發(fā)布時(shí)間：2018-04-26 06:06

  本文選題：綿羊 + Sox2基因��； 參考：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：哺乳動(dòng)物體細(xì)胞克隆技術(shù)自誕生以來,發(fā)展迅速且日益趨于成熟,但體細(xì)胞克隆效率低始終是制約其在畜牧業(yè)生產(chǎn)中應(yīng)用與發(fā)展的瓶頸。核不完全重編程及供體細(xì)胞的類型與克隆效率密切相關(guān)。表觀遺傳修飾中DNA甲基化、組蛋白乙�；怯绊懞酥鼐幊痰年P(guān)鍵因素。研究證實(shí),sox2基因?qū)S持細(xì)胞多能性及胚胎著床后的發(fā)育起關(guān)鍵的作用,以高表達(dá)sox2基因的神經(jīng)干細(xì)胞為核供體進(jìn)行核移植時(shí)具有較高的克隆效率。為此,本研究以內(nèi)蒙古地區(qū)優(yōu)勢(shì)畜種綿羊?yàn)閷?shí)驗(yàn)對(duì)象,選擇綿羊骨髓間充質(zhì)干細(xì)胞(Bone Marrow-derived Mesenchymal Stem Cells, BMSCs)、脂肪間充質(zhì)干細(xì)胞(Adipose-derived Stromal Cells,ADSCs)和皮膚成纖維細(xì)胞(Sheep Epidermal Fibroblast Cells, SEFCs)為核供體,經(jīng)電轉(zhuǎn)染將sox2基因?qū)隑MSCs和ADSCs和SEFCs,檢測(cè)sox2基因?qū)θ惡斯w細(xì)胞DNA甲基轉(zhuǎn)移酶1(DNA Methyl transferase 1，，DNMT1)和組蛋白去乙�；�2(Histone Deacetylase 2, HDAC2)基因的表達(dá)水平,分析了sox2基因?qū)θ惡斯w細(xì)胞表觀遺傳修飾的影響。同時(shí)以導(dǎo)入sox2基因的BMSCs為核供體構(gòu)建重構(gòu)胚,以SEFCs核供體構(gòu)建的重構(gòu)胚為對(duì)照,分析了sox2基因?qū)χ貥?gòu)胚表觀遺傳修飾及發(fā)育能力的影響。主要研究結(jié)果如下：(1)分離得到了綿羊BMSCs, ADSCs和SEFCs細(xì)胞系,三類細(xì)胞生長(zhǎng)曲線均呈“S”型;經(jīng)RT-PCR檢測(cè),BMSCs和ADSCs均表達(dá)了干細(xì)胞特異因子Sox2、Oct4和Nanog基因。(2)構(gòu)建了pEGFPS-N1-5bx2真核表達(dá)載體,對(duì)BMSCs、ADSCs和SEFCs三類細(xì)胞進(jìn)行了電轉(zhuǎn)染,經(jīng)篩選得到過表達(dá)sox2基因細(xì)胞株,經(jīng)實(shí)時(shí)定量PCR檢測(cè),三種過表達(dá)sox2基因細(xì)胞株中的DNMT1和HDAC2基因表達(dá)量表現(xiàn)出不同程度的下降,DNNT1分別下降多少68%、25%、43%；HDAC2下降了15%左右。(3)以過表達(dá)sox2基因BMSCs為核供體,構(gòu)建重構(gòu)胚159個(gè),以SEFCs為核供體構(gòu)建重構(gòu)胚182個(gè)。檢測(cè)兩類重構(gòu)胚中DNMT1和HDAC2的表達(dá)量,過表達(dá)sox2基因BMSCs為供體的克隆胚比以SEFCs構(gòu)建的克隆胚中DNMT1和HDAC2的表達(dá)量低50%左右,由此推測(cè)sox2基因的過表達(dá)可能有利于克隆胚核的重編程,進(jìn)而促進(jìn)重構(gòu)胚的發(fā)育。以上結(jié)果對(duì)闡明sox2基因影響不同核供體細(xì)胞表觀遺傳修飾的機(jī)制奠定了必要的基礎(chǔ),為通過選擇及對(duì)不同核供體細(xì)胞進(jìn)行遺傳修飾從而提高體細(xì)胞克隆效率提供了新的方法,進(jìn)而為日后利用轉(zhuǎn)基因體細(xì)胞克隆技術(shù)進(jìn)行優(yōu)質(zhì)綿羊種畜的改良及高效快繁提供了科學(xué)依據(jù)。
[Abstract]:Since the birth of mammalian somatic cell cloning technology, it has developed rapidly and become more and more mature, but the low efficiency of somatic cell cloning has always been the bottleneck of its application and development in animal husbandry. Incomplete nuclear reprogramming and donor cell types are closely related to cloning efficiency. DNA methylation and histone acetylation are key factors affecting nuclear reprogramming in epigenetic modification. It has been proved that sox2 gene plays a key role in maintaining the pluripotency of the cells and the development of embryos after implantation. The neural stem cells with high expression of sox2 gene are used as nuclear donors for nuclear transplantation with high cloning efficiency. Therefore, in this study, the dominant cattle sheep in Inner Mongolia were selected as nuclear donors, including bone