本文選題：Dazl基因 + DNA甲基化�。� 參考：《南京醫(yī)科大學(xué)》2015年博士論文

【摘要】：DAZL是高度保守的DAZ(Deleted in Azoospermia)家族成員之一,定位于脊椎動(dòng)物的常染色體,在PGC發(fā)育遷移、精原細(xì)胞分化、減數(shù)分裂起始及發(fā)展等過(guò)程中起重要作用,人DAZL蛋白可以促進(jìn)胚胎干細(xì)胞分化為精子,是配子生成的重要調(diào)控因子。DAZL同源基因在從硬骨魚(yú)到人類幾乎所有物種的生殖細(xì)胞中特定表達(dá),其表達(dá)持續(xù)存在于胚胎干細(xì)胞(ESCs)直至單倍體圓形精子。是什么機(jī)制調(diào)控了Daz1高度時(shí)空特異性表達(dá)?這是我們面臨的科學(xué)問(wèn)題,DNA甲基化在內(nèi)的表觀遺傳修飾是可能的表達(dá)調(diào)控機(jī)制。精子形成過(guò)程中,存在原始生殖細(xì)胞甲基化信息廣泛擦除和隨后生殖細(xì)胞甲基化的重新建立,這對(duì)保障精子的正常發(fā)育起重要作用,異常的表觀遺傳修飾尤其是DNA甲基化可能是導(dǎo)致男性生精障礙及精子活力下降的機(jī)制之一。生精相關(guān)基因Daz1啟動(dòng)子甲基化模式異常改變是否與男性生精障礙存在關(guān)聯(lián)?部分生殖特異表達(dá)基因在人類體細(xì)胞惡性腫瘤中異常激活稱為癌-生殖基因,研究生殖特定基因Daz1的表達(dá)調(diào)控機(jī)制以及鑒定癌-生殖基因能夠?yàn)閻盒阅[瘤相關(guān)基礎(chǔ)研究提供幫助。本研究分二部分：一、DNA甲基化參與調(diào)控Daz1生殖特異表達(dá)及其在物種間進(jìn)化上的保守性該部分研究首先檢測(cè)了Daz1在小鼠睪丸和體細(xì)胞組織中的表達(dá)差異,進(jìn)而測(cè)定Daz1啟動(dòng)子CpG島在不同組織類型中的甲基化模式,分析小鼠Daz1啟動(dòng)子甲基化模式與Daz1特定表達(dá)的關(guān)系；進(jìn)一步研究了解小鼠精子發(fā)生不同階段中Daz1表達(dá)是否和其甲基化模式存在關(guān)聯(lián)；在體細(xì)胞系(NIH3T3)中采用5-氮雜-2’-脫氧胞苷處理去甲基化后檢測(cè)Daz1表達(dá)情況；在其他哺乳動(dòng)物和脊椎動(dòng)物中選取不同物種進(jìn)行研究,了解Daz1轉(zhuǎn)錄調(diào)控機(jī)制在進(jìn)化上的保守性;設(shè)定計(jì)算法對(duì)Daz1啟動(dòng)子區(qū)域進(jìn)行生物信息學(xué)分析,預(yù)測(cè)可能的轉(zhuǎn)錄因子結(jié)合位點(diǎn)。通過(guò)以上實(shí)驗(yàn)力圖闡明Daz1生殖特異表達(dá)的轉(zhuǎn)錄調(diào)控機(jī)制。成果如下：1.小鼠Daz1基因表達(dá)存在時(shí)空特異性通過(guò)qRT-PCR方法檢測(cè)Daz1在成年雄性小鼠睪丸、肝臟、心臟、腸管、脾臟、肌肉、腎臟、胸腺、肺及腦組織中的表達(dá)。在體細(xì)胞組織中沒(méi)有檢測(cè)到Daz1mRNA的表達(dá),而在小鼠睪丸中高表達(dá)。繼續(xù)用qRT-PCR方法檢測(cè)Daz1在生后3天、6天、8天、12天、14天、16天、18天、20天、21天、5周小鼠睪丸中的表達(dá)。在不同發(fā)育階段的小鼠睪丸中,Daz1在3日齡小鼠睪丸中檢測(cè)出表達(dá),于出生后16-18天時(shí)達(dá)到峰值,其后有所降低。實(shí)驗(yàn)室前期進(jìn)行的小鼠睪丸冰凍切片免疫組化實(shí)驗(yàn)表明Daz1在精原細(xì)胞、前細(xì)線期精母細(xì)胞、偶線期精母細(xì)胞和早-中粗線期精母細(xì)胞中高表達(dá),此后表達(dá)漸減少,在圓形精子中有少量表達(dá),于長(zhǎng)形精子中未檢測(cè)到。通過(guò)以上研究,表明Daz1基因表達(dá)模式具有高度時(shí)空特異性。2.小鼠Daz1啟動(dòng)子CpG島的甲基化模式與基因表達(dá)呈現(xiàn)顯著負(fù)相關(guān)我們運(yùn)用Methyl Primer Expresss V1.0軟件對(duì)Daz1啟動(dòng)子進(jìn)行分析,預(yù)測(cè)出相應(yīng)區(qū)域CpG島,采用亞硫酸氫鹽修飾后測(cè)序PCR(Bisulfite sequencing PCR, BSP)法,分別檢測(cè)不同組織細(xì)胞類型Daz1啟動(dòng)子CpG島的甲基化狀態(tài)。結(jié)果顯示：在成年小鼠體細(xì)胞組織(腎臟、腦)中Daz1啟動(dòng)子CpG島高度甲基化,而在睪丸中Daz1啟動(dòng)子CpG島呈現(xiàn)低甲基化模式,附睪精子中甲基化程度相較睪丸組織更低。Daz1啟動(dòng)子CpG島的甲基化模式與其表達(dá)情況呈現(xiàn)明顯的負(fù)相關(guān),提示Daz1啟動(dòng)子區(qū)域甲基化可能參與了Daz1生殖細(xì)胞特異性表達(dá)的調(diào)控。通過(guò)對(duì)小鼠出生后不同時(shí)間點(diǎn)睪丸中Daz1啟動(dòng)子CpG島的甲基化檢測(cè),我們發(fā)現(xiàn)Daz1啟動(dòng)子區(qū)域在小鼠6-8日齡以及10-12日齡呈現(xiàn)低甲基化狀態(tài),而在出生后20-24天表現(xiàn)為甲基化程度升高,這一結(jié)果與小鼠Daz1表達(dá)于精原細(xì)胞、持續(xù)到圓形精子、以及后續(xù)階段的表達(dá)降低相一致。以上相關(guān)性結(jié)果提示DNA甲基化參與調(diào)控了小鼠Daz1時(shí)間(精子發(fā)生階段)空間(生殖細(xì)胞)特異性的表達(dá)。3.體細(xì)胞中進(jìn)行去甲基化處理激活Daz1異常表達(dá)上述實(shí)驗(yàn)顯示小鼠Daz1表達(dá)與其啟動(dòng)子甲基化狀態(tài)呈現(xiàn)顯著負(fù)相關(guān)關(guān)系,提示DNA甲基化參與調(diào)控小鼠Daz1的時(shí)空特異表達(dá),那么在Daz1不表達(dá)的體細(xì)胞中采用藥物去甲基化是否能夠誘導(dǎo)Daz1異常激活?為此,我們采用甲基化抑制劑5-Aza-CdR在體細(xì)胞系進(jìn)行去甲基化處理,通過(guò)qRT-PCR檢測(cè)5-Aza-CdR處理后Daz1 mRNA的水平。