猪卵核对核移植胚胎发育及其早期基因表达的影响

发布时间：2018-06-05 04:07

本文选题：再生医学 + 体细胞重编程　；参考：《广西大学》2016年博士论文

【摘要】：目前,哺乳动物体细胞核移植重编程效率仍旧很低,作为治疗性克隆的首选动物模型,猪的核移植胚胎干细胞系仍然没有建立。其技术瓶颈主要是体细胞核重编程不完全。卵母细胞核作为母源遗传物质,对胚胎发育甚至体细胞重编程影响巨大。因此,本研究以猪核移植体系为背景,观察在不去除卵母细胞核的情况下,核移植胚胎的体外发育情况,结合高通量测序技术,研究高效重编程核移植体系特有的基因表达模式,为体细胞核移植重编程机制以及功能性基因的深入研究奠定基础,以期找到提高体细胞重编程效率的突破点。本研究主要内容如下：1、猪卵核对核移植胚胎发育及体细胞重编程效率的影响首先构建猪不去核核移植胚胎,观察其发育潜能。我们一共注射了539枚卵丘细胞于完整的卵母细胞内,同时注射了461枚卵丘细胞于去核的卵母细胞内,两组重构胚分别融合激活后,有260枚多倍体重构胚发育至囊胚,同时仅有93枚去核重构胚发育至囊胚,相比去核组,不去核胚胎的卵裂率(81.15% vs 91.05%)、囊胚率(20.15% vs 48.29%)均显著提高。2、鉴定供体细胞、去核和不去核核移植囊胚染色体数目对供体细胞、去核核移植囊胚以及不去核核移植囊胚进行染色体制片,染色并统计各细胞染色体数目,最终确定各细胞染色体数目正常(38/38/57,正常猪染色体数目为38条)3、不去核核移植囊胚在形态及细胞数上与去核核移植囊胚、孤雌囊胚的差异以孤雌囊胚为对照,分别对去核、不去核核移植囊胚细胞进行荧光染色计数,发现孤雌囊胚平均细胞数最多(58.60),去核核移植囊胚平均细胞数最少(43.30),不去核核移植囊胚平均细胞数介于二者之间(56.95)。囊胚形态上,孤雌囊胚直径较大,颜色较浅,去核与不去核核移植囊胚直径较小,颜色较深,且不去核核移植囊胚多为孵化囊胚,呈8字形态,而去核核移植囊胚多为扩张囊胚,未孵化。4、卵母细胞机械损伤对核移植胚胎发育的影响考虑到去核过程会对卵母细胞造成机械损伤,因此,本实验通过对卵母细胞预先扎孔,并将卵核吸出再注回,模拟去核操作对卵母细胞造成的机械损伤,随后进行孤雌激活。同时将对照组卵母细胞仅做扎孔处理,不做卵核吸吐动作,模拟不去核核移植胚胎操作,观察两种胚胎发育情况,发现机械损伤导致孤雌胚胎卵裂率、囊胚率降低(卵裂率93.136% vs95.032%,P≈0.36；囊胚率56.14% vs 64.42%,P≈0.014),囊胚细胞数减少(52.65 vs 55.40),但抑制作用不显著。证明机械损伤这一单方面因素不是造成核移植胚胎发育能力低下的主要原因。5、染色体加倍对胚胎发育的影响以及不去核重构胚中的卵核能否被体细胞核所取代将不去核重构胚模型中的卵核用体细胞核进行置换,再移入另一枚体细胞重新构建多倍体胚胎,观察其发育情况,发现体细胞核+体细胞+卵胞质胚胎囊胚率仅有10.5%,远低于卵核+体细胞+卵胞质胚胎囊胚率50.6%,说明在不去核重构胚模型中,单纯性染色体数目加倍无法促进胚胎发育,卵母细胞核无法被体细胞核任意取代。6、卵巢类型与卵母细胞发育潜能的关系由于卵巢样品取自市内屠宰场,样品品系不确定,为保证测序样品间遗传背景一致,采取单卵巢采卵独立培养。根据卵巢表面有无红体将其分类,分别进行卵子采集、成熟,观察其发育潜能,发现有红体卵巢采卵效率高,所得卵子成熟率(75.77% vs 68.97%,P0.05)、卵裂率(95.33%vs 94.00%,P0.05)、囊胚率(68.67%vs 59.00%,P0.05)均高于没有红体的卵巢。但差异不显著,确定可以选择带有红体的卵巢进行采卵,然后构建核移植胚胎,用于后续基因表达图谱的构建实验。7、构建去核、不去核重构胚早期基因表达图谱为探究卵核对体细胞重编程作用的分子机制,了解两种重构胚特有的基因表达模式,本研究采集了去核以及不去核重构胚2cell(第一次卵裂)和4cell(发育阻滞期)时期胚胎进行基因表达谱测序(RNA sequencing)。成功构建两种重构胚各自特异基因表达图谱。发现去核重构胚2cell胚胎有大约28%的基因参与表达,4cell胚胎大约22%的基因参与表达；不去核重构胚2cell胚胎有大约30%的基因参与表达,4cell胚胎有大约27%的基因参与表达。8、去核、不去核重构胚阻滞前期差异表达基因筛选与分析将去核和不去核重构胚相同时期胚胎基因表达图谱进行比对,发现2cell阶段,不去核重构胚相比去核重构胚有1738条基因呈现表达上调,728条基因表达下调(｜log2Ratio｜≥5); 4cell阶段,不去核重构胚有2941条基因表达上调,1682条基因表达下调(｜log2Ratio｜≥5);这些差异基因WEGO分析中富集程度最深的基因簇分别集中在绑定调控、催化和分子转导活性上；其他在不去核重构胚中呈现高表达的基因则多数参与各种代谢过程。其中,Ribosome和Oxidative phosphorylation两条信号通路在去核重构胚发育趋势中以及不去核重构胚发育趋势中反复出现富集。在4cell阻滞期阶段,Protein processing in endoplasmic reticulum信号通路显著富集。由于内质网结构与细胞核结构紧密相连,并且附着大量核糖体结构,在核移植去核操作过程中可能连同细胞核一起被去除。因此推测内质网蛋白加工以及核糖体、氧化磷酸化功能的缺失可能是去核重构胚较之不去核重构胚发育劣势的主要原因。9、核定位转录因子差异表达模式分析去核、不去核重构胚差异表达基因中有267条转录活性相关基因在细胞核部位定位表达,其中绝大多数在去核重构胚中表达量微弱,但在不去核重构胚中表达显著上调。推测这些核定位表达的转录因子表达上调导致体细胞重编程加快,重编程效果更彻底。10、QRT-PCR基因表达量验证在RNA-seq差异基因中随机挑选了6个表达量较高的基因DNMT1、 FTL、POLRID、RPS3、RPS20、BUB3分别进行QRT-PCR检测。将检测结果与RNA-seq结果比对后发现,6个基因在两种检测方法下表达趋势基本一致。11、内质网荧光示踪检验使用内质网红色荧光探针ER-Tracker Red对成熟MⅡ期卵母细胞、去核后卵母细胞分别进行染色,比较两种细胞的内质网结构,检验卵母细胞内质网在核移植去卵核操作过程中是否被去除。
[Abstract]:At present, the efficiency of reprogramming of mammalian cell nuclear transfer is still very low. As the first animal model of therapeutic cloning, the pig's nuclear transplantation embryonic stem cell line is still not established. Its technical bottleneck is the incomplete reprogramming of somatic cell nucleus. The oocyte nucleus is a parent genetic material, and the embryo development or even somatic cell is reprogrammed. Therefore, this study, based on the porcine nuclear transplantation system, observed the development of the embryo in vitro, combined with high throughput sequencing technology, and studied the specific gene expression pattern of the high performance reprogramming nuclear transplantation system with high throughput sequencing technology without removing the nucleus of the oocyte. The main contents of this study are as follows: 1, the effects of porcine oocyte nucleation on embryo development and somatic reprogramming efficiency were first constructed to construct porcine non nuclear transplanting embryos and to observe their developmental potential. A total of 539 cumulus cells were injected into complete oocyte refinement. Within the cell, 461 cumulus cells were injected into the oocyte at the same time. After the two reconstructive embryos were fused and activated, 260 multiple weight structure