本文選題：誘導(dǎo)多能干細(xì)胞(iPS細(xì)胞)　切入點(diǎn)：電穿孔轉(zhuǎn)染　出處：《石河子大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：2006年,山中申彌利用病毒載體將四個(gè)轉(zhuǎn)錄因子(Oct4、Sox2、Klf4和c-Myc)感染小鼠體細(xì)胞,獲得了類胚胎干細(xì)胞的誘導(dǎo)多潛能性干細(xì)胞(i PS細(xì)胞),為細(xì)胞學(xué)研究提供了新的方向,也為再生醫(yī)學(xué)領(lǐng)域帶來了廣闊的應(yīng)用前景。動(dòng)物i PS細(xì)胞的出現(xiàn)對(duì)疾病模型的建立、細(xì)胞治療,種質(zhì)資源的保存、改善體細(xì)胞克隆動(dòng)物的生產(chǎn)效率等方面都有著重要的意義。目的:在無飼養(yǎng)層條件下建立電穿孔轉(zhuǎn)染五因子制備綿羊i PS細(xì)胞,掌握i PS細(xì)胞誘導(dǎo)這一技術(shù),為進(jìn)一步研究細(xì)胞重編程機(jī)制及未來i PS細(xì)胞的臨床應(yīng)用奠定基礎(chǔ)。方法:1本實(shí)驗(yàn)采用組織培養(yǎng)法,雙酶法分離出中國(guó)美利奴成纖維細(xì)胞,并進(jìn)行傳代培養(yǎng)。2將含有人的Oct4;Sox2、Klf4;l-Myc、lin28基因的3個(gè)質(zhì)粒,分別采用4-D電轉(zhuǎn)染儀中EH-100,EN-150,CZ-167,CA-137四個(gè)電轉(zhuǎn)程序轉(zhuǎn)染到綿羊成纖維細(xì)胞中,篩選出最適合綿羊成纖維細(xì)胞的電轉(zhuǎn)染程序;細(xì)胞密度和質(zhì)粒濃度均相同,質(zhì)粒濃度不同對(duì)AP染色陽(yáng)性(AP+)克隆的影響;細(xì)胞密度,質(zhì)粒濃度均一致,質(zhì)粒比例不同,對(duì)AP+克隆的影響;細(xì)胞密度,質(zhì)粒濃度,質(zhì)粒濃度均一致,培養(yǎng)液中有無添加Vc,對(duì)AP+克隆的影響,以選出最適合制備i PS的方案。3通過外形觀察、定量PCR、免疫熒光染色、轉(zhuǎn)錄組分析等來檢測(cè)所制備的綿羊類i PS細(xì)胞。結(jié)果與結(jié)論:(1)雙酶法快速成功分離出中國(guó)美利奴綿羊耳源成纖維細(xì)胞,為重編程的提供優(yōu)良的體細(xì)胞。(2)采用EH-100電轉(zhuǎn)程序,細(xì)胞密度為5×106個(gè)/m L,質(zhì)粒比例1:1:1,質(zhì)粒質(zhì)量濃度為0.1 mg/L,培養(yǎng)液中添加0.05 mg/L Vc條件下,AP染色陽(yáng)性(AP+)克隆形成率達(dá)14%,優(yōu)化的電轉(zhuǎn)染的方法和培養(yǎng)方案有效提升了綿羊類誘導(dǎo)性多潛能干細(xì)胞的制備效率。(3)在無飼養(yǎng)層條件下,通過電轉(zhuǎn)染法將五因子導(dǎo)入綿羊耳源成纖維細(xì)胞,并成功重編程為類i PS細(xì)胞,在外觀形態(tài)、多能基因的表達(dá)、轉(zhuǎn)錄組等方面的鑒定結(jié)果都表明其具有多潛能性,為以后進(jìn)一步研究綿羊多潛能干細(xì)胞機(jī)制奠定了基礎(chǔ)。
[Abstract]:In 2006, Yamanaka used virus vectors to infect mouse somatic cells with four transcription factors Oct4N Sox2Klf4 and c-Myc.Induced pluripotent stem cells were obtained from embryonic stem cells, which provided a new direction for cytological research. The emergence of animal I PS cells has brought about the establishment of disease models, cell therapy, and preservation of germplasm resources. It is of great significance to improve the productivity of somatic cloned animals. Objective: to establish electroporation transfection of five factors to prepare sheep iPS cells without feeding layer, and to master the technique of inducing iPS cells. In order to further study the mechanism of cell reprogramming and the clinical application of iPS cells in the future, Chinese merino fibroblasts were isolated by using tissue culture method and double enzymatic method. The three plasmids containing human Oct4nSox2Klf4nl-Myclin28 gene were transfected into sheep fibroblasts by four electrotransposes (EH-100, EN-150, CZ-167, CA-137) respectively, and the most suitable electrotransfection procedure was selected for ovine fibroblasts, and the results showed that the three plasmids containing human Oct4nSox2Klf4hl-Myclin28 gene were transfected into sheep fibroblasts by four electrotransposes (EH-100, EN-150, CZ-167, CA-137). The cell density and plasmid concentration are the same, the effect of plasmid concentration on AP clone is same, the cell density and plasmid concentration are the same, the proportion of plasmids is different, the cell density, plasmid concentration, cell density, plasmid concentration, The concentration of plasmids was the same, and the effect of Vcaddition on AP clone was found in the culture medium. In order to select the most suitable method for preparing iPS, the best method was observed by shape, quantitative PCR, immunofluorescence staining. Results and conclusion the Chinese Merino sheep ear derived fibroblasts were isolated by double enzyme method, which provided excellent somatic cells for reprogramming. Results and conclusion the EH-100 electrotransposition procedure was used. The cell density was 5 脳 106 / mL, the plasmid ratio was 1: 1: 1, the plasmid concentration was 0.1 mg / L, and the colony forming rate was 14% under the condition of adding 0.05 mg/L VC to the culture medium. Preparation efficiency of inducible multipotential stem cells. Five factors were introduced into sheep ear fibroblasts by electrotransfection and successfully reprogrammed as iPS cells. The identification results of appearance, expression of pluripotent genes and transcriptome showed that they had multipotential. It will lay a foundation for further study of sheep multipotential stem cell mechanism.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S826

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本文編號(hào)：1574140