發(fā)布時間：2018-01-31 06:14

  本文關(guān)鍵詞： 印跡基因 人類卵母細(xì)胞 植入前胚胎 mRNA表達(dá) 印跡疾病 輔助生殖技術(shù)(ART) 單細(xì)胞巢式RT-PCR Prader-Willi綜合征 熒光原位雜交技術(shù)(FISH) 甲基化特異性PCR(MS-PCR)　出處：《中南大學(xué)》2007年博士論文　論文類型：學(xué)位論文

【摘要】： 基因印跡(gene imprinting))是指親源依賴性單等位基因表達(dá),即只有一個父源性或母源性等位基因表達(dá),也稱親代印跡(parentalimprinting)。具有這種現(xiàn)象的基因被稱為印跡基因(imprinted gene)。傳統(tǒng)的孟德爾規(guī)律指出:胚胎從父親和母親遺傳的兩個拷貝,即等位基因(alleles)均有同等的機(jī)會表達(dá),與其親代來源無關(guān)。基因印跡不遵循經(jīng)典的孟德爾遺傳,是一種非遺傳性基因調(diào)控方式,為一種表觀遺傳修飾現(xiàn)象,其修飾方式主要為DNA甲基化。基因印跡發(fā)生于配子形成過程中,可影響卵泡成熟,胚胎形成、發(fā)育、胎兒生長及胎盤分化,印跡中心(imprinting centre,IC)的印跡異常則導(dǎo)致流產(chǎn)、死胎、畸型及智力障礙等一系列遺傳性印跡疾病,以及兒童和成人與基因印跡相關(guān)的腫瘤。目前,在卵母細(xì)胞和胚胎早期印跡基因的表達(dá)、印跡形成、輔助生殖技術(shù)(assisted reproductive technology,ART)的體外培養(yǎng)和顯微操作等技術(shù)是否干擾了印跡基因的甲基化印跡狀態(tài)等問題,已成為生殖醫(yī)學(xué)及遺傳學(xué)研究的熱點(diǎn)。 自首例體外受精(in vitro fertilization,IVF)嬰兒1978年在英國誕生后,胞漿內(nèi)單精子注射(intracytoplasmic sperm injection,ICSI)于1992年成功應(yīng)用于治療男性不育,ART做為治療不孕的重要技術(shù)已在世界范圍內(nèi)廣泛應(yīng)用。近年來美、英、德等很多國家研究提出ART的體外培養(yǎng)和顯微操作可能干擾了卵母細(xì)胞或早期胚胎印跡基因的甲基化印跡狀態(tài),從而導(dǎo)致基因印跡疾病,如:韋-伯綜合征(Beckwith-Wiedemannsyndrome,BWS)和安吉爾曼綜合征(Angelman syndrome,AS)等遺傳性印跡疾病,Halliday等比較1316500例自然懷孕出生的孩子和14894例經(jīng)ART出生的孩子的發(fā)病率,發(fā)現(xiàn)BWS的發(fā)病風(fēng)險ART人群比正常人群高9倍,同時報(bào)道7例經(jīng)ART出生的BWS患者,其中6例存在父源性印跡基因KCNQ1OT1或H19的印跡缺失。Orstavik和Cox等報(bào)道經(jīng)ICSI出生的3例AS患兒,都是由于母源性表達(dá)的基因UBE3A在染色體15q11-13區(qū)域的印跡丟失而發(fā)病。在ART出生患兒中70%的BWS和100%的AS患者存在母源性等位基因甲基化異常,導(dǎo)致印跡丟失,而在普通患者中僅有50%-60%的PWS和5%的AS患者各存在甲基化異常。另外,人類胚胎干細(xì)胞體外培養(yǎng),是否干擾了胚胎干細(xì)胞的基因印跡狀態(tài)也需要進(jìn)一步研究。 由于人類卵母細(xì)胞和胚胎的來源及相關(guān)倫理問題的限制,且標(biāo)本僅含單個細(xì)胞至數(shù)個細(xì)胞,mRNA含量極微,實(shí)驗(yàn)室條件要求高,技術(shù)難度大,其相關(guān)研究多以動物為主,對人類的研究,僅國外少量文獻(xiàn)報(bào)道了IGF2、H19等數(shù)條印跡基因在卵母細(xì)胞及植入前胚胎中的表達(dá)。而與PWS相關(guān)的印跡基因SNRPN、NDN,與AS相關(guān)的印跡基因UBE3A,與BWS相關(guān)的印跡基因KvLQT、MASH2,與SRS相關(guān)的印跡基因PEG1等,在人類卵母細(xì)胞及植入前胚胎的表達(dá)情況報(bào)道極少,與SRS相關(guān)的候選印跡基因PEG10、ASB4,目前國內(nèi)外尚未見報(bào)道,本研究應(yīng)用單細(xì)胞巢式RT-PCR技術(shù),對SNRPN、NDN、UBE3A、KvLQT1、MASH2、PEG1、PEG10、ASB4等與印跡疾病相關(guān)的8個印跡基因進(jìn)行mRNA檢測,旨在了解人類印跡基因在卵母細(xì)胞和植入前胚胎的表達(dá)情況,以對其各種異常表達(dá)情況、ART與印跡疾病的關(guān)系和印跡基因調(diào)控機(jī)制進(jìn)行初步探討,為遺傳性印跡疾病的PGD開展奠定理論和實(shí)驗(yàn)基礎(chǔ)。