本文選題：豬 + 豬類胚胎干細(xì)胞�。� 參考：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：胚胎干細(xì)胞是研究基因功能、建立疾病模型和促進(jìn)再生醫(yī)學(xué)領(lǐng)域發(fā)展的一種重要工具。豬由于生理結(jié)構(gòu)與人類相似,長期以來被人們作為疾病模型廣泛的應(yīng)用于臨床研究中,因此豬胚胎干細(xì)胞的建立對于生物醫(yī)學(xué)領(lǐng)域有著重大意義。不僅如此,豬作為農(nóng)業(yè)經(jīng)濟(jì)動物,其胚胎干細(xì)胞的建立可以在基因工程水平上來幫助改善豬的健康水平和生長性狀提高經(jīng)濟(jì)效益。但在豬胚胎干細(xì)胞的研究中,人們始終沒有獲得一株真正的胚胎干細(xì)胞系,同時豬胚胎干細(xì)胞所需要的培養(yǎng)條件也不能確定,在2014年我們實驗組獲得了一株豬類胚胎干細(xì)胞系,512506具有一定的干細(xì)胞特點,其培養(yǎng)液(MXV培養(yǎng)液),經(jīng)研究是一組比較適合豬類胚胎干細(xì)胞生長的干細(xì)胞培養(yǎng)液。但是由于其成分過于復(fù)雜,操作性差,并不利于豬胚胎干細(xì)胞的進(jìn)一步研究。在豬胚胎干細(xì)胞培養(yǎng)條件的探索中,人們依據(jù)大鼠胚胎干細(xì)胞的成功,2i體系也得到了廣泛的應(yīng)用。但卻存在著相反的兩種作用效果。本實驗針對MXV培養(yǎng)液的缺點,我們對其進(jìn)行了優(yōu)化,找出MXV培養(yǎng)液中必需成分。并探索了2i對豬胚胎干細(xì)胞建系的影響。首先是針對MXV培養(yǎng)液的五種主要成分bFGF,hLif,BSA,N2B27,KOSR,進(jìn)行逐一簡化,分別通過半量降低這五種成分的培養(yǎng)液和全量降低這五種成分的培養(yǎng)液分別培養(yǎng)512506細(xì)胞系最終可以去掉MXV培養(yǎng)液中的不重要成分。得到成分簡化的MXV培養(yǎng)液,隨后,我們對MXV培養(yǎng)液中基礎(chǔ)培養(yǎng)液進(jìn)行優(yōu)化,X中的基礎(chǔ)培養(yǎng)液為三種基礎(chǔ)培養(yǎng)液Neurobasal,KO-DMEM,和DMEM/F1的混合物。我們用單一的培養(yǎng)液成分來替代這個混合體系。最后可以獲得簡化的MXV培養(yǎng)液。為了進(jìn)一步確定簡化的MXV培養(yǎng)液是否可以完全替代MXV培養(yǎng)液,本實驗探索了簡化MXV培養(yǎng)液對豬類胚胎干細(xì)胞建系效率的影響。同時針對CHIR99021,PD0325901簡稱2i對于有豬多能性細(xì)胞研究中相對立的研究結(jié)果,我們探究了2i對豬胚胎干細(xì)胞建系效率的影響。經(jīng)過上述內(nèi)容研究的結(jié)果顯示:KOSR可以從MXV培養(yǎng)液中去除獲得MXV-K培養(yǎng)液,不添加KOSR的優(yōu)化MXV培養(yǎng)液可以使512506的細(xì)胞狀態(tài)更好,細(xì)胞排列緊密,脂滴消失,形態(tài)更接近人胚胎干細(xì)胞系。其他三組主要成分雖然沒有bFGF對MXV培養(yǎng)液那么大的影響,但去除后,512506細(xì)胞系狀態(tài)仍然會變差。所以在五種成分中我們只能將KOSR去除.而針對基礎(chǔ)培養(yǎng)液的簡化,最終可獲得兩組單一的基礎(chǔ)培養(yǎng)液KO-DMEM和DMEM/F12的優(yōu)化MXV培養(yǎng)液,可以完全代替MXV培養(yǎng)液培養(yǎng)512506細(xì)胞系,獲得的512506P5K和512506P5D在形態(tài)上比512506細(xì)胞系更接近人胚胎干細(xì)胞系,多能性狀態(tài)與512506細(xì)胞系相似。在建系效率的研究中,實驗結(jié)果顯示發(fā)育至6d的體外受精胚胎在KO-DMEM培養(yǎng)液中貼壁率和原代克隆形成率顯著高于DMEM/F12培養(yǎng)液,添加2i后兩種培養(yǎng)液中的胚胎貼壁率和原代克隆形成率均下降。在KO-DMEM培養(yǎng)液中可獲得穩(wěn)定傳代的細(xì)胞系,細(xì)胞形態(tài)與512506細(xì)胞系不同,無脂滴,細(xì)胞排列緊密,形態(tài)更類似與人胚胎干細(xì)胞系。細(xì)胞系呈堿性磷酸酶陽性,核型正常,表達(dá)Oct4、Sox2和Nanog,不表達(dá)Cdx2。細(xì)胞系可成功進(jìn)行轉(zhuǎn)基因操作。結(jié)果表明以KO-DMEM為基礎(chǔ)培養(yǎng)液的優(yōu)化MXV培養(yǎng)液體系可以獲得豬類胚胎干細(xì)胞,2i不利于胚胎貼壁和原代克隆形成。該研究為豬胚胎干細(xì)胞建系培養(yǎng)體系選擇與推斷豬胚胎干細(xì)胞多能性細(xì)胞信號調(diào)控通路提供了重要的實驗依據(jù)。
[Abstract]:Embryonic stem cells (SSCs) are an important tool to study gene function, establish disease models and promote the development of regenerative medicine. Pigs have been widely used as disease models in clinical research for a long time because of their similar physiological structure. Therefore, the establishment of pig embryonic stem cells is of great significance in the field of biomedicine. In this case, as an agricultural economy animal, the establishment of embryonic stem cells can help improve the health level and growth traits of pigs at the level of genetic engineering. However, in the study of pig embryonic stem cells, people have never obtained a real embryonic stem cell line, and the need for the cultivation of pig embryonic stem cells. The conditions were also undetermined. In 2014, a pig like embryonic stem cell line was obtained in our experimental group. 