DEHP致隱睪癥跨代遺傳的基因組印記修飾機制
發(fā)布時間：2018-08-03 14:01
【摘要】：研究背景：兒童時期泌尿生殖畸形中最常見的疾病便是隱睪(Cryptorchidism),足月生產(chǎn)男嬰中隱睪的發(fā)病率高達3%-4%,近些年研究提示在歐洲、美洲和我國部分工業(yè)發(fā)達的地區(qū),泌尿生殖畸形包括隱睪的發(fā)生率明顯增加。越來越多證據(jù)表明隱睪癥的發(fā)生、發(fā)展與環(huán)境污染、機體內(nèi)分泌、家族遺傳等因素密切相關,環(huán)境因素的刺激和變化可能在其中起到主要的作用。隱睪癥能引起男性生殖功能障礙導致不育,睪丸癌變幾率也比正常睪丸增加了20-60倍。但是環(huán)境內(nèi)分泌因子等究竟是通過什么途徑對機體造成的改變和影響目前還不能明確的闡述說明,因此,研究隱睪癥的發(fā)病機制具有十分重要的現(xiàn)實意義。人類很早就認識到很多的人工合成化學污染物、添加劑通過干擾生物的內(nèi)分泌系統(tǒng)從而影響泌尿生殖系統(tǒng)的發(fā)育和功能,這些因子廣泛存在于環(huán)境中,人類很容易接觸到它們,現(xiàn)已經(jīng)將這類物質(zhì)統(tǒng)稱為環(huán)境內(nèi)分泌干擾因子(Environmental Endocrine Disruptors, EED)。鄰苯二甲酸二乙基己酯(Di(2-Ethylhexyl) Phthalate, DEHP)是鄰苯二甲酸酯類化合物(phthalate esters, PAEs)中重要的一員,它已經(jīng)被證實為環(huán)境內(nèi)分泌干擾物。DEHP通稱塑化劑,在我國廣泛應用于各行各業(yè),產(chǎn)耗量極大,大量用于塑料產(chǎn)品制造,最常見如食品包裝材料、醫(yī)用材料、玩具制造等。DEHP也是很多化工產(chǎn)品的重要組成成分,不易降解,長期存在于自然界,可以通過多種途徑對環(huán)境造成嚴重的“白色”污染,無形之中危害到人類身體健康。研究證實：使親代(F0)孕期暴露于DEHP,能誘導出子代(F1)隱睪癥發(fā)生,隱睪癥的發(fā)病率高達57%,病變睪丸體積明顯縮小,睪丸組織曲細精管發(fā)育不良,附睪組織管腔內(nèi)精子數(shù)量減少,生育能力下降明顯。親代(FO)孕期暴露DEHP雖然沒有造成F1代基因突變或者DNA序列改變,但是能導致F1代DNA甲基化修飾狀態(tài)的改變,具體表現(xiàn)：(1)F1代基因組DNA甲基化水平較正常對照組增高約10%；(2)DNA甲基化轉(zhuǎn)移酶的表達均有不同程度的增強,分別是Dnmt1、Dnmt3a、Dnmt3b。研究結(jié)果提示表觀遺傳學研究范疇的DNA甲基化修飾模式改變是DEHP導致F1代隱睪發(fā)生的機制之一。同時,在前期研究中觀察到：染毒孕鼠F0代所產(chǎn)F1代雄性仔鼠與正常雌鼠交配所生F2代雄性小鼠隱睪的發(fā)生率較正常對照組明顯升高,F2代雄性小鼠隱睪的發(fā)生率約為12.5%。環(huán)境因素雖然沒有造成基因突變或者DNA序列改變,但是所引起的DNA甲基化修飾模式等表觀遺傳學改變卻能促使親代疾病的發(fā)生,更為重要的是理論上這種“獲得性”表觀遺傳修飾改變可以通過生殖細胞(基因印記)遺傳給后代,導致子代疾病的發(fā)生。研究提示：環(huán)境污染源DEHP所致的“獲得性”隱睪能夠從F1代遺傳給F2代。然而,DEHP是如何通過DNA甲基化轉(zhuǎn)移酶影響基因組印記甲基化修飾模式的改變,從而抑制雄性生殖系統(tǒng)發(fā)育關鍵基因的表達,誘導隱睪癥發(fā)生跨代遺傳?