同時,本研究針對臨床疑為PWS患兒,對該患兒及其父母從染色體及基因水平對15q11-13區(qū)帶的印跡基因SNRPN是否微缺失、易位、單親二倍體等進(jìn)行檢測,對其病因從分子水平做出精確診斷,以對其父母再次妊娠作出遺傳指導(dǎo),避免PWS患兒再次出生,從產(chǎn)前水平甚至從胚胎植入前水平阻斷PWS的發(fā)生提供實(shí)驗(yàn)依據(jù)。 第一章印跡基因在人類卵母細(xì)胞及植入前胚胎mRNA表達(dá) 目的:研究印跡基因在人類卵母細(xì)胞和植入前胚胎中的mRNA表達(dá),對其生物學(xué)意義、印跡調(diào)控機(jī)制、ART與印跡疾病的關(guān)系的探討,并為開展胚胎植入前遺傳學(xué)診斷提供理論和實(shí)驗(yàn)依據(jù)。 方法:選擇北大深圳醫(yī)院輔助生殖醫(yī)學(xué)中心2005年6-9月IVF、ICSI廢用的GV、MⅠ、MⅡ的卵母細(xì)胞,2、4、8細(xì)胞胚胎及囊胚。或者經(jīng)IVF/ICSI-ET治療后已出生健康兒、自愿捐獻(xiàn)多余凍存的胚胎。無遺傳病家族史。經(jīng)患者同意及醫(yī)院醫(yī)學(xué)倫理協(xié)會同意。在倒置顯微鏡下去除透明帶,細(xì)胞裂解,cDNA制備,應(yīng)用單細(xì)胞巢式PCR技術(shù),對SNRPN、NDN、UBE3A、KvLQT1、MASH2、PEG1、PEG10、ASB4等與印跡疾病相關(guān)的8個印跡基因進(jìn)行mRNA檢測,并對其中SNRPN、UBE3A基因進(jìn)行PCR產(chǎn)物純化、連接轉(zhuǎn)化、搖菌提取質(zhì)粒測序驗(yàn)證。 結(jié)果: 1.SNRPN、PEG1基因卵母細(xì)胞在GV、MⅠ、MⅡ期,胚胎在2、4、8細(xì)胞及囊胚階段均有mRNA的表達(dá) 2.UBE3A基因自卵母細(xì)胞MⅠ期開始表達(dá),維持表達(dá)至整個植入前胚胎 3.NDN基因表達(dá)于卵母細(xì)胞GV、MⅠ、MⅡ及胚胎8細(xì)胞與囊胚階段 4.KvLQT1基因各期卵母細(xì)胞及植入前胚胎均無mRNA表達(dá) 5.MASH2基因僅MⅡ期卵母細(xì)胞、8細(xì)胞胚胎及囊胚中出現(xiàn)表達(dá) 6.PEG10、ASB4基因在MⅡ期卵母細(xì)胞、4、8細(xì)胞胚胎及囊胚中有表達(dá) 結(jié)論: 1.國內(nèi)首次證實(shí)與PWS相關(guān)的染色體15q11-13上父源性印跡基因SNRPN在卵母細(xì)胞GV、MⅠ、MⅡ期及植入前胚胎有表達(dá),NDN表達(dá)于8細(xì)胞與囊胚及卵母細(xì)胞GV、MⅠ、MⅡ期,提示SNRPN、NDN促進(jìn)卵母細(xì)胞成熟,早期胚胎生長發(fā)育。 2.國內(nèi)首次檢測了與AS密切相關(guān)的染色體15q11-13上母源性印跡基因UBE3A表達(dá)于卵母細(xì)胞MⅠ、MⅡ期及植入前胚胎各期,提示其印跡狀態(tài)受ART某些環(huán)節(jié)的干擾導(dǎo)致AS發(fā)病率增高提供理論依據(jù)。 3.首次證實(shí)7號染色體父源表達(dá)的原癌基因PEG10表達(dá)于MⅡ期卵母細(xì)胞及4、8細(xì)胞與囊胚中,提示ART可能干擾其印跡狀態(tài),導(dǎo)致PEG10的活化,發(fā)生SRS。并督促我們必需長期隨訪ART出生人群,監(jiān)測SRS及腫瘤的發(fā)生率。 4.首次證實(shí)7號染色體上新近證實(shí)的父源印跡的基因ASB4表達(dá)于MⅡ期卵母細(xì)胞及除2細(xì)胞外的植入前胚胎。 5.國內(nèi)首次證實(shí)在11號染色體上母源表達(dá)、與胎盤發(fā)育有關(guān)的印跡基因MASH2在人類卵母細(xì)胞MⅡ期和8胚胎細(xì)胞階段出現(xiàn)表達(dá)。 6.證實(shí)了印跡基因表達(dá)在卵母細(xì)胞和胚胎的不同階段,具有時間的特異性,提示各個基因組的印跡抹除、重建及維持也具有時間的特異性。 7.單細(xì)胞巢式RT-PCR擴(kuò)增成功率為81.0%(64/79),證實(shí)該技術(shù)準(zhǔn)確性較高、穩(wěn)定性較強(qiáng),為進(jìn)一步開展印跡疾病的PGD打下牢固基礎(chǔ)。 