512506 had a certain characteristic of stem cells. The culture solution (MXV Culture) was a group of stem cell culture medium suitable for the growth of pig embryonic stem cells. But because of its complexity and poor operability, it was not beneficial to pig embryos. Further research on stem cells. In the exploration of the culture conditions of the pig embryonic stem cells, the 2I system has also been widely used on the basis of the success of the rat embryonic stem cells. But there are two kinds of effects. In this experiment, we have optimized the MXV culture solution to find out the essential components in the MXV culture solution. The effect of 2I on the construction of pig embryonic stem cell line was explored. First, the five main components of MXV culture solution, bFGF, hLif, BSA, N2B27, KOSR, were simplified one by one. The culture solution of the five components was reduced by half quantity and the 512506 cell lines of the five components were cultured respectively to remove the MXV culture solution. The important ingredient. We obtained the simplified MXV culture solution. Then, we optimized the basic culture medium in the MXV culture. The basic medium in X was the mixture of three basic cultures, Neurobasal, KO-DMEM, and DMEM/F1. We used a single culture medium to replace the mixture. Finally, we can obtain a simplified MXV culture. It is further determined whether the simplified MXV culture solution can completely replace the MXV culture solution. This experiment explored the effect of the simplified MXV culture on the establishment of the pig embryonic stem cell line construction efficiency. At the same time, we explored the establishment of 2I on the construction of pig embryonic stem cells according to the relative research results of CHIR99021 and PD0325901 for abbreviation of 2I in the study of porcine pluripotent cells. The results of this study showed that KOSR could remove MXV-K culture from MXV culture, and the optimized MXV culture solution without KOSR could make the 512506 cell state better, the cells were arranged closely, the lipid droplets disappeared, and the form was closer to the human embryonic stem cell line. The other three main components were not bFGF to MX. The effect of V culture is so great, but after removal, the state of the 512506 cell line will still be worse. So we can only remove the KOSR in the five components. For the simplification of the basic culture solution, we can finally obtain two sets of single basic culture medium KO-DMEM and DMEM/F12 optimized MXV culture, which can completely replace the MXV culture medium to cultivate 512506 cell lines. The obtained 512506P5K and 512506P5D were much closer to the human embryonic stem cell line than the 512506 cell line, and the pluripotent state was similar to that of the 512506 cell line. In the study of the building efficiency, the experimental results showed that the adherence rate and the primary clone formation rate of the in vitro fertilized embryos developed to 6D were significantly higher than that of the DMEM/F12 culture. After adding 2I, the rate of adherence to the embryo and the formation rate of primary clones in the two cultures decreased. In the KO-DMEM culture, a stable cell line was obtained. The cell morphology was different from that of the 512506 cell line. The cell line was closely related to the human embryonic stem cell line. The cell line was similar to the human embryonic stem cell line. The cell lines were alkaline phosphatase, the karyotype was normal, the expression of Oct4, S Ox2 and Nanog did not express the Cdx2. cell line successfully. The results showed that the optimized MXV culture liquid system with KO-DMEM as the base medium could obtain the pig embryonic stem cells, and 2I was not conducive to the formation of the embryo and the primary clones. The regulation of sex cell signaling provides an important experimental basis.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S828

