当前位置:主页 > 医学论文 > 畜牧兽医论文 >

绵羊诱导多能性干细胞的制作与鉴定研究

发布时间:2018-03-06 08:50

  本文选题:诱导多能干细胞(iPS细胞) 切入点:电穿孔转染 出处:《石河子大学》2015年硕士论文 论文类型:学位论文


【摘要】:2006年,山中申弥利用病毒载体将四个转录因子(Oct4、Sox2、Klf4和c-Myc)感染小鼠体细胞,获得了类胚胎干细胞的诱导多潜能性干细胞(i PS细胞),为细胞学研究提供了新的方向,也为再生医学领域带来了广阔的应用前景。动物i PS细胞的出现对疾病模型的建立、细胞治疗,种质资源的保存、改善体细胞克隆动物的生产效率等方面都有着重要的意义。目的:在无饲养层条件下建立电穿孔转染五因子制备绵羊i PS细胞,掌握i PS细胞诱导这一技术,为进一步研究细胞重编程机制及未来i PS细胞的临床应用奠定基础。方法:1本实验采用组织培养法,双酶法分离出中国美利奴成纤维细胞,并进行传代培养。2将含有人的Oct4;Sox2、Klf4;l-Myc、lin28基因的3个质粒,分别采用4-D电转染仪中EH-100,EN-150,CZ-167,CA-137四个电转程序转染到绵羊成纤维细胞中,筛选出最适合绵羊成纤维细胞的电转染程序;细胞密度和质粒浓度均相同,质粒浓度不同对AP染色阳性(AP+)克隆的影响;细胞密度,质粒浓度均一致,质粒比例不同,对AP+克隆的影响;细胞密度,质粒浓度,质粒浓度均一致,培养液中有无添加Vc,对AP+克隆的影响,以选出最适合制备i PS的方案。3通过外形观察、定量PCR、免疫荧光染色、转录组分析等来检测所制备的绵羊类i PS细胞。结果与结论:(1)双酶法快速成功分离出中国美利奴绵羊耳源成纤维细胞,为重编程的提供优良的体细胞。(2)采用EH-100电转程序,细胞密度为5×106个/m L,质粒比例1:1:1,质粒质量浓度为0.1 mg/L,培养液中添加0.05 mg/L Vc条件下,AP染色阳性(AP+)克隆形成率达14%,优化的电转染的方法和培养方案有效提升了绵羊类诱导性多潜能干细胞的制备效率。(3)在无饲养层条件下,通过电转染法将五因子导入绵羊耳源成纤维细胞,并成功重编程为类i PS细胞,在外观形态、多能基因的表达、转录组等方面的鉴定结果都表明其具有多潜能性,为以后进一步研究绵羊多潜能干细胞机制奠定了基础。
[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.
【学位授予单位】:石河子大学
【学位级别】:硕士
【学位授予年份】:2015
【分类号】:S826

【参考文献】

相关期刊论文 前2条

1 覃兆鲜;潘天彪;谢炳坤;;猪成纤维细胞转染方法的比较[J];江苏农业科学;2011年03期

2 钱锋,肖成组;影响细胞电穿孔效率的因素[J];生物技术;1998年05期



本文编号:1574140

资料下载
论文发表

本文链接:https://www.wllwen.com/yixuelunwen/dongwuyixue/1574140.html


Copyright(c)文论论文网All Rights Reserved | 网站地图 |

版权申明:资料由用户7834c***提供,本站仅收录摘要或目录,作者需要删除请E-mail邮箱bigeng88@qq.com