本文選題：再生醫(yī)學(xué) + 體細(xì)胞重編程��； 參考：《廣西大學(xué)》2016年博士論文

【摘要】：目前,哺乳動(dòng)物體細(xì)胞核移植重編程效率仍舊很低,作為治療性克隆的首選動(dòng)物模型,豬的核移植胚胎干細(xì)胞系仍然沒有建立。其技術(shù)瓶頸主要是體細(xì)胞核重編程不完全。卵母細(xì)胞核作為母源遺傳物質(zhì),對(duì)胚胎發(fā)育甚至體細(xì)胞重編程影響巨大。因此,本研究以豬核移植體系為背景,觀察在不去除卵母細(xì)胞核的情況下,核移植胚胎的體外發(fā)育情況,結(jié)合高通量測(cè)序技術(shù),研究高效重編程核移植體系特有的基因表達(dá)模式,為體細(xì)胞核移植重編程機(jī)制以及功能性基因的深入研究奠定基礎(chǔ),以期找到提高體細(xì)胞重編程效率的突破點(diǎn)。本研究主要內(nèi)容如下：1、豬卵核對(duì)核移植胚胎發(fā)育及體細(xì)胞重編程效率的影響首先構(gòu)建豬不去核核移植胚胎,觀察其發(fā)育潛能。我們一共注射了539枚卵丘細(xì)胞于完整的卵母細(xì)胞內(nèi),同時(shí)注射了461枚卵丘細(xì)胞于去核的卵母細(xì)胞內(nèi),兩組重構(gòu)胚分別融合激活后,有260枚多倍體重構(gòu)胚發(fā)育至囊胚,同時(shí)僅有93枚去核重構(gòu)胚發(fā)育至囊胚,相比去核組,不去核胚胎的卵裂率(81.15% vs 91.05%)、囊胚率(20.15% vs 48.29%)均顯著提高。2、鑒定供體細(xì)胞、去核和不去核核移植囊胚染色體數(shù)目對(duì)供體細(xì)胞、去核核移植囊胚以及不去核核移植囊胚進(jìn)行染色體制片,染色并統(tǒng)計(jì)各細(xì)胞染色體數(shù)目,最終確定各細(xì)胞染色體數(shù)目正常(38/38/57,正常豬染色體數(shù)目為38條)3、不去核核移植囊胚在形態(tài)及細(xì)胞數(shù)上與去核核移植囊胚、孤雌囊胚的差異以孤雌囊胚為對(duì)照,分別對(duì)去核、不去核核移植囊胚細(xì)胞進(jìn)行熒光染色計(jì)數(shù),發(fā)現(xiàn)孤雌囊胚平均細(xì)胞數(shù)最多(58.60),去核核移植囊胚平均細(xì)胞數(shù)最少(43.30),不去核核移植囊胚平均細(xì)胞數(shù)介于二者之間(56.95)。囊胚形態(tài)上,孤雌囊胚直徑較大,顏色較淺,去核與不去核核移植囊胚直徑較小,顏色較深,且不去核核移植囊胚多為孵化囊胚,呈8字形態(tài),而去核核移植囊胚多為擴(kuò)張囊胚,未孵化。4、卵母細(xì)胞機(jī)械損傷對(duì)核移植胚胎發(fā)育的影響考慮到去核過程會(huì)對(duì)卵母細(xì)胞造成機(jī)械損傷,因此,本實(shí)驗(yàn)通過對(duì)卵母細(xì)胞預(yù)先扎孔,并將卵核吸出再注回,模擬去核操作對(duì)卵母細(xì)胞造成的機(jī)械損傷,隨后進(jìn)行孤雌激活。同時(shí)將對(duì)照組卵母細(xì)胞僅做扎孔處理,不做卵核吸吐動(dòng)作,模擬不去核核移植胚胎操作,觀察兩種胚胎發(fā)育情況,發(fā)現(xiàn)機(jī)械損傷導(dǎo)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),囊胚細(xì)胞數(shù)減少(52.65 vs 55.40),但抑制作用不顯著。證明機(jī)械損傷這一單方面因素不是造成核移植胚胎發(fā)育能力低下的主要原因。5、染色體加倍對(duì)胚胎發(fā)育的影響以及不去核重構(gòu)胚中的卵核能否被體細(xì)胞核所取代將不去核重構(gòu)胚模型中的卵核用體細(xì)胞核進(jìn)行置換,再移入另一枚體細(xì)胞重新構(gòu)建多倍體胚胎,觀察其發(fā)育情況,發(fā)現(xiàn)體細(xì)胞核+體細(xì)胞+卵胞質(zhì)胚胎囊胚率僅有10.5%,遠(yuǎn)低于卵核+體細(xì)胞+卵胞質(zhì)胚胎囊胚率50.6%,說明在不去核重構(gòu)胚模型中,單純性染色體數(shù)目加倍無法促進(jìn)胚胎發(fā)育,卵母細(xì)胞核無法被體細(xì)胞核任意取代。6、卵巢類型與卵母細(xì)胞發(fā)育潛能的關(guān)系由于卵巢樣品取自市內(nèi)屠宰場(chǎng),樣品品系不確定,為保證測(cè)序樣品間遺傳背景一致,采取單卵巢采卵獨(dú)立培養(yǎng)。根據(jù)卵巢表面有無紅體將其分類,分別進(jìn)行卵子采集、成熟,觀察其發(fā)育潛能,發(fā)現(xiàn)有紅體卵巢采卵效率高,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)均高于沒有紅體的卵巢。