本文選題：透明帶基因 + 卵細胞　； 參考：《山東大學》2017年碩士論文

【摘要】：研究背景成熟的卵細胞是保證正常受精,胚胎發(fā)育以及著床的關鍵,是妊娠維持及胎兒生長的重要影響及決定因素。ZP(zonapellucida,透明帶)基因屬于卵細胞特異性表達基因,編碼ZP蛋白,人類的ZP蛋白包括ZP1,ZP2,ZP3和ZP4;小鼠的ZP僅包括ZP1,ZP2和ZP3,與人的高度同源,小鼠的ZP結構也與人的相似。ZP最早在初級卵泡中合成,在卵細胞成熟過程中,ZPmRNAs的拷貝數(shù)顯著增加,ZP厚度隨著卵細胞的生長增加。敲除雌鼠的任何一個ZP基因,均可導致其生育障礙和/或卵細胞形態(tài)異常,說明ZP基因在卵細胞成熟和受精過程中起著至關重要的作用。在輔助生殖實驗室中,我們發(fā)現(xiàn)有部分較為罕見的原發(fā)性不孕患者,在不同的促排卵周期中均表現(xiàn)為卵細胞成熟障礙或形態(tài)異常,無法正常受精。因此我們推測:ZP基因突變是否與這部分患者卵細胞異常有關。研究目的分析ZP(ZP1,ZP2,ZP3和ZP4)基因突變是否參與卵細胞成熟阻滯或者形態(tài)異常的發(fā)病。研究方法選擇多周期重復表現(xiàn)為卵細胞異常的輔助生殖助孕患者作為研究對象,自2011年至2015年共收集92例(屬較為罕見病例),其中卵細胞成熟阻滯(組Ⅰ)49例,表現(xiàn)為卵細胞成熟阻滯(GV期和/或MI期阻滯);卵細胞形態(tài)異常(組Ⅱ)43例,表現(xiàn)為卵細胞胞質異�；蛲该鲙У犬惓�。納入的研究對象同時滿足:(1)21歲≤年齡≤40歲;(2)染色體核型均為正常(46,XX);(3)多個(不少于兩個)IVF/ICSI周期中卵細胞異常(成熟阻滯或形態(tài)異常)表現(xiàn)一致。對照組選擇行輔助生殖助孕的,卵細胞形態(tài)無異常且正常受精,并生育過至少一個正常孩子的女性,共373例。提取所有受試者的外周血DNA,對其ZP1-ZP4四個透明帶編碼基因的外顯子及其側翼序列進行基因突變篩查,分析ZP1-ZP4基因的外顯子及其側翼序列是否存在單個堿基的改變(點突變)、缺失、插入、重復或移碼突變。結果在組Ⅰ的49例卵細胞成熟阻滯患者中,除了已知的單核苷酸多態(tài)性(SNP)位點,未發(fā)現(xiàn)ZP1-ZP4有新的變異。在組Ⅱ的43例卵細胞形態(tài)異�；颊咧�,發(fā)現(xiàn)了四處變異,其中兩處位于ZP1基因:c.247TC(p.W83R)和c.1413GA(P.W471X);兩處位于 ZP2 基因:c.1599GT(p.R533S)和 c.1696TC(p.C566R)。這四處位點共在四個不同的病例中檢測到,在373例對照組中未檢測到,同時在常用數(shù)據(jù)庫(dbSNP、ExAC、1000 Genomes Project Database、NHLBI GO ESP)中進行篩查亦未見報道。這四處變異位點在各個物種之間高度保守;利用生物信息學軟件預測,發(fā)現(xiàn)四處位點均為有害突變。含有ZP基因變異的病例大約占組Ⅱ的9%(4/43),均為原發(fā)性不孕患者。其中患者#224同時攜帶有c.247TC(ZP1)和c.1413GA(ZP1)兩處變異,該患者表現(xiàn)為兩個周期卵全部退變、碎裂�；颊�#006攜帶有c.1413GA(ZP1),該變異為無義突變,ZP1翻譯提前終止,透明帶結構域(ZPD)部分缺失,該患者三個促排卵周期所獲卵全部退變�；颊�#189攜帶c.1599GT(ZP2),在三個促排卵周期中,所獲卵多數(shù)退變、碎裂。患者#198攜帶c.1696TC(ZP2),在三個不同方案的促排卵周期中,所獲卵多數(shù)退變、ZP異常。結論ZP基因變異可能與卵細胞退變或透明帶等異常導致的原發(fā)性不孕有關,這些變異位點所造成的功能影響需要進一步實驗證實。
[Abstract]:The mature egg cell is the key to ensure normal fertilization, embryo development and implantation. It is an important influence and determinant of pregnancy maintenance and fetal growth..ZP (Zonapellucida, zona pellucida) gene belongs to the specific expression gene of egg cell, encoding ZP protein, and human ZP protein including ZP1, ZP2, ZP3 and ZP4; ZP only includes ZP1, ZP in mice. 2 and ZP3, highly homologous to human, the ZP structure of mice is also similar to that of human.ZP in the primary follicle. In the process of egg maturation, the number of copies of ZPmRNAs increases significantly, and the thickness of ZP increases with the growth of the egg cells. The knockout of any one of the ZP genes of the female rats can lead to their fertility disorder and / or the abnormal morphology of the egg cells, indicating ZP Genes play a vital role in the maturation and fertilization of egg cells. In the auxiliary reproductive laboratory, we have found that some of the relatively rare patients with primary infertility have an ooocyte maturation disorder or abnormal morphology during the different cycle of ovulation, and can not be fertilized normally. Therefore, we speculate whether the ZP gene mutation is associated with this The purpose of this study is to analyze whether ZP (ZP1, ZP2, ZP3, and ZP4) gene mutations participate in the pathogenesis of