小分子化合物BrdU促进化合物诱导重编程的研究

发布时间：2018-06-02 11:55

本文选题：5-溴脱氧尿嘧啶核苷 + BrdU　；参考：《中国科学院上海药物研究所》2016年博士论文

【摘要】：胚胎干细胞(Embryonic Stem Cells,ESC)来源于胚胎囊胚期内细胞团,具有自我更新和多向分化能力,在再生医学领域有广泛的应用前景。但胚胎干细胞的获取需要破坏胚胎,存在伦理问题,且应用时存在免疫排斥问题,这两个问题限制了胚胎干细胞的应用。2006年,Yamanaka等利用逆转录病毒介导Oct4、Sox2、Klf4和c-Myc 4个多能性相关的转录因子(OKSM)表达,成功将体细胞重编程,获得了诱导性多能干细胞(induced pluripotent stem cells,iPSC)。诱导性多能干细胞避免了伦理问题和免疫排斥问题,在再生医学领域的应用前景令人瞩目。但iPS技术一直受困于重编程方法的安全隐患和低效率两大难题,不能真正用于临床治疗。应用小分子化合物促进重编程则是解决以上难题的可能途径。小分子化合物不仅能提高重编程的效率或替代某些转录因子,还能帮助我们探究重编程的机制。2013年,化合物诱导性多能干细胞(chemical induced pluripotent stem cells,CiPSC)的成功,解决了iPS的安全性问题,小分子化合物也成为iPS研究的热点。本论文的研究目标是发现能促进重编程的小分子化合物,并通过探究其作用机制来研究重编程机制,为iPS最终走向再生医学应用做出贡献。应用本课题组建立的促进四因子诱导iPS化合物筛选体系,我们在化合物库中筛选到常用的生物试剂5-溴脱氧尿嘧啶核苷(BrdU)。在Oct4,Sox2,klf4,c-Myc四因子诱导条件下,BrdU能显著提高iPS诱导效率,加快重编程的进程,且加入BrdU得到的iPS与胚胎干细胞的特性高度相似。BrdU还能够提高Oct4,Sox2和klf4三因子和Oct4,klf4两因子的诱导效率。进一步研究发现,BrdU能代替Oct4,与Sox2,klf4和c-Myc诱导重编程,并成功得到了SKM-iPS细胞系。在CiPS诱导实验中,BrdU在与已报到的化合物组合时,分别能够提高7个化合物VC6PFZT和4个化合物组合C6FZ的诱导效率,得到多能性与胚胎干细胞接近的CiPS。我们还发现了新的化合物组合:BrdU与C6F(CHIR99021,6616452,Forskolin)可诱导CiPS,且得到的CiPS与与胚胎干细胞的形态和功能十分接近。在机制研究中我们发现BrdU可能通过降低基因组整体的甲基化水平来推动重编程。本论文的研究为安全、高效的获得iPS提供了新的途径,为研究全化合物诱导人类iPS提供了重要的候选化合物和组合,为iPS早日应用于人类临床治疗做出了贡献。
[Abstract]:Embryonic Stem cells ESCs derived from the cell mass in blastocyst stage have the ability of self-renewal and multi-differentiation, so they have a wide application prospect in the field of regenerative medicine. But the acquisition of embryonic stem cells needs to destroy the embryo, there are ethical problems, and the application of the problem of immune rejection, These two problems limited the application of embryonic stem cells. In 2006, Yamanaka et al successfully reprogrammed the somatic cells by using retrovirus-mediated expression of Oct4G Sox2G Klf4 and c-Myc four transcription factors related to pluripotency, and obtained induced pluripotent stem cells of inducible pluripotent stem cells. Inducible pluripotent stem cells avoid ethical problems and immune rejection problems, so the application prospect of induced pluripotent stem cells in regenerative medicine is attractive. However, iPS technology has been trapped in the safety and low efficiency of reprogramming methods, which can not be used in clinical treatment. The application of small molecular compounds to promote reprogramming is a possible way to solve these problems. Small molecular compounds can not only improve the efficiency of reprogramming or replace some transcription factors, but also help us to explore the mechanism of reprogramming. In 2013, the success of compound induced pluripotent stem cell chemical induced pluripotent stem cells solved the security problem of iPS. Small molecular compounds have also become the focus of iPS research. The aim of this paper is to find small molecular compounds that can promote reprogramming, and to study the reprogramming mechanism by exploring the mechanism of reprogramming, and to contribute to the application of iPS to regenerative medicine. Using the screening system of iPS compounds induced by four factors established by our team, we screened the commonly used biological reagents, 5-bromodeoxyuridine (5-bromodeoxyuridine) BrdUX, in the compound library. Under the condition of Oct4N Sox2Klf4nc-Myc induction, BrdU can significantly improve the induction efficiency of iPS and speed up the process of reprogramming. The iPS obtained by adding BrdU is highly similar to embryonic stem cells. BrdU can also improve the induction efficiency of Oct4N Sox2 and klf4 three factors and Oct4Klf4. It was further found that BrdU can replace Oct4 and reprogram with Sox2chlf4 and c-Myc, and the SKM-iPS cell line was successfully obtained. In the CiPS induction experiment, BrdU can improve the induction efficiency of 7 compounds VC6PFZT and 4 compound combinations C6FZ respectively when combined with the reported compounds, and obtain Cipps with pluripotency similar to that of embryonic stem cells. We also found that a new compound combination of: BrdU and C6FNCHIR99021n6616452Forskolin could induce CiPS, and the CiPS obtained was very close to the morphology and function of embryonic stem cells. In the study of mechanism, we found that BrdU may promote reprogramming by reducing the methylation level of genome as a whole. The research in this paper provides a new way to obtain iPS safely and efficiently, provides important candidate compounds and combinations for the study of human iPS induced by whole compounds, and contributes to the early application of iPS in human clinical therapy.
【学位授予单位】：中国科学院上海药物研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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