結(jié)果顯示Daz1在去甲基化處理后的體細(xì)胞中表達(dá)升高,作為對(duì)照的Tex19.1和RHOX2亦異常升高,而DAZ家族另一成員Boule卻無(wú)明顯變化。這一結(jié)果提示體細(xì)胞中Daz1的表達(dá)沉默由其基因啟動(dòng)子甲基化模式調(diào)控,而B(niǎo)oule表達(dá)調(diào)控機(jī)制可能更為復(fù)雜多元。4.DNA甲基化機(jī)制參與調(diào)控Daz1生殖特異表達(dá)在物種進(jìn)化上存在高度保守性。實(shí)驗(yàn)采用qRT-PCR對(duì)收集的人睪丸及體細(xì)胞組織(結(jié)腸、前列腺、胃、膽囊及脾臟)DAZL mRNA表達(dá)水平進(jìn)行檢測(cè),結(jié)果顯示DAZL mRNA在人睪丸組織中高表達(dá),而在體細(xì)胞組織中不表達(dá)。進(jìn)一步研究DAZL啟動(dòng)子甲基化模式是否與此相關(guān),實(shí)驗(yàn)提取人睪丸、精子、體細(xì)胞組織(前列腺、脾臟)DNA進(jìn)行甲基化檢測(cè),發(fā)現(xiàn)在體細(xì)胞組織中DAZL啟動(dòng)子為高甲基化狀態(tài)而在睪丸中顯示為低甲基化,精子中顯示為更低的甲基化程度。相似的實(shí)驗(yàn)結(jié)果在另一種哺乳動(dòng)物豬中同樣存在,表明DNA甲基化機(jī)制參與調(diào)控Daz1生殖特異表達(dá)存在于哺乳動(dòng)物中。鑒于Daz1最早表達(dá)于脊椎動(dòng)物生殖細(xì)胞,我們進(jìn)一步實(shí)驗(yàn)了解這一調(diào)控機(jī)制在進(jìn)化上是否在更低級(jí)別的脊椎動(dòng)物中存在。我們提取了斑馬魚(yú)和萊航雞性腺及體細(xì)胞組織的RNA及基因組DNA,實(shí)驗(yàn)結(jié)果顯示Daz1 mRNA在性腺中高表達(dá)而Daz1啟動(dòng)子呈低甲基化狀態(tài),體細(xì)胞組織中Daz1啟動(dòng)子呈高甲基化而無(wú)Daz1 mRNA表達(dá)。Daz1同源基因表達(dá)從硬骨魚(yú)類開(kāi)始出現(xiàn)到哺乳動(dòng)物直至人類,以上實(shí)驗(yàn)表明啟動(dòng)子區(qū)差異化的甲基化模式參與調(diào)控Daz1生殖特異性表達(dá),支持這一調(diào)控機(jī)制古老并且在物種進(jìn)化上維持保守性的假說(shuō)。5. Daz1啟動(dòng)子區(qū)轉(zhuǎn)錄因子可能結(jié)合位點(diǎn)的生物信息學(xué)預(yù)測(cè)分析預(yù)測(cè)轉(zhuǎn)錄因子結(jié)合位點(diǎn)是研究基因轉(zhuǎn)錄調(diào)控的重要組成部分。前述研究明確了Daz1在生殖細(xì)胞和體細(xì)胞中差異化的甲基化模式和其生殖特異表達(dá)密切相關(guān),且這種關(guān)聯(lián)貫穿Daz1整個(gè)基因進(jìn)化歷程,表明DNA甲基化調(diào)控機(jī)制在Daz1基因表達(dá)調(diào)控中保守存在。那么,Dazl啟動(dòng)子區(qū)域是否存在保守的順式作用元件參與基因的轉(zhuǎn)錄調(diào)控?我們檢索人、小鼠、豬及大鼠Dazl Exonl上游3.5kb的序列,設(shè)定計(jì)算法檢測(cè)長(zhǎng)度大于7個(gè)堿基的保守序列,結(jié)果顯示在上述物種中共同存在12個(gè)由7個(gè)核苷酸組成的相同序列,數(shù)據(jù)庫(kù)檢索發(fā)現(xiàn)這些保守存在的相似序列可能是一些轉(zhuǎn)錄因子的結(jié)合位點(diǎn)。二、DAZL表達(dá)調(diào)控異常與男性生精障礙及腫瘤相關(guān)性研究。1.DAZL啟動(dòng)子異常的甲基化模式與男性生精障礙有關(guān)。精子發(fā)生過(guò)程經(jīng)歷了基因組范圍內(nèi)的去甲基化和重新甲基化過(guò)程,男性精子甲基化的研究工作將從表觀遺傳學(xué)角度為男性生精障礙臨床診療提供幫助。為此,我們比較了正常男性與少弱精子癥患者精子DAZL啟動(dòng)子甲基化譜差異。收集精液標(biāo)本后進(jìn)行精液常規(guī)檢查和精子形態(tài)測(cè)定；采用密度梯度離心法去除體細(xì)胞污染,提取精子基因組DNA；亞硫酸氫鹽處理后PCR體外擴(kuò)增并純化,與pCR2.1載體連接及酶切驗(yàn)證,挑取陽(yáng)性克隆測(cè)序；分析不同樣本精子DNA甲基化程度差異。和對(duì)照相比,少弱精癥患者DAZL啟動(dòng)子甲基化率明顯升高,結(jié)果存在統(tǒng)計(jì)學(xué)差異。這一結(jié)果提示DAZL啟動(dòng)子區(qū)異常的甲基化模式可能與男性生精障礙有關(guān)。2.DAZL在部分惡性腫瘤中異常表達(dá)。癌-生殖基因或癌-睪丸基因是指一類其抗原蛋白表達(dá)于人類多種組織來(lái)源惡性腫瘤,而在正常組織中只存在于睪丸、卵巢或胎盤(pán)組織。上研究顯示Dazl生殖細(xì)胞特定表達(dá)與其基因調(diào)控區(qū)甲基化模式差異密切相關(guān),其是否類似于癌-生殖基因在惡性腫瘤中異常表達(dá)?我們?cè)赥CGA數(shù)據(jù)庫(kù)中搜索發(fā)現(xiàn)DAZL在頭頸部腫瘤、肺癌和乳腺癌中表達(dá),尤其在后者中表達(dá)較明顯。但和其他經(jīng)典癌-生殖基因相比,DAZL在腫瘤中表達(dá)量仍較低,提示DAZL在部分腫瘤中異常表達(dá)可能和腫瘤細(xì)胞基因組整體異常低甲基化有關(guān)。