oocyte maturation block or morphological abnormalities. The research method selected 92 cases from 2011 to 2015. There were 49 cases of oocyte maturation block (group I), which showed mature oocyte block (GV phase and / or MI phase block); abnormal oocyte morphology (Group II), 43 cases of abnormal oocyte cytoplasm or zona pellucida. The subjects included (1) 21 years of age < < < < 40 years'; (2) chromosome karyotype were normal (46, XX); (3) multiple (no less than two) of the IVF/ICSI cycle of abnormal oocyte (mature or morphologically abnormal). The control group chose to assist reproductive pregnancy with no abnormal and normal fertilization, and had 373 women who had had at least one normal child. A total of 373 cases were extracted from all the subjects' peripheral blood, and four of their ZP1-ZP4 zona pellucida were encoded. Gene mutation screening for gene exons and their flanking sequences to analyze whether there is a single base change (point mutation), deletion, insertion, repetition or code mutation in the exons and flanking sequences of the ZP1-ZP4 gene. Results in 49 cases of egg cell maturity block in group I, except the known single nucleotide polymorphisms (SNP) sites, have not been found. There are new variations in ZP1-ZP4. In 43 patients with abnormal oocyte morphologic abnormalities in group II, two of them are located in the ZP1 gene: c.247TC (p.W83R) and c.1413GA (P.W471X); two loci are located in the ZP2 gene: c.1599GT (p.R533S) and c.1696TC (p.C566R). These loci are detected in four different cases and in 373 control groups. It was not detected, and the screening was not reported in the common database (dbSNP, ExAC, 1000 Genomes Project Database, NHLBI GO ESP). The four heterotopic sites were highly conserved among the species. Using bioinformatics software, the sites were found to be catastrophic mutations. The cases containing the ZP gene mutation accounted for about 9% of the group II. 4/43), all of which were primary infertility. The patient #224 also carried two variations of c.247TC (ZP1) and c.1413GA (ZP1). The patient showed all two cycles of degeneration and fragmentation. The patient #006 carried c.1413GA (ZP1), the mutation was a nonsense mutation, the ZP1 translation was terminated, the zona pellucida domain (ZPD) partial deletion, and three ovulation in the patient. All the eggs obtained from the cycle were degenerated. The patient #189 carried c.1599GT (ZP2), and in the three ovulation cycle, most of the eggs were degenerated and fractured. The patient #198 carried c.1696TC (ZP2), and during the ovulation cycle of the three different schemes, the majority of the eggs were degenerated and ZP was abnormal. Conclusion the genetic variation of ZP may be caused by abnormal changes in the oocyte or the clear zone of the oocyte. Infertility is related to the functional effects of these mutation sites. Further experiments are needed to confirm this.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R714.8

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