通过重编程方法将大鼠肝细胞诱导为多能干细胞的研究

发布时间：2018-01-01 19:40

本文关键词：通过重编程方法将大鼠肝细胞诱导为多能干细胞的研究　出处：《河南师范大学》2012年硕士论文　论文类型：学位论文

【摘要】：诱导性多能干细胞(iPSCs)是利用特异的诱导因子组合，如重编程因子组合或结合某些小分子化合物等将成熟的体细胞去分化为类似于胚胎干细胞的一种多潜能细胞。这些细胞具有胚胎干细胞的特征，并能分化为三种胚层细胞。有关iPSCs的研究极大地促进了细胞的分化调控、再生医学、细胞发育等基础研究，并有极高的临床应用前景。本文利用慢病毒介导的基因转染技术将重编程因子组合Sox2、Oct4、c-Myc和Klf4导入原代培养的大鼠肝细胞，对其进行诱导去分化。肝细胞在含四种重编程因子的重组慢病毒感染7天后，其形态开始发生改变。将其转移到饲养层细胞(MEFs)上培养11天后，部分细胞聚集在一起，呈致密克隆状生长，边界清晰且致密整齐，形成与大鼠胚胎干细胞相似的克隆。这些克隆细胞较小、增殖迅速，且在高倍镜下观察到细胞核较大，核质比率较高。从克隆形态初步判断为大鼠iPSCs克隆，，碱性磷酸酶染色结果显示，这些细胞克隆表达了高水平的碱性磷酸酶。采用逆转录PCR(RT-PCR)分别分析诱导的细胞克隆、大鼠原代培养肝细胞和大鼠4.5天胚胎中的基因表达状况。结果显示外源重编程因子(Oct4、Sox2、c-Myc和Klf4)仅在诱导的细胞克隆中表达；大鼠胚胎干细胞标志基因Sox2、Oct4、Nanog、Fgf4、Lin28和Ncam在诱导的细胞克隆和4.5天大鼠胚胎中均有表达。进一步的免疫荧光染色检测结果显示，上述诱导的细胞克隆表达了大鼠胚胎干细胞的标志蛋白SSEA-1和Nanog。综上结果表明，本实验成功地将大鼠原代培养的肝细胞诱导去分化成了大鼠肝细胞来源的iPSCs。
[Abstract]:Induced pluripotent stem cells (iPSCs) is utilized to induce specific factor combination, such as reprogramming factors or a combination with some small molecules will mature somatic cells to differentiate into a pluripotent cells similar to embryonic stem cells. These cells have the characteristics of embryonic stem cells, and can be differentiated into three types germ cell. The research on iPSCs has greatly promoted the differentiation of cells in regenerative medicine, on the basis of cell development, clinical application and high.
In this paper, using lentivirus mediated gene transfection of reprogramming factors Sox2, Oct4, c-Myc and Klf4 into primary cultured rat liver cells, which were induced to differentiation of liver cells 7 days after infection in containing four kinds of reprogramming factors of recombinant lentivirus, the morphology changed to. The transfer to the feeder cells (MEFs) cultured for 11 days, some cells together, a dense colony like growth, boundary clear and dense neat, and formation of rat embryonic stem cell clones similar. These clones were small, rapid proliferation, and at high magnification to observe large nuclei, karyoplasmic ratio high. From the preliminary judgment for clonal morphology of rat iPSCs clone, alkaline phosphatase staining showed that these cells were cloned and expressed high levels of alkaline phosphatase.
Using reverse transcription PCR (RT-PCR) were analyzed and induced cell clone, expression in primary cultured rat hepatocytes and rat embryos in 4.5 days. The results showed that exogenous gene reprogramming factors (Oct4, Sox2, c-Myc and Klf4) expressed only in cell clones induced; rat embryonic stem cell marker gene Sox2 Oct4, Nanog, Fgf4, Lin28, and Ncam were expressed in cell clones induced and 4.5 day rat embryos. Further immunofluorescence staining showed that these clones induced expression of rat embryonic stem cell marker proteins SSEA-1 and Nanog. in conclusion, this experiment successfully primary rat cultured liver cells induced to differentiate into liver cells from rat iPSCs.

【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【共引文献】