但差異不顯著,確定可以選擇帶有紅體的卵巢進(jìn)行采卵,然后構(gòu)建核移植胚胎,用于后續(xù)基因表達(dá)圖譜的構(gòu)建實(shí)驗(yàn)。7、構(gòu)建去核、不去核重構(gòu)胚早期基因表達(dá)圖譜為探究卵核對(duì)體細(xì)胞重編程作用的分子機(jī)制,了解兩種重構(gòu)胚特有的基因表達(dá)模式,本研究采集了去核以及不去核重構(gòu)胚2cell(第一次卵裂)和4cell(發(fā)育阻滯期)時(shí)期胚胎進(jìn)行基因表達(dá)譜測(cè)序(RNA sequencing)。成功構(gòu)建兩種重構(gòu)胚各自特異基因表達(dá)圖譜。發(fā)現(xiàn)去核重構(gòu)胚2cell胚胎有大約28%的基因參與表達(dá),4cell胚胎大約22%的基因參與表達(dá)；不去核重構(gòu)胚2cell胚胎有大約30%的基因參與表達(dá),4cell胚胎有大約27%的基因參與表達(dá)。8、去核、不去核重構(gòu)胚阻滯前期差異表達(dá)基因篩選與分析將去核和不去核重構(gòu)胚相同時(shí)期胚胎基因表達(dá)圖譜進(jìn)行比對(duì),發(fā)現(xiàn)2cell階段,不去核重構(gòu)胚相比去核重構(gòu)胚有1738條基因呈現(xiàn)表達(dá)上調(diào),728條基因表達(dá)下調(diào)(｜log2Ratio｜≥5); 4cell階段,不去核重構(gòu)胚有2941條基因表達(dá)上調(diào),1682條基因表達(dá)下調(diào)(｜log2Ratio｜≥5);這些差異基因WEGO分析中富集程度最深的基因簇分別集中在綁定調(diào)控、催化和分子轉(zhuǎn)導(dǎo)活性上；其他在不去核重構(gòu)胚中呈現(xiàn)高表達(dá)的基因則多數(shù)參與各種代謝過程。其中,Ribosome和Oxidative phosphorylation兩條信號(hào)通路在去核重構(gòu)胚發(fā)育趨勢(shì)中以及不去核重構(gòu)胚發(fā)育趨勢(shì)中反復(fù)出現(xiàn)富集。在4cell阻滯期階段,Protein processing in endoplasmic reticulum信號(hào)通路顯著富集。由于內(nèi)質(zhì)網(wǎng)結(jié)構(gòu)與細(xì)胞核結(jié)構(gòu)緊密相連,并且附著大量核糖體結(jié)構(gòu),在核移植去核操作過程中可能連同細(xì)胞核一起被去除。因此推測(cè)內(nèi)質(zhì)網(wǎng)蛋白加工以及核糖體、氧化磷酸化功能的缺失可能是去核重構(gòu)胚較之不去核重構(gòu)胚發(fā)育劣勢(shì)的主要原因。9、核定位轉(zhuǎn)錄因子差異表達(dá)模式分析去核、不去核重構(gòu)胚差異表達(dá)基因中有267條轉(zhuǎn)錄活性相關(guān)基因在細(xì)胞核部位定位表達(dá),其中絕大多數(shù)在去核重構(gòu)胚中表達(dá)量微弱,但在不去核重構(gòu)胚中表達(dá)顯著上調(diào)。推測(cè)這些核定位表達(dá)的轉(zhuǎn)錄因子表達(dá)上調(diào)導(dǎo)致體細(xì)胞重編程加快,重編程效果更徹底。10、QRT-PCR基因表達(dá)量驗(yàn)證在RNA-seq差異基因中隨機(jī)挑選了6個(gè)表達(dá)量較高的基因DNMT1、 FTL、POLRID、RPS3、RPS20、BUB3分別進(jìn)行QRT-PCR檢測(cè)。將檢測(cè)結(jié)果與RNA-seq結(jié)果比對(duì)后發(fā)現(xiàn),6個(gè)基因在兩種檢測(cè)方法下表達(dá)趨勢(shì)基本一致。11、內(nèi)質(zhì)網(wǎng)熒光示蹤檢驗(yàn)使用內(nèi)質(zhì)網(wǎng)紅色熒光探針ER-Tracker Red對(duì)成熟MⅡ期卵母細(xì)胞、去核后卵母細(xì)胞分別進(jìn)行染色,比較兩種細(xì)胞的內(nèi)質(zhì)網(wǎng)結(jié)構(gòu),檢驗(yàn)卵母細(xì)胞內(nèi)質(zhì)網(wǎng)在核移植去卵核操作過程中是否被去除。
[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.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S814

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