二相法诱导多能干细胞及其分子通路研究

发布时间：2018-01-04 06:03

本文关键词：二相法诱导多能干细胞及其分子通路研究　出处：《南京大学》2012年博士论文　论文类型：学位论文

【摘要】：诱导性多能干细胞(iPS细胞)可以通过在成体细胞内转导入Oct4, Sox2, Klf4和c-Myc进行重编程得到。iPS细胞的出现使获得到病人自身的多能干细胞进行治疗具有了可实现性。尽管这是干细胞研究和治疗领域的一个重大突破,但是在实现iPS细胞的医疗应用之前,仍然有许多问题需要解决。这些问题包括iPS细胞诱导的低效率,基因组中的随机突变以及重编程因子的副作用。为了解决这些问题,我们需要解析重编程过程中的分子机制。然而目前对重编程因子引发的分子相互作用过程的了解仍然很少。阻碍这些分子机制解读的障碍是iPS细胞诱导过程的随机性造成的混杂细胞群落。在比较iPS细胞系和胚胎干细胞(ES细胞)系后,我们从一株异常的iPS细胞系中发现了一个中间相。根据这个结果我们提出了一个假设：在体细胞相和多能干细胞相之间还存在一个稳定的中间相,并且这个稳定相能够为分子机制分析提供单一细胞群落。在本文的第一部分,我们证明了重编程过程能够被二相法诱导同步化：开始的稳定中间相由Oct4, Klf4,和c-Myc转导得到,然后由Sox2的过表达得到最终的多能干细胞相。二相法诱导多能干细胞方法的建立使我们能够通过分析实时的基因表达谱鉴定出Sox2下游中重编程必须的基因。在比较了初始稳定中间相和终末多能干细胞相后,我们能够通过使用二相法证实由Sox2也下调的TGF-β信号通路对重编程过程必须的。这证明了我们通过该方法研究重编程过程中分子机制的可行性。综上所述,本文提出了一种新颖的解析iPS细胞诱导过程内在细节的方法。该方法在重编程机制研究领域中有重要作用,并且能为其它重编程机制的研究提供启发。
[Abstract]:Induced pluripotent stem cells (iPS cells) in adult cells can turn into Oct4, Sox2, Klf4 and c-Myc were reprogrammed.IPS cells appear to have been treated with the realization of the patient's own stem cells. Although this is a major breakthrough in the field of stem cell research and treatment but, before the medical application of iPS cells, there are still many problems need to be solved. These problems include the low efficiency of iPS cells induced by random mutations in the genome, and the side effects of reprogramming factors. In order to solve these problems, we need to analysis the molecular mechanism during the reprogramming process. However, the molecular interaction process the reprogramming factors caused the understanding of the molecular mechanism is still very small. These hinder the interpretation of the obstacle is the hybrid cell community random process induced by iPS cells caused.
In comparison to iPS cells and embryonic stem cell (ES cell line), we iPS cell lines from a strain anomaly was found in an intermediate phase. According to this result, we propose a hypothesis that in somatic cells and pluripotent stem cells are still exist between a stable intermediate phase, and the stable phase can provide community to analyze the molecular mechanism of single cell. In the first part of this paper, we prove that the reprogramming process can be synchronized by two-phase method: stable middle phase by Oct4, Klf4, and c-Myc transduction by overexpression of Sox2, and then get the final phase of pluripotent stem cells.
A two-phase method of induced pluripotent stem cells methods so that we can real-time analysis of gene expression profiles identified must be reprogrammed in the downstream of the Sox2 gene. In comparison with the initial stability of intermediate phase and end phase of pluripotent stem cells, we can use two phase method confirmed that TGF- beta signaling pathway by Sox2 also lowered the weight must be on the process of programming. This proves the feasibility of this method through study on molecular mechanism of our reprogramming process. In summary, this paper presents a method for analysis of iPS cell induced by novel process internal details. This method has an important role in the field of reprogramming mechanism, research and can provide inspiration for other heavy programming mechanism.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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