人诱导性多潜能干细胞（iPS）系的建立
发布时间：2019-06-22 13:17
【摘要】： 2006年8月,Yamanaka小组将24种转录因子基因排列组合导入小鼠成纤维细胞,最终确定最少有4种转录因子组合——3ct4、Sox2、c-Myc和Klf4即可将成纤维细胞重编程为诱导性多潜能干(iPS)细胞,2007年11～12月,Yamanaka小组和Thomson小组先后将人的体细胞重编程为iPS细胞。这之后iPS细胞的研究和关注度呈爆炸式增长,且取得了一些突破性进展,如建立了疾病特异的人iPS细胞、借助转座子介导的转基因方法高效制备了virus-free iPS细胞以及成功地从所获得的iPS细胞中移除先前导入的转录因子基因。在利用特定小分子化合物的情况下,更少的外源基因导入即可高效率获得iPS细胞,这向制备无遗传修饰的iPS细胞方面迈出了一大步；此外,亦建立了大鼠和猴等的iPS细胞系,这些成绩将iPS细胞在临床上的实际应用又大大向前推进了一步,iPS细胞研究和应用将有望成为21世纪最伟大的医学生物学成就之一。 研究肿瘤细胞重编程的经典方法有：①利用胚胎微环境,②囊胚腔注射,③利用胚胎干细胞生长微环境。iPS细胞技术的问世为研究肿瘤细胞重编程提供了一种新的思路和手段。利用Oct4、Sox2、C-myc和Klf4即可将肿瘤细胞重编程,2009年7月Hochedlinger课题组用Oct4、Sox2、c-Myc和Klf4将小鼠黑色素瘤细胞重编程为iPS细胞。iPS技术为在体外研究肿瘤细胞重编程提供了重要的技术平台,为研究肿瘤发病机制及肿瘤治疗等方面提供一种新思路。 本课题借助慢病毒基因投递法将Oct4、Sox2、Klf4和c-Myc四种基因导入人皮肤成纤维(CCD)细胞,进而将其重编程为iPS细胞,从而实现以下目的：①为体外研究肿瘤细胞重编程构筑技术平台；②为后续研究[如iPS细胞生物学特性和行为(如自我复制、增殖和分化等)调控机制研究]奠定基础。 目的： 借助慢病毒载体法将Oct4、Sox2、Klf4和c-Myc四种基因导入CCD细胞,进而将其重编程为iPS细胞,以实现多种目的(见上)。 方法： 1)携带Oct4、Sox2、C-Myc和Klf4基因的慢病毒载体鉴定 酶切鉴定分别用BamHⅠ/KpnⅠ和EcoRⅠ/EcoRⅤ进行酶切,酶切产物进行1%琼脂糖凝胶电泳。 PCR鉴定根据Oct4、Klf4、c-Myc和Sox2序列设计引物分别扩增Oct4、Klf4、c-Myc和Sox2基因；PCR扩增后,取5μl反应液进行2%琼脂糖凝胶电泳。 2)慢病毒生产与滴度测定 慢病毒包装按标准程序进行慢病毒包装(脂质体介导的转染)。 慢病毒滴度测定用5μl病毒上清感染15万293T细胞,48h后4%多聚甲醛固定,DAPI染色,荧光显微镜下计数EGFP阳性细胞和总细胞数,将其带入公式：病毒滴度(IU/ml)=EGFP阳性细胞数／总细胞数÷5×1.5x105×103。 3)慢病毒载体法重编程CCD细胞为iPS细胞 a)取一定量的病毒上清过滤后悬浮感染CCD细胞。 b)病毒感染24小时后,将CCD细胞消化铺于feeder细胞上,换为hES细胞培养基,第十天开始换为条件培养基。 c)20天左右开始挑取克隆,选择AP阳性细胞继续后续工作。 4)人iPS细胞建系及其生物学特性鉴定 a)iPS细胞克隆形态观察； b)细胞免疫荧光检测人ES细胞标志性抗原表达； c)提取iPS细胞总RNA,进而RT-PCR检测人ES细胞标志性基因表达； d)人iPS细胞核型分析； e)悬浮培养观察拟胚体形成及体外分化； f)iPS细胞接种至SCID小鼠皮下,观察畸胎瘤形成及体内分化情况。 结果： 1)携带Oct4、Sox2、C-Myc和Klf4基因的慢病毒载体鉴定 酶切鉴定携带Oct4、Sox2、C-Myc和Klf4基因的慢病毒载体分别经BamH I/KpnⅠ和EcoRⅠ／EcoRⅤ酶切,酶切产物经1%琼脂糖凝胶电泳均可见两条预期大小的条带。 PCR鉴定分别以携带Oct4、Sox2、C-Myc和Klf4基因的慢病毒载体为模板,扩增Oct4、Klf4、c-Myc和Sox2基因,PCR产物大小均与预期值相符。 2)慢病毒生产与滴度测定 携带Oct4、Sox2、C-Myc和Klf4基因的慢病毒载体与病毒包装质粒共转染293T细胞,24h后倒置荧光显微镜下可见绿色荧光,预示转染成功。按照上述.方法进行慢病毒滴度测定,病毒滴度值都在1×106IU/ml以上。 3)慢病毒载体法重编程CCD细胞为iPS细胞 用携带Oct4、Sox2、C-Myc和Klf4基因的慢病毒感染CCD细胞,24h后种植于饲养层(feeder)细胞上,并换为hES细胞培养基,48-72h在倒置荧光显微镜下观察到绿色荧光,第十天左右开始换为条件培养基。 4)人iPS细胞建系及其生物学特性鉴定 a)第7-8天可见CCD细胞形态发生变化,由长梭形变为圆形并聚集；第20天左右挑取克隆； b)其中只有1株iPS细胞AP染色呈阳性,且具有典型的hES细胞克隆形态； c)1株人iPS细胞系表达hES细胞特有的标志性基因； d)1株人iPS细胞系表达hES细胞特有的标志性抗原：SSAE-4(+), TRA-1-60(+),TRA-1-81(+),Oct4(+),SSAE-1(-)； e) 1株人iPS细胞核型分析未见异常； f) 1株人iPS细胞系具有体外分化形成囊性拟胚体的能力,并能表达各胚层的标志性基因； g)畸胎瘤实验目前还在进行中。 结论： 1.运用Oct4、Sox2、c-Myc和Klf4四种基因成功将CCD细胞重编程为iPS细胞。 2.1株人iPS细胞系具有人ES细胞的一些生物学特,且在体外长期传代中能够维持此特性,初步证明建立了人iPS细胞系。
[Abstract]:In August 2006, the Yamanaka group introduced 24 transcription factor genes into the mouse fibroblast, and finally determined the least four transcription factor combinations _ 3ct4, Sox2, c-Myc and Klf4 to reprogram the fibroblasts to the induced pluripotent stem (iPS) cells, in November to December 2007, The Yamanaka group and the Thomson group have reprogrammed human body cells to iPS cells. After this, the research and attention of iPS cells is explosive, and some breakthrough progress has been made, such as the establishment of a disease-specific human iPS cell, The virus-free iPS cells were efficiently prepared by the transposon-mediated transgene method and the previously introduced transcription factor genes were successfully removed from the obtained iPS cells. In the case of using a specific small molecule compound, an iPS cell can be obtained with high efficiency with less foreign gene introduction, which is a significant step in the preparation of iPS cells without genetic modification; in addition, an iPS cell line such as a rat and a monkey is also established, These results have further advanced the practical application of iPS cells, and the research and application of iPS cells will be expected to be one of the greatest medical biological achievements in the 21 st century. The classical methods to study the reprogramming of the tumor cells are: the use of the embryo microenvironment, the injection of the embryo cavity, the growth of