第二章印跡疾病PWS分子診斷的研究 目的:旨在從染色體及基因水平對臨床疑為PWS的患兒及其父母進(jìn)行印跡基因SNRPN的檢測,以作出PWS及其病因的確診,為其父母再次生育提供理論依據(jù),指導(dǎo)臨床遺傳咨詢、治療及產(chǎn)前診斷。 方法:采用甲基化特異性PCR(methylation specific PCR,MS-PCR)技術(shù)研究染色體上15q11-13區(qū)與PWS密切相關(guān)的印跡基因SNRPN外顯子alpha區(qū)上的19個CG位點(diǎn)的父母等位基因的甲基化狀況。其作用原理基于DNA經(jīng)亞硫酸氫鈉修飾后,來自父方非甲基化序列的胞嘧啶(C)轉(zhuǎn)變?yōu)槟蜞奏?U),而來自母方甲基化序列保持不變,隨后用甲基化和非甲基化兩對具有高度特異性的引物進(jìn)行擴(kuò)增,根據(jù)有無相應(yīng)的特異產(chǎn)物對其病因做出診斷,并采用FISH技術(shù)從染色體水平上很直觀的進(jìn)一步精確確診。 結(jié)果: 1.患兒 MS-PCR結(jié)果:無父源非甲基化擴(kuò)增條帶,證實(shí)SNRPN基因外顯子alpha區(qū)父源等位基因缺失或?yàn)槟冈磫斡H二倍體(maternal uniparentaldiplont,matUPD),確診患兒為PWS。 FISH結(jié)果:證實(shí)為15號染色體15q11-13片段SNRPN基因的微缺失。結(jié)合MS-PCR結(jié)果,該P(yáng)WS患兒病因系15號父源染色體15q11-13片段SNRPN基因的微缺失,排除matUPD(15)。 2.患兒父母與正常成人無異常。 3.PCR產(chǎn)物測序結(jié)果證實(shí)SNRPN基因外顯子alpha區(qū)擴(kuò)增的基因片斷存在19個甲基化位點(diǎn)(-CG-)。其母源為甲基化,父源為非甲基化。 結(jié)論: 1.FISH技術(shù)從染色體水平及MS-PCR技術(shù)從基因水平能對PWS等印跡疾病做出準(zhǔn)確、快速的診斷,可作為印跡疾病確診及產(chǎn)前印跡疾病篩查的常規(guī)技術(shù)。 2.MS-PCR技術(shù)結(jié)合其PCR產(chǎn)物測序結(jié)果,可以了解某些基因CpG島的甲基化狀況,為在植入前胚胎逆轉(zhuǎn)異常甲基化位點(diǎn),阻斷及治療印跡疾病帶來新的思路。
[Abstract]:Gene imprinting (gene imprinting)) refers to parent of origin dependent monoallelic expression, which is only a paternal or maternal allele expression, also known as parental imprinting (parentalimprinting). This phenomenon is called gene imprinting gene (imprinted gene). It is pointed out that the traditional Mendel rule: embryos from the two copies of the father and the mother is genetic, allele (alleles) expressed the same opportunities related to the parental origin of genetic imprinting. Mendel does not follow the classic, is a kind of non genetic regulation of genes, as an epigenetic modification, the modification is mainly DNA methyl. Gene betides gametogenesis, can affect oocyte maturation, embryo formation, development, growth and differentiation of fetal placenta, imprinting Center (imprinting centre IC) is abnormal imprinting of induced abortion, fetal death, malformation and mental disorders A series of genetic imprinted diseases and tumors in