發(fā)布時間：2018-06-05 02:51

  本文選題：小鼠 + 卵子�。� 參考：《廣西大學(xué)》2008年博士論文

【摘要】： 基因組印跡在調(diào)節(jié)胎兒生長、發(fā)育、胎盤功能和出生后的行為中發(fā)揮著重要的作用。一旦甲基化印跡建立起來,它們甚至在受精之后發(fā)生表觀遺傳重編程過程中當(dāng)基因組DNA發(fā)生整體去甲基化的時候都會得到維持,從而使得親本特異性甲基化印跡可以在基因組重編程過程中發(fā)揮重要的作用,這也是決定發(fā)育潛能的一個重要因素。正常的印跡一旦被破壞,將會導(dǎo)致發(fā)育異常和印跡缺陷相關(guān)的疾病。排卵后卵子老化和ICSI操作都有可能干預(yù)正常的甲基化印跡。我們進(jìn)行下面的試驗(yàn)探討這些問題。 試驗(yàn)一:延長排卵后卵子停留在輸卵管或者是體外培養(yǎng)到相對長的時間而不進(jìn)行受精的情況下會導(dǎo)致卵子的老化。這些已老化的卵子受精后會影響著床前和著床后胚胎/胎兒的發(fā)育。本研究中,我們使用重亞硫酸鹽測序法和COBRA法檢測兩個母本印跡基因,Snrpn和Peg1/Mest甲基化差異區(qū)在排卵后卵子的體內(nèi)和體外老化過程中的甲基化狀況。結(jié)果顯示,卵子排放后在體內(nèi)和裸卵在體外培養(yǎng)至注射HCG之后29 h,Snrpn的差異甲基化區(qū)域發(fā)生了去甲基化。然而,Peg1/Mest無論在體內(nèi)或是體外的環(huán)境下從HCG注射之后21-29 h都沒有發(fā)生去甲基化。這些數(shù)據(jù)表明,Snrpn差異甲基化區(qū)域在小鼠排卵后卵子的老化過程中會發(fā)生時間依賴性的去甲基化。 試驗(yàn)二:胞質(zhì)內(nèi)單精子注射(ICSI)在十九世紀(jì)九十年代初首次獲得成功之后,一直被廣泛應(yīng)用于治療男性不育癥。但是人們對包括IVF和ICSI在內(nèi)的輔助生殖技術(shù)(ART)的安全性仍存有顧慮,尤其是近來有報(bào)導(dǎo)指出ART可能與基因組印跡缺陷有關(guān)。然而,目前我們還不了解ICSI對囊胚的印跡基因甲基化是否會造成影響。為了評價(jià)ICSI是否影響囊胚印跡基因的正常甲基化,我們使用基因組測序法檢測了來自體內(nèi)囊胚,體內(nèi)受精-體外培養(yǎng)囊胚,體內(nèi)MII卵子ICSI囊胚以及IVM-ICSI囊胚H19差異甲基化區(qū)域的DNA甲基化情況。結(jié)果顯示,體內(nèi)囊胚“忠實(shí)”遺傳了配子的甲基化模式;KSOM培養(yǎng)液沒有造成囊胚H19甲基化異常;ICSI體內(nèi)MII卵子所得囊胚發(fā)生H19甲基化的丟失和甲基化模式的異常;ICSI聯(lián)合IVM同樣導(dǎo)致了H19甲基化的丟失和甲基化模式的異常。與以往使用多囊胚進(jìn)行的研究相比較,我們的研究強(qiáng)調(diào)了個體之間存在的高度差異,這對臨床來說更具有實(shí)用意義。 從我們的實(shí)驗(yàn)結(jié)果可以看出,外界環(huán)境或者是ART的操作都會導(dǎo)致配子和胚胎印跡基因甲基化的異常,臨床上有必要充分考慮這些問題。
[Abstract]:Genomic imprinting plays an important role in regulating fetal growth, development, placental function and postnatal behavior. Once methylated imprinting is established, they are maintained even during epigenetic reprogramming after fertilization, when global demethylation of genomic DNA occurs. Therefore, parental specific methylation imprinting can play an important role in genome reprogramming, which is also an important determinant of developmental potential. Normal imprinting, once destroyed, can lead to dysplasia and imprinting related diseases. Egg aging after ovulation and ICSI manipulation may interfere with normal methylation imprinting. We are going through the following experiments to explore these problems. Experiment 1: prolonged ovulation after the egg stays in the fallopian tube or in vitro culture for a relatively long time without fertilization will lead to egg aging. Fertilization of these aged eggs affects the development of preimplantation and postimplantation embryos / fetuses. In this study, we used the method of bisulfite sequencing and COBRA to detect the methylation of two female imprinted genes, SNPN and Peg1/Mest, during the in vivo and in vitro aging of ovulation eggs. The results showed that demethylation occurred in the differential methylation region of Snrpn 29 h after HCG injection in vivo and naked eggs. However, Peggy / Mest had no demethylation 21-29 h after injection of HCG in vivo or in vitro. These data suggest that the differential methylation region of Snrpn occurs time-dependent demethylation during the aging of mouse oocytes after ovulation. Experiment 2: ICSII has been widely used in the treatment of male infertility since its first success in the early 1890s. But there are still concerns about the safety of assistive reproductive technologies, including IVF and ICSI, especially recent reports that ART may be related to genomic imprinting defects. However, we do not know whether ICSI affects blastocyst imprinting gene methylation. To evaluate whether ICSI affects the normal methylation of blastocyst imprinted genes, we used genomic sequencing to detect blastocysts from in vivo, fertilization and in vitro culture of blastocysts. In vivo DNA methylation of ICSI blastocyst and IVM-ICSI blastocyst H 19 in MII eggs. The results show that In vivo blastocyst "faithfully" inherited the methylation pattern of gametes. KSOM culture medium did not cause abnormal H19 methylation of blastocyst. Loss of H19 methylation and abnormal methylation pattern of blastocyst derived from MII eggs in ICSI The loss of H 19 methylation and the abnormality of methylation pattern were induced. Compared with previous studies using polyblastocysts, our study emphasizes the high level of individual differences, which is more practical for clinical use. From our experimental results, we can see that the outside environment or the operation of ART can lead to abnormal methylation of gametes and embryo imprinting genes, and it is necessary to consider these problems in clinic.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R321

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 袁韋娜;體外培養(yǎng)對小鼠生精細(xì)胞印跡基因H19和IGF2r甲基化狀態(tài)的影響[D];安徽醫(yī)科大學(xué);2011年

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本文編號：1980123