[Abstract]:DAZL is one of the highly conserved members of the DAZ (Deleted in Azoospermia) family. It is located in the autosomes of vertebrates. It plays an important role in the process of PGC development and migration, spermatogonial differentiation, meiosis initiation and development. Human DAZL protein can promote the differentiation of embryonic stem cells into sperm, which is an important regulatory factor of gamete generation,.DAZL. The source genes are expressed in the germ cells of almost all species from the hard bone fish to the human species, and their expression persists in the embryonic stem cells (ESCs) until the haploid round sperm. What mechanism regulates the high temporal and spatial specific expression of Daz1? This is the scientific problem we face, and the epigenetic modification, including DNA methylation, is a possible table. In the process of sperm formation, there is a widespread erasure of the methylation information of the primitive germ cells and the reestablishment of the subsequent methylation of the germ cells, which plays an important role in ensuring the normal development of sperm. Abnormal epigenetic modification, especially DNA methylation, may be the mechanism leading to male spermatogenesis disorder and the decline of sperm motility. 1. Is the abnormal change of the Daz1 promoter methylation pattern associated with male spermatogenesis disorders? The abnormal activation of partial reproductive specific genes in human body cell malignant tumors is called the cancer reproductive gene, studies the regulation mechanism of the expression of the specific reproductive gene Daz1 and the identification of the cancer reproductive genes for malignant tumors. The relevant basic research provides help. This study is divided into two parts: first, DNA methylation participates in the regulation of Daz1 reproductive specific expression and its conservatism in interspecies evolution. This part of the study first detected the difference in the expression of Daz1 in mouse testis and somatic cells, and then measured the methylation of the Daz1 promoter CpG island in different tissue types. Model analysis of the relationship between Daz1 promoter methylation and Daz1 specific expression in mice; further study whether Daz1 expression in different stages of spermatogenesis in mice was associated with its methylation pattern; in somatic cell line (NIH3T3), 5- n-heterozygous -2 'deoxycytidine demethylation was used to detect the expression of Daz1; The selection of different species in mammals and vertebrates is conducted to understand the evolutionary conservatism of the Daz1 transcriptional regulation mechanism; to set a computational method for the bioinformatics analysis of the Daz1 promoter region and to predict the possible transcription factor binding site. Through the above experiments, the transcriptional regulation mechanism of the specific expression of Daz1 is clarified. As follows: 1. the expression of Daz1 gene in mice was specific by qRT-PCR method to detect the expression of Daz1 in the testis, liver, heart, intestines, intestines, spleen, muscle, kidney, thymus, lung and brain tissue of adult