embryos developed to blastocysts, and only 93 reconstructive embryos developed to blastocysts. Compared with the nucleus removal group, the cleavage rate of non nuclear embryos (81.15% vs 91.05%) and the blastocyst rate (20.15% vs 48.29%) were significantly raised. High.2, identification of donor cells, nuclear and non nuclear nuclear transplantation blastocysts chromosome number to donor cells, nuclear transplantation blastocyst and non nuclear nuclear transplantation blastocysts for chromosome production, dyeing and statistics the number of chromosomes of each cell, and ultimately determine the number of chromosomes of each cell (38/38/57, the number of normal pigs is 38) 3, no nuclear The difference between the nucleus transplantation blastocyst and the nucleus transplanting blastocyst and the parthenogenetic blastocyst was compared with the parthenogenetic blastocyst, which was compared with the blastocyst of the parthenogenetic blastocyst. The average number of cells in the parthenogenetic blastocyst was the most (58.60), and the average number of cells in the nucleus removed blastocyst was least (43.30), and the nuclear transplant sac was not removed. The average number of cells in the embryo is between two (56.95). The blastocyst morphology of the blastocyst, the diameter of the parthenogenetic blastocyst is larger, the color is lighter, the blastocyst of the nucleus transplant and the non nuclear nucleus transplantation is smaller and the color is deeper, and the blastocyst of the nuclear nucleus transplantation is 8 word shape, and the blastocyst of the nucleus nucleus transplantation is mostly dilated blastocyst, not hatching.4, and the mechanical damage to oocyte is the same. The effect of nuclear transplantation on embryo development takes into account that the process of nuclear removal can cause mechanical damage to oocytes. Therefore, in this experiment, the oocyte was beforehand and the oocyte was sucked out and reinjected to simulate the mechanical damage to oocyte and then parthenogenetic activation was performed, and the control group of oocytes was treated only with a ligation of the oocytes. In the action of oocytic ememation, the embryo development of two kinds of embryos was observed, and the rate of parthenogenetic embryo cleavage and blastocyst rate decreased (cleavage rate 93.136% vs95.032%, P 0.36; blastocyst rate 56.14% vs 64.42%, P 0.014), and blastocyst fine cell number decreased (52.65 vs 55.40), but the inhibition effect was not significant. The unilateral factor of mechanical injury is not the main cause of the low developmental ability of the embryo,.5, the effect of chromosome doubling on the development of the embryo and the replacement of the nucleus of the nucleus in the non nuclear restructured embryo model by the replacement of the nucleus in the non nuclear reconstructive embryo by the nucleus of the non nuclear reconstructive embryo, and then retransferring to another somatic cell. A polyploid embryo was constructed and its development was observed. It was found that the rate of somatic cell nucleus + somatic + oocyst blastocyst was only 10.5%, far below the rate of egg nucleus + somatic cell + oocyst blastocyst 50.6%, which indicated that in the non nuclear reconstructive embryo model, the number of simple chromosomes could not promote the development of embryo, and the oocyte nucleus could not be free from the nucleus of the body. Instead of.6, the relationship between ovarian types and oocyte developmental potential is due to ovarian samples taken from the municipal slaughterhouse, and the sample lines are uncertain. In order to ensure the consistent genetic background between the sequencing samples, the ovaries are independently cultured with single ovaries. The ovum extraction efficiency of erythrocyte was high, the rate of egg maturation (75.77% vs 68.97%, P0.05), cleavage rate (95.33%vs 94%, P0.05), blastocyst rate (68.67%vs 59%, P0.05) were higher than those without red body, but the difference was not significant. It was determined that the