目前機制尚不明確,國內(nèi)外資料也未見報道。本研究擬通過將DEHP于性腺發(fā)育的關鍵時期作用于孕鼠(FO),研究Fl-F4代隱睪跨代遺傳的演變情況,篩選雄性生殖系統(tǒng)發(fā)育過程中的關鍵性印記基因,以期探明基因組印記修飾在隱睪癥跨代遺傳發(fā)生、發(fā)展過程中的作用和機制,為科學有效干預阻斷隱睪癥的發(fā)生和未來生物制藥作用靶位點的選擇,提高國民人口素質(zhì),提供科學的實驗依據(jù)。本文的主要研究內(nèi)容及結(jié)果如下：第一部分“獲得性”隱睪癥跨代遺傳模型的建立目的：親代孕期暴露DEHP誘導子代隱睪癥的發(fā)生,“獲得性”隱睪癥跨代遺傳模型建立。方法：1.動物分組與給藥：妊娠SD大鼠隨機分為2組：正常對照組和DEHP實驗組,實驗組自妊娠第7d到19d持續(xù)經(jīng)口予以DEHP 750 mg/kg·d灌胃,觀察子代隱睪發(fā)生情況。2.隱睪的發(fā)生情況和生殖系統(tǒng)的組織學檢查：觀察子代隱睪發(fā)生情況,雌鼠受孕率；記錄大鼠體重和睪丸、附睪重量以及AGD值,觀察精子數(shù)量和質(zhì)量；觀察連續(xù)4代大鼠睪丸組織形態(tài)的演變情況。3.胰島素樣因子3、睪酮(T)表達的檢測。結(jié)果：1.成功建立隱睪癥跨代遺傳模型,F1代隱睪發(fā)生率為30%,F2代隱睪發(fā)生率為12.5%,F3、F4代未見隱睪發(fā)生。2.交配實驗顯示F1代使雌鼠受孕率為50%,F2代為75%,F3、F4代100%；HE染色發(fā)現(xiàn)F1代睪丸生精上皮明顯萎縮,生精細胞少,F2代有所改善,F3、F4代形態(tài)趨于正常。3.胰島素樣因子3、睪酮表達在F1代明顯減少。第二部分“獲得性”隱睪癥基因組印記機制研究目的：探討基因組甲基化狀態(tài)改變和基因組印記甲基化修飾差異是否為“獲得性”隱睪癥發(fā)生跨代遺傳的機制?方法：1. Real-Time PCR 和 Western Blot檢測DNA甲基化轉(zhuǎn)移酶mRNA和蛋白質(zhì)的表達2.甲基化測序檢測基因組甲基化水平和CpG甲基化差異檢測3.印記基因篩選和鑒定結(jié)果：1.Real Time-PCR、免疫組化和Western Blot表明DNA甲基化轉(zhuǎn)移酶的表達是一個動態(tài)變化的過程,在F1代表達水平明顯增加,隨著傳代數(shù)增加,表達水平逐漸降低,至F4代與正常對照組無明顯差異。2.全基因組甲基化測序提示Fl代與F4代及正常對照組的甲基化水平有著明顯的差別。3.FGF9、RN5-8S基因啟動子區(qū)甲基化差異明顯與隱睪癥的發(fā)生相關。結(jié)論：DEHP導致大鼠雄性生殖功能受損,且損傷程度隨著遺傳代數(shù)的增加逐漸減弱。這可能是由于甲基化轉(zhuǎn)移酶表達受到調(diào)控繼而導致DNA甲基化修飾模式產(chǎn)生改變,產(chǎn)生基因組印記并遺傳給下一代,從而使子代雄性生殖系統(tǒng)發(fā)育的關鍵性印記基因作用失衡,并最終導致子代產(chǎn)生隱睪,可能是導致生殖系統(tǒng)損害的重要毒理機制之一。隱睪癥跨代遺傳有自我修復的趨勢,是否與DNA去甲基化有關尚需要進一步研究。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R726.9
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R726.9
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