male mice. The expression of Daz1mRNA was not detected in somatic tissue and high expression in mouse testis. QRT-PCR method continued to be used. The expression of Daz1 was detected in mice testis at 3 days, 6 days, 8 days, 12 days, 14 days, 16 days, 18 days, 20 days, 21 days and 5 weeks. In mice testis at different developmental stages, Daz1 was detected in the testicles of 3 days of age in mice, and reached their peak value after birth and then decreased. The frozen section of testis in the early laboratory of mice was immunized. The results showed that Daz1 was highly expressed in spermatogonia, pre fine line spermatocyte, dipinogenic spermatocyte, and early middle roughen stage spermatocyte, and then decreased gradually. There was a small amount of expression in round sperm and not detected in long spermatozoa. Through the above study, the Daz1 gene expression pattern has a highly spatio-temporal specific.2. mouse Daz1 startup. The methylation pattern of the sub CpG island has a significant negative correlation with the gene expression. We use Methyl Primer Expresss V1.0 software to analyze Daz1 promoter, predict the corresponding region CpG Island, use the hydrogen sulfite modified sequence PCR (Bisulfite sequencing PCR, BSP) method, and separate the different tissue cell types to detect the promoter Island. Methylation status. The results showed that the Daz1 promoter CpG island was highly methylation in the adult mouse somatic tissue (kidney, brain), and the Daz1 promoter CpG island in the testis showed a low methylation pattern. The methylation level in the epididymal sperm was lower than that of the testicular tissue, and the methylation pattern of the.Daz1 promoter CpG island was obviously negative. Correlation, suggesting that methylation of the Daz1 promoter region may be involved in the regulation of the specific expression of Daz1 germ cells. By methylation of the Daz1 promoter CpG island in the testicles at different time points after birth, we found that the Daz1 promoter region showed low methylation status at 6-8 days and 10-12 days of age in mice, and 20-24 days after birth. The results showed that the degree of methylation was elevated, which was consistent with the expression of Daz1 in spermatogonial cells, round spermatozoa and subsequent stages. The above correlation results suggest that DNA methylation participates in the regulation of Daz1 time (spermatogenesis) space (spermatogenic cell) specific expression of.3. somatic cells in mice. The above experiments of activation of Daz1 showed that the expression of Daz1 in mice was negatively correlated with the methylation status of the promoter, suggesting that DNA methylation participates in the time and space specific expression of Daz1 in mice. Then, whether the use of drug demethylation in Daz1 non expressed somatic cells can induce abnormal