小分子化合物激活中间态细胞KMEG-prCs内源多能性基因表达的研究

发布时间：2018-05-15 01:14

本文选题：小分子化合物 + 多能性基因　；参考：《内蒙古大学》2017年硕士论文

【摘要】：本研究在前期实验研究中获得一种由四因子c-Myc,Klf4,E-cadherin,Glis1诱导的,形态、体外增殖和体内外分化能力类似于胚胎干细胞,但不具有嵌合能力的部分重编程细胞,KMEG-prCs。该细胞虽具有一定多能性,但oct4,sox2,nanog,AKP并不表达,且SSEA1只在部分细胞中被激活。经转录物组分析显示,KMEG-prCs处于重编程的中间状态。该细胞可以作为一个新的细胞模型来研究重编程过程中内源多能性信号网络激活的分子机制。本研究旨在利用KMEG-prCs细胞,筛选可激活该细胞中内源多能性基因表达的小分子化合物或组合,为进一步探讨内源多能性调控网络的激活和发现新的小分子化合物组合建立新的小分子诱导体系奠定理论和技术基础。通过流式细胞分析显示,重编程不同阶段的表面标记蛋白Thy1(CD90)完全下调,CD54(ICAM-1)完全上调,CD44和SSEA1分别部分上调,CD31仅少数上调。结合转录物组的分析,进一步确定KMEG-prCs是一个处于重编程中段的中间态细胞。分选获得SSEA1+KMEG-prCs和SSEA1-KMEG-prCs,并用含有不同小分子化合物的培养液进行培养,内源多能性基因激活显示,加入HDAC抑制剂TSA、VPA、Oct4激活剂(OAC1)与腺苷酸环化酶活化剂(Forskolin)后激活了多能性基因AKP的表达,并且使一部分SSEA1阴性细胞转变为SSEA1阳性细胞。随后加入EZH2蛋白胞内增强子抑制剂(DZNeP)进一步诱导后,在小分子组合PCA+FD处理下,SSEA1+KMEG-prCs 和 SSEA1-KMEG-prCs 内源 oct4 均被激活,SSEA1+KMEG-prCs中oct4高水平表达。获得了一个新的细胞状态,KMEG-prCs-O+,该细胞仍然保持体内外分化能力。随后,虽然将PCA+FD中的A-83-01替换为E-616452,并联合LiCl,构成PCE+FD+Li小分子化合物组合可有效激活nanog的表达,但oct4的表达被下调。综上所述,KMEG-prCs是一个处于重编程中段的中间态细胞,通过小分子化合物的处理其内源多能性调控网络可以被激活。该研究为探讨小分子化合物对重编程的作用机理以及进一步获得完全重编程的iPSCs奠定了基础,同样对于细胞在重编程过程中信号通路,代谢途径,表观遗传修饰等的研究提供依据。
[Abstract]:In this study, we obtained a partial reprogramming cell (KMEG-prCs), which was induced by four-factor c-Mycf4E-cadherin1, was similar to embryonic stem cells in vitro, proliferation and differentiation in vitro and in vivo, but had no chimeric ability. Although this cell has certain pluripotency, it is not expressed in oct4nsox2, and SSEA1 is only activated in some cells. Transcriptome analysis showed that KMEG-prCs were in the intermediate state of reprogramming. This cell can be used as a new cell model to study the molecular mechanism of activation of endogenous pluripotent signal network during reprogramming. The aim of this study was to screen small molecular compounds or combinations that could activate the expression of endogenous pluripotent genes in KMEG-prCs cells. It lays a theoretical and technical foundation for further discussion on the activation of endogenous pluripotent regulatory networks and the discovery of new combinations of small molecular compounds for the establishment of new small molecular induction systems. Flow cytometry analysis showed that the surface marker protein Thy1 (CD90) completely down-regulated CD54 ICAM-1) in different stages of reprogramming, and up-regulated CD44 and SSEA1, respectively. Only a few of them upregulated CD31, respectively. Combined with transcriptome analysis, it was further confirmed that KMEG-prCs is an intermediate cell in the middle of reprogramming. SSEA1 KMEG-prCs and SSEA1-KMEG-prCswere isolated and cultured in medium containing different small molecular compounds. The activation of endogenous pluripotent gene showed that the expression of AKP was activated by adding HDAC inhibitor TSA-VPA-Oct4 activator OAC _ 1 and adenylate cyclase activator Forskolin. Some SSEA1 negative cells were transformed into SSEA1 positive cells. After further induction with the addition of EZH2 protein intracellular enhancer inhibitor (DZNeP), both KMEG-prCs and SSEA1-KMEG-prCs endogenous oct4 were activated in the high level of oct4 expression in SSA1 KMEG-prCs under the treatment of small molecular combination PCA FD. A new cell state, KMEG-prCs-O, was obtained, which still maintained the ability of differentiation in vitro and in vivo. Subsequently, although A-83-01 in PCA FD was replaced with E-616452 and LiCl-LiCl was used to form a combination of PCE FD Li small molecule compounds, the expression of nanog was effectively activated, but the expression of oct4 was down-regulated. In conclusion, KMEG-prCs is an intermediate state cell in the middle of reprogramming, and its endogenous pluripotent regulatory network can be activated through the treatment of small molecular compounds. This study laid a foundation for exploring the action mechanism of small molecular compounds on reprogramming and for further obtaining fully reprogrammed iPSCs, and also for the signal pathway and metabolic pathway of cells during reprogramming. The study of epigenetic modification and so on provides the basis.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q23

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