children and adults associated with gene imprinting. At present, the expression in oocytes and early embryos of imprinted gene imprinting, assisted reproductive technology (assisted reproductive technology, ART) in vitro and micro manipulation technology is the problem of interference of imprinted gene methylation the imprinting status, has become a hot topic in medical genetics and reproduction.
The first case of in vitro fertilization (in vitro, fertilization, IVF) the baby was born in Britain in 1978 after intracytoplasmic sperm injection (intracytoplasmic sperm, injection, ICSI) in 1992 successfully used in the treatment of male infertility, ART as an important technique in the treatment of infertility has been in the world wide application. In recent years the United States, Britain, Germany many other countries of the in vitro culture and micromanipulation ART may interfere with the methylation imprinting status of oocytes or early embryos of imprinted genes, resulting in gene imprinting diseases, such as: Beckwith Wiedemann syndrome (Beckwith-Wiedemannsyndrome, BWS) and Angelman syndrome (Angelman syndrome, AS) and other genetic imprinted diseases, Halliday comparison of 1316500 cases of pregnant naturally born children and 14894 cases of ART children born in the incidence of BWS, found that the risk of ART than those of normal group was 9 times higher, while 7 cases reported by ART The birth of the BWS patients, including 6 cases of loss of imprinting of.Orstavik exist paternally imprinted gene KCNQ1OT1 or H19 and Cox reported the 3 cases of children with AS ICSI was born, are due to maternal expression of UBE3A gene in chromosome 15q11-13 loss of imprinting and disease. Born in ART were 70% BWS and 100% the AS patients have maternal allele methylation, resulting in loss of imprinting, and in common with only 50%-60% PWS and 5% AS patients with abnormal methylation. In addition, in vitro culture of human embryonic stem cells, embryonic stem cells are interfering with gene imprinting status also need further research.