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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2 Chunjing Feng;Yun-Dan Jia;Xiao-Yang Zhao;;Pluripotency of Induced Pluripotent Stem Cells[J];Genomics,Proteomics & Bioinformatics;2013年05期

3 Guangjun Xi;Pingfang Hu;Cunye Qu;Shenfeng Qiu;Chang Tong;Qi-Long Ying;;Induced Neural Stem Cells Generated from Rat Fibroblasts[J];Genomics,Proteomics & Bioinformatics;2013年05期

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7 王喜艷;潘曉燕;李質(zhì)馨;王雪楠;;哺乳動物囊胚孵化調(diào)控機(jī)制的研究進(jìn)展[J];軍事醫(yī)學(xué);2014年09期

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相關(guān)博士學(xué)位論文 前10條

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2 鄭喜邦;穩(wěn)定表達(dá)牛Nanog基因的成纖維細(xì)胞制作、多能性分析及轉(zhuǎn)基因克隆胚胎構(gòu)建[D];西北農(nóng)林科技大學(xué);2008年

3 叢義梅;豬原始生殖細(xì)胞體內(nèi)跟蹤、體外培養(yǎng)及遺傳印記的研究[D];東北農(nóng)業(yè)大學(xué);2013年

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8 趙宇航;ZFNs介導(dǎo)的牛MSTN位點的基因打靶研究[D];內(nèi)蒙古大學(xué);2014年

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10 楊翌;應(yīng)用類轉(zhuǎn)錄激活因子效應(yīng)物核酸酶（TALEN）定點修飾豬基因組[D];吉林大學(xué);2014年

相關(guān)碩士學(xué)位論文 前10條

1 張曼玲;豬單倍體孤雌胚胎和體外受精胚胎干細(xì)胞培養(yǎng)的研究[D];內(nèi)蒙古大學(xué);2011年

2 陳博;LIF和bFGF對維持綿羊類胚胎干細(xì)胞自我更新和多潛能性候選基因的影響[D];新疆農(nóng)業(yè)大學(xué);2011年

3 陳濤;豬胚胎體外生產(chǎn)和孤雌囊胚發(fā)育能力的檢測[D];安徽農(nóng)業(yè)大學(xué);2009年

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5 宋志強(qiáng);豬胚胎干細(xì)胞分離培養(yǎng)與OCT4-eGFP示蹤體系細(xì)胞篩選[D];東北農(nóng)業(yè)大學(xué);2013年

6 李少維;從原始卵泡培育MⅡ卵制備胚胎干細(xì)胞的可行性研究[D];福建醫(yī)科大學(xué);2013年

7 黃子明;肝腫瘤細(xì)胞內(nèi)Sox17基因mRNA的表達(dá)水平與細(xì)胞遷移、侵襲力的相關(guān)性研究[D];安徽醫(yī)科大學(xué);2013年

8 艾志營;CHIR99021通過激活Wnt通路維持小鼠F9細(xì)胞中Klf4表達(dá)[D];西北農(nóng)林科技大學(xué);2013年

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10 趙無恙;胚胎干細(xì)胞在肝臟微環(huán)境下成瘤性的研究[D];浙江理工大學(xué);2014年

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