Marrow-derived Mesenchymal Stem Cells, BMSCs, adipose mesenchymal stem cells (Adipose-derived Stromal CellsCellsCellsASCs) and skin fibroblasts (Sheep Epidermal Fibroblast Cells, SEFCs) from sheep bone marrow mesenchymal stem cells (BMSCs), adipose mesenchymal stem cells (Adipose-derived Stromal cells) and skin fibroblasts (Sheep Epidermal Fibroblast Cells, SEFCs). Sox2 gene was transfected into BMSCs, ADSCs and SEFCs.The expression of sox2 gene on DNA methyltransferase 1(DNA Methyl transferase 1 (DNMT1) and histone deacetylase 2(Histone Deacetylase 2 (HDAC2) in three kinds of nuclear donor cells was detected. The effect of sox2 gene on epigenetic modification of three nuclear donor cells was analyzed. At the same time, the effect of sox2 gene on the epigenetic modification and developmental ability of reconstructed embryos was analyzed by using BMSCs as nuclear donor and SEFCs nuclear donor as control. The main results were as follows: (1) Sheep BMSCs, ADSCs and SEFCs cell lines were isolated, and the three cell growth curves were all "S" type, and the pEGFPS-N1-5bx2 eukaryotic expression vector was constructed by RT-PCR detection of the stem cell specific factor Sox2Oct4 and Nanog gene. After electrotransfection of BMSCs cells and SEFCs cells, overexpression of sox2 gene was obtained and detected by real time quantitative PCR. The expression of DNMT1 and HDAC2 genes in three kinds of over-expressed sox2 gene cell lines showed a decrease of 68%, and that of DNNT1 decreased by about 15%, respectively. The over expressed sox2 gene BMSCs was used as nuclear donor to construct 159 reconstructed embryos. 182 reconstructed embryos were constructed with SEFCs as core donor. The expression of DNMT1 and HDAC2 in two kinds of reconstructed embryos was detected. The expression of DNMT1 and HDAC2 in cloned embryos with overexpression of sox2 gene BMSCs was about 50% lower than that of cloned embryos constructed with SEFCs, which suggested that the overexpression of sox2 gene might be beneficial to the nuclear reprogramming of cloned embryos. Furthermore, it promotes the development of reconstructed embryos. These results provide a necessary basis for elucidating the mechanism of sox2 gene affecting epigenetic modification of different nuclear donor cells, and provide a new method for improving the cloning efficiency of somatic cells through selection and genetic modification of different nuclear donor cells. It provides a scientific basis for the improvement and efficient rapid propagation of high quality sheep breeds by using transgenic somatic cell cloning technology in the future.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q813

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