activation of Daz1? For this reason, Methylation inhibitor 5-Aza-CdR was used to demethylation in somatic cell lines, and the level of Daz1 mRNA after 5-Aza-CdR treatment was detected by qRT-PCR. The results showed that the expression of Daz1 was increased in the somatic cells after demethylation treatment, and the control Tex19.1 and RHOX2 also increased, but the Boule of the other member of the DAZ family did not change significantly. The results suggest that the expression of Daz1 in somatic cells is regulated by the methylation mode of its gene promoter, and the regulatory mechanism of Boule expression may be more complex and multiple.4.DNA methylation mechanism involved in the regulation of Daz1 reproduction specific expression in the species evolution. The experimental qRT-PCR was used to collect human testis and somatic tissue (colon,) The expression level of DAZL mRNA in the prostate, stomach, gallbladder and spleen was detected. The results showed that DAZL mRNA was highly expressed in human testicular tissue, but was not expressed in somatic tissue. Further study of the correlation between the Zi Jiaji mode of DAZL initiation and the experimental extraction of DNA from human testis, spermatozoa and somatic cells (prostate, spleen) DNA was detected. It was found that the DAZL promoter was methylation in the somatic tissue and showed low methylation in the testis, and the sperm showed a lower degree of methylation. Similar experimental results were found in another mammal pig, indicating that the DNA methylation mechanism involved in the regulation of Daz1 reproductive specific expression in mammals. In view of Daz1 It was first expressed in vertebrate germ cells. We further studied whether this regulatory mechanism existed in lower levels of vertebrates. We extracted the RNA and genomic DNA of the gonads and somatic tissues of zebrafish and leihang chickens. The experimental results showed that the Daz1 mRNA was highly expressed in the gonads and the Daz1 promoter was low. In the basic state, the Daz1 promoter in somatic tissue is hypermethylation and no Daz1 mRNA expression.Daz1 homologous gene expression from the bone fishes to mammalian and human. The above experiments show that the differential methylation mode of the promoter region participates in the regulation of Daz1 reproductive specificity, and supports this regulatory mechanism in ancient and in species. Evolutionary conservatism hypothesis.5. Daz1 promoter region transcription factor potential binding site bioinformatics prediction analysis and prediction of transcription factor binding site is an important component of gene transcription regulation. The previous study clarified the differential methylation pattern and its reproductive specific expression in Daz1 in germ cells and somatic cells. Closely related, and this association runs through the