As the source of human oocytes and embryos and related ethical issues, and the specimens containing only single cell or a plurality of cells, the mRNA content is minimal, laboratory conditions, technical difficulty, the related research on animal based research on humans, only a small amount of foreign literature reported that IGF2, H19 etc. a number of imprinted gene expression in embryonic development of oocytes and preimplantation. Imprinted genes SNRPN, PWS and NDN related imprinted genes UBE3A and AS related to the imprinted gene KvLQT associated with BWS, MASH2, and SRS related imprinted genes PEG1, in human oocytes and preimplantation embryos the expression is rarely reported, candidate imprinted genes PEG10, SRS and related ASB4, has not been reported so far, the research and application of single cell nested RT-PCR technique, SNRPN, NDN, UBE3A, KvLQT1, MASH2, PEG1, PEG10, ASB4 and other 8 related imprinting and imprinting diseases The gene was detected by mRNA, in order to understand the expression of imprinted genes in human oocytes and preimplantation embryos, with the abnormal expression of imprinted genes, and the regulatory mechanism of ART and imprinting disease was discussed, lay a theoretical and experimental basis for genetic imprinted diseases PGD development. At the same time, this study aimed at clinical suspected PWS children of the imprinted gene SNRPN in children and their parents with the area of 15q11-13 from the chromosome and gene level whether the deletion, translocation, uniparental disomy was detected on the etiology and make accurate diagnosis from the molecular level, genetic guidance to pregnancy on their parents to avoid PWS children from birth again. The level of prenatal even from preimplantation level block to provide experimental basis for the occurrence of PWS.
Chapter 1 the expression of imprinted gene in human oocyte and preimplantation embryo mRNA
Objective: To study the expression of mRNA in human oocytes and preimplantation embryos, and to explore its biological significance, imprinting regulation mechanism, and the relationship between ART and imprinted diseases, and to provide theoretical and experimental evidence for preimplantation genetic diagnosis.
Methods: North Shenzhen hospital assisted reproductive medical center in 2005 6-9 months IVF, ICSI waste with the GV, M I and M II oocytes, 2,4,8 cell embryos and blastocyst. Or after the treatment of IVF/ICSI-ET have been born healthy, voluntary donation of extra frozen embryos. No family history of genetic disease. With the consent of the medical patient consent and hospital ethics Association. Under inverted microscope, zona free, cell lysis, cDNA preparation, application of single cell nested PCR technique, SNRPN, NDN, UBE3A, KvLQT1, MASH2, PEG1, PEG10, mRNA, ASB4 were detected in 8 imprinted genes associated with imprinting diseases, and the SNRPN. UBE3A gene PCR product purification, ligation and transformation, shaked bcteria andextracted plasmids sequencing.
Result:
1.SNRPN, PEG1 gene oocyte in GV, M I, M II stage, and the expression of mRNA in 2,4,8 and blastocyst stages
The expression of 2.UBE3A gene from the M I period of oocyte to the whole preimplantation embryo
The expression of 3.NDN gene in oocyte GV, M I, M II, and embryo 8 cells and blastocyst stage
No mRNA expression was found in all stages of oocyte and preimplantation embryo of 4.KvLQT1 gene
5.MASH2 gene is only M II oocyte, and the expression of 8 cell embryos and blastocysts
The expression of 6.PEG10, ASB4 gene in M II oocytes, 4,8 cell embryos and blastocysts
Conclusion:
1. domestic for the first time that chromosome 15q11-13 associated with PWS on the paternal imprinted gene SNRPN in oocytes of GV M, M I, II and preimplantation embryos expressed NDN expression in 8 cells and blastocysts and oocytes of GV M, M I, II, SNRPN, NDN promote oocyte mature, early embryo growth and development.
