本文選題：人多能干細(xì)胞　切入點(diǎn)：多梳家族蛋白PHC1　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景再生醫(yī)學(xué)界領(lǐng)袖之一 Richard.GOSS將再生、生命、死亡三者之間的關(guān)系解釋為:"如果沒(méi)有再生,就沒(méi)有生命,如果處處再生,就沒(méi)有死亡",可見(jiàn)再生是個(gè)體存活維持組織功能的重要機(jī)制。基于人干細(xì)胞尤其是人多能干細(xì)胞(human pluripotent stem cells,hPSCs)含人胚胎干細(xì)胞(human embryonic stem cells,hESCs)和人誘導(dǎo)多能干細(xì)胞(induced pluripotent stem cells,iPSCs)的再生醫(yī)學(xué)為發(fā)展個(gè)體化醫(yī)療帶來(lái)了前所未有機(jī)遇和前景。hPSCs具有在體外無(wú)限復(fù)制和向各種譜系細(xì)胞分化的潛能,為治療多個(gè)系統(tǒng)的退行性病變提供可能的種子細(xì)胞用于移植。但是hPSCs應(yīng)用于臨床還存在很多瓶頸,例如研究表明體細(xì)胞重編程過(guò)程中過(guò)表達(dá)重編程因子后引起的DNA復(fù)制壓力和氧化自由基應(yīng)激導(dǎo)致的DNA損傷可以破壞iPSCs的基因組產(chǎn)生基因突變,另一方面hPSCs在快速增殖過(guò)程中伴隨著由細(xì)胞代謝和DNA復(fù)制活躍產(chǎn)生的活性氧自由基(ROS)也可以導(dǎo)致DNA損傷從而產(chǎn)生基因突變,移植攜帶基因突變的細(xì)胞到體內(nèi)后可能產(chǎn)生腫瘤。因此研究hPSCs如何維持基因組的穩(wěn)定性對(duì)于獲得更安全的hPSCs用于細(xì)胞治療十分重要。相比于體細(xì)胞DNA損傷修復(fù)的研究,關(guān)于hESCs和iPSCs如何維持基因組穩(wěn)定性的報(bào)道卻不多,目前的研究發(fā)現(xiàn)hPSCs維持基因組穩(wěn)定性的主要機(jī)制為:一.ESCs內(nèi)線粒體數(shù)量較低,同時(shí)ESCs主要通過(guò)不依賴于線粒體氧化磷酸化的糖酵解代謝方式獲得能量,因此產(chǎn)生的ROS水平很低,另外ESCs高表達(dá)抗氧化相關(guān)基因,因此具備更強(qiáng)的抗.ROS導(dǎo)致的DNA損傷能力;二.ESCs在DNA損傷時(shí)會(huì)發(fā)生分化和凋亡清除無(wú)法完成損傷DNA修復(fù)的細(xì)胞群體。本課題研究了多梳家族蛋白PHC1在hESCs基因組穩(wěn)定性維持中的作用。我們的研究結(jié)果顯示利用shRNA沉默PHC1的表達(dá)后可以降低NANOG的表達(dá)水平,提示PHC1可能參與hESCs的干性維持;進(jìn)一步通過(guò)紫外輻射和阿霉素處理誘導(dǎo)DNA損傷后,敲降PHC1后的hESCs對(duì)DNA損傷更敏感。另外,以前的研究表明過(guò)表達(dá)重編程因子引起的DNA損傷反應(yīng)是體細(xì)胞重編程的一個(gè)重要障礙。我們的結(jié)果顯示在人體細(xì)胞重編程過(guò)程中敲降PHC1的表達(dá)后引起細(xì)胞凋亡增加、周期阻滯,從而降低重編程的效率,提示PHC1參與了多能性形成過(guò)程中細(xì)胞的DNA損傷反應(yīng)。第一部分PHC1在hESCs干性維持中的作用目的:研究PHC1對(duì)于多能性維持的作用。方法:利用qPCR和western blot檢測(cè)PHC1在hPSCs與分化細(xì)胞中的表達(dá)差異,構(gòu)建PHC1 shRNA在hESCs中沉默PHC1基因表達(dá)后檢測(cè)多能性基因的表達(dá)變化。結(jié)果:PHC1在hPSCs中的表達(dá)高度富集。成功構(gòu)建了在HFF和hESCs中有良好敲降效果的PHC1 shRNA。hESCs中沉默PHC1基因表達(dá)后多能性基因OCT4和NANOG在轉(zhuǎn)錄水平?jīng)]有明顯差異,但NANOG蛋白水平表達(dá)有一定降低。結(jié)論:PHC1對(duì)于hESCs多能性維持具有一定的作用。第二部分PHC1對(duì)于維持hESCs基因組穩(wěn)定性的作用目的:建立誘導(dǎo)hESCs DNA損傷的模型,探索敲降PHC1后hESCs對(duì)DNA損傷修復(fù)的反應(yīng)。方法:沉默PHC1基因表達(dá)后,分別采用紫外(UV)輻射,阿霉素(Dox)處理誘導(dǎo)hESCs DNA損傷模型,檢測(cè)細(xì)胞凋亡水平變化細(xì)胞增殖情況。結(jié)果:對(duì)比載體對(duì)照組,敲降PHC1可以造成hESCs基因組穩(wěn)定性下降,對(duì)DNA損傷敏感度增加。結(jié)論:PHC1對(duì)hESCs基因組穩(wěn)定性維持具有一定作用。第三部分PHC1在人體細(xì)胞重編程中的作用目的:探討PHC1在過(guò)表達(dá)轉(zhuǎn)錄因子介導(dǎo)的體細(xì)胞重編程中的作用。方法:建立利用慢病毒載體過(guò)表達(dá)OCT4,SOX2,KLF4,c-MYC重編程人成纖維細(xì)胞為iPSCs的體系,檢測(cè)沉默PHC1表達(dá)后對(duì)重編程效率的影響。結(jié)果:重編程過(guò)程中沉默PHC1后引起細(xì)胞周期阻滯、細(xì)胞凋亡增加以及重編程效率降低。結(jié)論:PHC1參與人體細(xì)胞重編程過(guò)程中發(fā)生的DNA損傷反應(yīng),從而影響重編程效率。
[Abstract]:One of the background of regenerative medicine leaders Richard.GOSS regeneration, life, the relationship between the three death explanation: "if there is no regeneration, there is no life everywhere, if regeneration, there is no death", is an important mechanism for the survival of regenerated individuals maintain the tissue function. Based on human stem cells especially human pluripotent stem cells (human pluripotent stem cells, hPSCs) containing human embryonic stem cells (human embryonic stem cells, hESCs) and human induced pluripotent stem cells (induced pluripotent stem cells, iPSCs) in regenerative medicine for the development of personalized medicine has brought hitherto unknown opportunities and prospects of.HPSCs with in vitro replication and differentiation into various lineages of infinite cell potential, to provide seed cells for transplantation for the treatment of multiple system degeneration. But the clinical application of hPSCs there are still many bottlenecks, such as the reprogramming of somatic cells too In the process of over expression of DNA damage caused by reprogramming factors after DNA replication stress and oxidation of free radicals caused by stress can damage the genome of iPSCs mutation, on the other hand, along with the active hPSCs produced by the cell metabolism and DNA replication of the active oxygen free radical in the process of rapid proliferation (ROS) can cause DNA damage to produce a gene mutation, carrying the mutant cells transplantation into the body may produce tumors. Therefore the research of hPSCs how to maintain genomic stability for secure hPSCs for cell therapy is very important. The research compared to somatic cell injury and repair of DNA, hESCs and iPSCs on how to maintain genome stability report does not, at present the study found that the main mechanism of hPSCs maintenance of genome stability: a low number of mitochondria in.ESCs and ESCs, mainly through does not depend on the mitochondrial oxidation Phosphorylation of glycolytic energy obtained, resulting in very low ROS levels, and ESCs high expression of antioxidant related genes, so it has stronger ability of anti DNA damage caused by.ROS; two.ESCs will be unable to complete the differentiation and apoptosis of clear cell population in DNA damage repair DNA damage. This study role of Polycomb family protein PHC1 hESCs in the maintenance of genome stability. Our results showed that the expression of shRNA by PHC1 silencing can reduce the expression level of NANOG, suggesting that PHC1 may be involved in the hESCs of the dry maintenance; further through ultraviolet radiation and doxorubicin induced DNA damage, after knocking down PHC1 hESCs sensitive to DNA damage. In addition, previous research showed that over expression of DNA damage response caused by reprogramming factors is an important obstacle to the reprogramming of somatic cells. Our results showed that in the cells of the body. The expression of cellular reprogramming in knockdown of PHC1 induced cell cycle arrest, apoptosis, thereby reducing the efficiency of reprogramming, suggesting that PHC1 is involved in the formation process of pluripotent cells in response to DNA damage. The first part of PHC1 in hESCs Objective: To study the stemness of PHC1 for the maintenance of pluripotency role. Methods: the expression of qPCR and Western by blot PHC1 hPSCs in the detection and differentiation of the cells in the construction of PHC1 shRNA in hESCs silencing of PHC1 gene expression to detect the expression changes of genes. Results: the expression of PHC1 in hPSCs was successfully constructed. Highly enriched in HFF and hESCs have a good knock down effect PHC1 shRNA.hESCs silencing of PHC1 gene expression can have no obvious difference in the transcription level of gene OCT4 and NANOG, but NANOG protein expression was reduced to a certain extent. Conclusion: PHC1 for hESCs to maintain a certain The role of PHC1 hESCs. The second part for the maintenance of genome stability effect Objective: to establish the hESCs induced DNA damage model, exploration after knocking down PHC1 hESCs to repair DNA damage response. Methods: the expression of PHC1 gene silencing, respectively by ultraviolet radiation (UV), adriamycin (Dox) induced hESCs DNA injury model, apoptosis to detect the level of cell cell proliferation. Results: compared with vector control group, knockdown of PHC1 can decrease the hESCs genomic stability and increased sensitivity to DNA damage. Conclusion: PHC1 has a certain effect on hESCs. The third part to maintain genomic stability in human PHC1 cell reprogramming in objective: To investigate the PHC1 expression in somatic cells transcription factor mediated reprogramming in vitro. Methods: using lentiviral vector to establish the expression of OCT4, SOX2, KLF4, c-MYC reprogramming of human fibroblasts for iPSCs detection system, silencing of PHC 1, the effect of expression on reprogramming efficiency. Results: during the reprogramming, silence PHC1 caused cell cycle arrest, increased cell apoptosis and reprogramming efficiency. Conclusion: PHC1 is involved in DNA damage response during reprogramming of human cells, thereby affecting reprogramming efficiency.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R329.2

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本文編號(hào)：1634790