entire genetic evolution of Daz1, indicating that the regulatory mechanism of DNA methylation exists conservatively in the regulation of Daz1 gene expression. Then, is there a conservative cis acting element in the Dazl promoter region to participate in gene transcription regulation? We retrieve the sequence of 3.5kb in the upstream of human, mice, pigs and rat Dazl Exonl. The results showed that there were 12 identical sequences of 7 nucleotides in the above species, and the database retrieval found that these conserved similar sequences might be the binding sites of some transcription factors. Two, the abnormal regulation of DAZL expression was associated with male spermatogenesis and cancer. The methylation pattern of the.1.DAZL promoter anomaly is related to the male spermatogenesis disorder. The process of spermatogenesis has undergone the process of demethylation and re methylation within the genome range. The study of male sperm methylation will help the clinical diagnosis and treatment of male spermatogenesis from the epigenetic point of view. Sperm DAZL promoter methylation spectrum difference between male and oligozoospermia patients. Semen specimens were collected for semen routine examination and sperm morphometry; density gradient centrifugation was used to remove somatic cell pollution and to extract sperm genome DNA; PCR was amplified and purified in vitro after hydrogen sulphite treatment, connected with pCR2.1 vector and verified by enzyme digestion. The positive clones were sequenced and the difference of DNA methylation in different samples of sperm was analyzed. Compared with the control, the methylation rate of DAZL promoter was significantly higher in the patients with oligoasthenospermia. The results suggested that the abnormal methylation pattern in the DAZL promoter region may be associated with the male spermatogenesis disorders associated with.2.DAZL in some malignant tumors. Abnormal expression. Cancer - reproductive gene or cancer - testicular gene is a class of antigen protein expressed in a variety of human malignant tumors, but in normal tissues only in the testis, ovaries or placenta tissues. The previous study showed that the specific expression of Dazl germ cells was closely related to the differences in the methylation patterns of the gene regulatory region. We found that DAZL is expressed in head and neck tumors, lung cancer and breast cancer in the TCGA database, especially in the latter, but the expression of DAZL in the tumor is still low compared with other classic cancer and reproductive genes, suggesting that the abnormal expression of DAZL in some tumors is likely to be swollen. The overall abnormal hypomethylation of the tumor cell genome is related.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R698;R73

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本文編號(hào)：2096994