egg nests with red body could be selected for ovum extraction, and then the nuclear transplant embryos were constructed for subsequent gene expression. The construction of.7, the construction of the nucleus and the molecular mechanism of the early gene expression map of the non nuclear reconfiguration to explore the molecular mechanism of the reprogramming of the egg nucleus to the somatic cell, and understand the specific gene expression patterns of two reconstructive embryos. This study collected the base and the non nuclear reconstructive embryo 2cell (the first cleavage) and the 4cell (development block) period embryo. The specific gene expression profiles of two reconstructed embryos were successfully constructed by the expression spectrum sequencing (RNA sequencing). It was found that about 28% of the genes involved in the reconstructive embryo 2cell embryos were expressed, and about 22% of the 4cell embryos were involved in the expression; about 30% of the 2cell embryos in the non nuclear reconstructive embryo were expressed in the gene, and the 4cell embryo had about 27% genes. The expression of differentially expressed genes in the prophase of the embryo block in the non nuclear reconfiguration was selected and analyzed for the expression of the gene expression of the embryos at the same period in the same period of non nuclear reconstructive embryos. It was found that there were 1738 genes in the 2cell stage, and 1738 genes were up regulated and the expression of 728 genes were down. 5): in the 4cell stage, 2941 genes were up-regulated in the non nuclear reconstructive embryo and 1682 genes were down down (log2Ratio > 5). The most enriched gene clusters in the WEGO analysis of these differentially expressed genes were concentrated in binding regulation, catalytic and molecular transduction activity, and other genes with high expression in the non nuclear reconstructive embryos were most of the ginseng. With various metabolic processes, the two signal pathways of Ribosome and Oxidative phosphorylation were enriched in the developmental trend of nuclear reconstructive embryos and in the development trend of non nuclear reconstructive embryos. In the phase of the 4cell block, the Protein processing in endoplasmic reticulum signaling pathway was significantly enriched. The structure is closely connected with a large number of ribosome structures and may be removed with the nucleus in the process of nuclear transplantation and nuclear removal. Therefore, it is presumed that the deletion of the endoplasmic reticulum protein and the ribosome, the deficiency of the acidification function may be the main reason for the nuclear reconstructive embryo compared with the disadvantage of the non nuclear reconstructive embryo,.9. The expression of 267 transcriptional activity related genes in the non nuclear reconstructive gene expressed in the nuclear site, most of which were expressed in the nucleus reconstructive embryo, but the expression of the gene was up to up in the non nuclear reconstructive embryo. The reprogramming of somatic cells was accelerated, the effect of reprogramming was more thorough.10, and the QRT-PCR gene expression was verified in the RNA-seq differential gene randomly selected 6 high expression genes DNMT1, FTL, POLRID, RPS3, RPS20, and BUB3 respectively for QRT-PCR detection. The detection results were compared with the RNA-seq results, and the 6 genes were expressed in the two detection methods. The potential of the.11 was basically consistent with the endoplasmic reticulum fluorescence tracer test using the endoplasmic reticulum red fluorescence probe ER-Tracker Red for mature M II oocytes, and the oocytes were stained respectively after the nucleation, and the endoplasmic reticulum of the two cells was compared to determine whether the oocyte endoplasmic reticulum was removed during the operation of nuclear transplantation to the oocyte nucleus.
【学位授予单位】：广西大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S814

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