the embryo stem cells, The advent of iPS cell technology provides a new way to study the reprogramming of tumor cells And the tumor cells can be reprogrammed by using Oct4, Sox2, C-myc and Klf4, and the mouse melanoma cells are reprogrammed into the iP by the Hochedlinger's research group in July 2009 with Oct4, Sox2, c-Myc and Klf4. The S-cell. iPS technique provides an important technical platform for the in vitro study of the reprogramming of the tumor cells, and provides a method for studying the pathogenesis of the tumor and the treatment of the tumor. In this paper, the four genes of Oct4, Sox2, Klf4 and c-Myc were introduced into human skin fibroblast (CCD) cells by means of slow viral gene delivery, and then the cells were reprogrammed to iPS cells. A platform for the construction of a technology; a regulatory mechanism for follow-up studies, such as the biological characteristics and behavior of iPS cells, such as self-replication, proliferation and differentiation. [Study] The purpose of this paper is to introduce the four genes of Oct4, Sox2, Klf4 and c-Myc into CCD cells by means of lentiviral vector, and then to re-program them as iP. PS缁，
本文编号：2504617
[Abstract]:In August 2006, the Yamanaka group introduced 24 transcription factor genes into the mouse fibroblast, and finally determined the least four transcription factor combinations _ 3ct4, Sox2, c-Myc and Klf4 to reprogram the fibroblasts to the induced pluripotent stem (iPS) cells, in November to December 2007, The Yamanaka group and the Thomson group have reprogrammed human body cells to iPS cells. After this, the research and attention of iPS cells is explosive, and some breakthrough progress has been made, such as the establishment of a disease-specific human iPS cell, The virus-free iPS cells were efficiently prepared by the transposon-mediated transgene method and the previously introduced transcription factor genes were successfully removed from the obtained iPS cells. In the case of using a specific small molecule compound, an iPS cell can be obtained with high efficiency with less foreign gene introduction, which is a significant step in the preparation of iPS cells without genetic modification; in addition, an iPS cell line such as a rat and a monkey is also established, These results have further advanced the practical application of iPS cells, and the research and application of iPS cells will be expected to be one of the greatest medical biological achievements in the 21 st century. The classical methods to study the reprogramming of the tumor cells are: the use of the embryo microenvironment, the injection of the embryo cavity, the growth of the embryo stem cells, The advent of iPS cell technology provides a new way to study the reprogramming of tumor cells And the tumor cells can be reprogrammed by using Oct4, Sox2, C-myc and Klf4, and the mouse melanoma cells are reprogrammed into the iP by the Hochedlinger's research group in July 2009 with Oct4, Sox2, c-Myc and Klf4. The S-cell. iPS technique provides an important technical platform for the in vitro study of the reprogramming of the tumor cells, and provides a method for studying the pathogenesis of the tumor and the treatment of the tumor. In this paper, the four genes of Oct4, Sox2, Klf4 and c-Myc were introduced into human skin fibroblast (CCD) cells by means of slow viral gene delivery, and then the cells were reprogrammed to iPS cells. A platform for the construction of a technology; a regulatory mechanism for follow-up studies, such as the biological characteristics and behavior of iPS cells, such as self-replication, proliferation and differentiation. [Study] The purpose of this paper is to introduce the four genes of Oct4, Sox2, Klf4 and c-Myc into CCD cells by means of lentiviral vector, and then to re-program them as iP. PS缁，
本文编号：2504617
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