本文選題：小分子化合物 + 多能性基因�。� 參考：《內(nèi)蒙古大學(xué)》2017年碩士論文

【摘要】：本研究在前期實(shí)驗(yàn)研究中獲得一種由四因子c-Myc,Klf4,E-cadherin,Glis1誘導(dǎo)的,形態(tài)、體外增殖和體內(nèi)外分化能力類似于胚胎干細(xì)胞,但不具有嵌合能力的部分重編程細(xì)胞,KMEG-prCs。該細(xì)胞雖具有一定多能性,但oct4,sox2,nanog,AKP并不表達(dá),且SSEA1只在部分細(xì)胞中被激活。經(jīng)轉(zhuǎn)錄物組分析顯示,KMEG-prCs處于重編程的中間狀態(tài)。該細(xì)胞可以作為一個(gè)新的細(xì)胞模型來(lái)研究重編程過(guò)程中內(nèi)源多能性信號(hào)網(wǎng)絡(luò)激活的分子機(jī)制。本研究旨在利用KMEG-prCs細(xì)胞,篩選可激活該細(xì)胞中內(nèi)源多能性基因表達(dá)的小分子化合物或組合,為進(jìn)一步探討內(nèi)源多能性調(diào)控網(wǎng)絡(luò)的激活和發(fā)現(xiàn)新的小分子化合物組合建立新的小分子誘導(dǎo)體系奠定理論和技術(shù)基礎(chǔ)。通過(guò)流式細(xì)胞分析顯示,重編程不同階段的表面標(biāo)記蛋白Thy1(CD90)完全下調(diào),CD54(ICAM-1)完全上調(diào),CD44和SSEA1分別部分上調(diào),CD31僅少數(shù)上調(diào)。結(jié)合轉(zhuǎn)錄物組的分析,進(jìn)一步確定KMEG-prCs是一個(gè)處于重編程中段的中間態(tài)細(xì)胞。分選獲得SSEA1+KMEG-prCs和SSEA1-KMEG-prCs,并用含有不同小分子化合物的培養(yǎng)液進(jìn)行培養(yǎng),內(nèi)源多能性基因激活顯示,加入HDAC抑制劑TSA、VPA、Oct4激活劑(OAC1)與腺苷酸環(huán)化酶活化劑(Forskolin)后激活了多能性基因AKP的表達(dá),并且使一部分SSEA1陰性細(xì)胞轉(zhuǎn)變?yōu)镾SEA1陽(yáng)性細(xì)胞。隨后加入EZH2蛋白胞內(nèi)增強(qiáng)子抑制劑(DZNeP)進(jìn)一步誘導(dǎo)后,在小分子組合PCA+FD處理下,SSEA1+KMEG-prCs 和 SSEA1-KMEG-prCs 內(nèi)源 oct4 均被激活,SSEA1+KMEG-prCs中oct4高水平表達(dá)。獲得了一個(gè)新的細(xì)胞狀態(tài),KMEG-prCs-O+,該細(xì)胞仍然保持體內(nèi)外分化能力。隨后,雖然將PCA+FD中的A-83-01替換為E-616452,并聯(lián)合LiCl,構(gòu)成PCE+FD+Li小分子化合物組合可有效激活nanog的表達(dá),但oct4的表達(dá)被下調(diào)。綜上所述,KMEG-prCs是一個(gè)處于重編程中段的中間態(tài)細(xì)胞,通過(guò)小分子化合物的處理其內(nèi)源多能性調(diào)控網(wǎng)絡(luò)可以被激活。該研究為探討小分子化合物對(duì)重編程的作用機(jī)理以及進(jìn)一步獲得完全重編程的iPSCs奠定了基礎(chǔ),同樣對(duì)于細(xì)胞在重編程過(guò)程中信號(hào)通路,代謝途徑,表觀遺傳修飾等的研究提供依據(jù)。
[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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q23

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Induced pluripotent stem cells(iPSCs)——a new era of reprogramming[J];遺傳學(xué)報(bào);2010年07期

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本文編號(hào)：1890308