation did not occur.2.. The mating experiment showed that the pregnant rate of the F1 generation was 50%, the F2 generation was 75%, the F3, and the F4 generation 100%. The HE staining found that the testicular spermatogenic epithelium of the F1 generation atrophied, the spermatogenic cell was less, the F2 generation improved, the F3, the F4 generation tended to normal.3. insulin-like factor 3, and the expression of testosterone was significantly reduced in the F1 generation. Second parts of the genomic imprinting mechanism of "acquired" cryptorchidism Purpose: To investigate whether the difference of genomic methylation status and genomic imprint methylation modification is a cross generation mechanism of "acquired" cryptorchidism by 1. Real-Time PCR and Western Blot detection of DNA methyltransferase mRNA and protein expression by 2. methylation sequencing and detection of genomic methylation level and CpG Methylation difference detection 3. imprinted gene screening and identification results: 1.Real Time-PCR, immunohistochemistry and Western Blot showed that the expression of DNA methyltransferase was a dynamic process, and the level of F1 representative increased obviously. With the increase of the transmission algebra, the expression level decreased gradually, and there was no significant difference between the F4 generation and the normal control group, and there was no significant difference between the.2. whole group and the normal control group. Methylation indicated that the methylation level of Fl generation and F4 generation and normal control group had a significant difference.3.FGF9. The difference of methylation in the promoter region of RN5-8S gene was significantly related to the occurrence of cryptorchidism. Conclusion: DEHP resulted in impaired male reproductive function and the degree of damage decreased with the increase of genetic algebra. The regulation of the expression of methyltransferase leads to changes in the DNA methylation modification pattern, producing genomic imprinting and hereditary to the next generation, thus making the key imprinting genes in the offspring male reproductive system unbalanced and eventually leading to the generation of cryptorchidism, which may be an important toxicological mechanism that causes reproductive system damage. (1) cryptorchidism has a trend of self repair, and whether it is related to DNA demethylation needs further study.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R726.9

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