2., for the first time in China, the maternal imprinting gene UBE3A expressed on chromosome 15q11-13 closely related to AS was expressed in oocyte M I, M II and preimplantation embryos, suggesting that its imprinting status is influenced by some links of ART, which can provide a theoretical basis for the incidence of AS.
For the first time in 3. confirmed the expression of chromosome 7 paternal proto oncogene PEG10 expression in M II oocytes and 4,8 cells and blastocysts, suggesting that ART may interfere with the imprinting status, leads to the activation of PEG10, SRS. and ART must urge us to long-term follow-up of birth population, the incidence of SRS and tumor monitoring.
4. it was first confirmed that the newly confirmed parent imprinted gene ASB4 on chromosome 7 was expressed in M II oocytes and the preimplantation embryos except 2 cells.
5., for the first time in China, the expression of MASH2, a marker related to placenta development, was first identified on chromosome 11, and it was expressed in human M phase II and 8 embryo cell stage.
6., it is confirmed that the expression of imprinted genes in different stages of oocytes and embryos has time specificity, suggesting that the genome blotting, reconstruction and maintenance are time specific.
7. the successful rate of single cell nested RT-PCR was 81% (64/79), which confirmed the accuracy and stability of the technology, and laid a solid foundation for further developing PGD of imprinted diseases.
The second chapter of the study of PWS molecular diagnosis of imprinted disease
Objective: to detect the imprinted gene SNRPN in children and their parents suspected of PWS by chromosome and gene level, so as to make the diagnosis of PWS and its causes, provide theoretical basis for the re birth of their parents, and guide the clinical genetic counseling, treatment and prenatal diagnosis.
Methods: methylation specific PCR (methylation specific PCR, MS-PCR). The methylation status of 19 CG loci in alpha region on the parental alleles of imprinted gene SNRPN on chromosome 15q11-13 region and the technology of PWS is closely related to the outside. Its function is based on the principle of DNA modified by Sodium Bisulfite, from the father of non methylation of cytosine into uracil (C) (U), but the maternal methylation sequence remains unchanged, then amplified by methylation and non methylation of two highly specific primers according to the specific product is there to make a diagnosis of the etiology, diagnosis and further refined by FISH from the technical level of chromosomes is intuitive.
Result:
1. children
MS-PCR results: there was no parental non methylation amplification band, which confirmed that the SNRPN gene exon alpha loci were deleted or the parental maternal alleles were maternal uniparentaldiplont (matUPD). The diagnosis was PWS..
FISH results: the deletion of SNRPN gene was found in chromosome 15q11-13 of chromosome 15. Combined with MS-PCR results, the etiology of PWS was microdeletion of SNRPN gene of paternal chromosome 15q11-13 15, and matUPD (15) was excluded.
2. parents had no abnormality with normal adults.
3.PCR products sequencing confirmed that there were 19 methylation sites (-CG-) in the SNRPN gene exon alpha fragment, and the parent was methylation and the parent was non methylated.
Conclusion:
1.FISH technology can accurately and quickly diagnose the imprinted diseases such as PWS from chromosome level and MS-PCR technology. It can be used as a routine technology for the diagnosis of imprinted diseases and the screening of prenatal imprinted diseases.
2.MS-PCR technology combined with the sequencing results of PCR products, we can understand the methylation status of CpG islands of some genes, and provide new ideas for reversing aberrant methylation sites, blocking and treating imprinted diseases in preimplantation embryos.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R714.8;R394

【共引文獻(xiàn)】

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1 王凱;樸云峰;丁大勇;馮野;;印跡基因PEG10在胃腺癌組織中的表達(dá)及意義[J];吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2008年02期

2 黃錦;林菊生;董旭e，

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