多梳家族蛋白PHC1在人多能干细胞基因组稳定性中的作用研究

发布时间：2018-03-19 15:05

  本文选题：人多能干细胞　切入点：多梳家族蛋白PHC1　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景再生医学界领袖之一 Richard.GOSS将再生、生命、死亡三者之间的关系解释为:"如果没有再生,就没有生命,如果处处再生,就没有死亡",可见再生是个体存活维持组织功能的重要机制。基于人干细胞尤其是人多能干细胞(human pluripotent stem cells,hPSCs)含人胚胎干细胞(human embryonic stem cells,hESCs)和人诱导多能干细胞(induced pluripotent stem cells,iPSCs)的再生医学为发展个体化医疗带来了前所未有机遇和前景。hPSCs具有在体外无限复制和向各种谱系细胞分化的潜能,为治疗多个系统的退行性病变提供可能的种子细胞用于移植。但是hPSCs应用于临床还存在很多瓶颈,例如研究表明体细胞重编程过程中过表达重编程因子后引起的DNA复制压力和氧化自由基应激导致的DNA损伤可以破坏iPSCs的基因组产生基因突变,另一方面hPSCs在快速增殖过程中伴随着由细胞代谢和DNA复制活跃产生的活性氧自由基(ROS)也可以导致DNA损伤从而产生基因突变,移植携带基因突变的细胞到体内后可能产生肿瘤。因此研究hPSCs如何维持基因组的稳定性对于获得更安全的hPSCs用于细胞治疗十分重要。相比于体细胞DNA损伤修复的研究,关于hESCs和iPSCs如何维持基因组稳定性的报道却不多,目前的研究发现hPSCs维持基因组稳定性的主要机制为:一.ESCs内线粒体数量较低,同时ESCs主要通过不依赖于线粒体氧化磷酸化的糖酵解代谢方式获得能量,因此产生的ROS水平很低,另外ESCs高表达抗氧化相关基因,因此具备更强的抗.ROS导致的DNA损伤能力;二.ESCs在DNA损伤时会发生分化和凋亡清除无法完成损伤DNA修复的细胞群体。本课题研究了多梳家族蛋白PHC1在hESCs基因组稳定性维持中的作用。我们的研究结果显示利用shRNA沉默PHC1的表达后可以降低NANOG的表达水平,提示PHC1可能参与hESCs的干性维持;进一步通过紫外辐射和阿霉素处理诱导DNA损伤后,敲降PHC1后的hESCs对DNA损伤更敏感。另外,以前的研究表明过表达重编程因子引起的DNA损伤反应是体细胞重编程的一个重要障碍。我们的结果显示在人体细胞重编程过程中敲降PHC1的表达后引起细胞凋亡增加、周期阻滞,从而降低重编程的效率,提示PHC1参与了多能性形成过程中细胞的DNA损伤反应。第一部分PHC1在hESCs干性维持中的作用目的:研究PHC1对于多能性维持的作用。方法:利用qPCR和western blot检测PHC1在hPSCs与分化细胞中的表达差异,构建PHC1 shRNA在hESCs中沉默PHC1基因表达后检测多能性基因的表达变化。结果:PHC1在hPSCs中的表达高度富集。成功构建了在HFF和hESCs中有良好敲降效果的PHC1 shRNA。hESCs中沉默PHC1基因表达后多能性基因OCT4和NANOG在转录水平没有明显差异,但NANOG蛋白水平表达有一定降低。结论:PHC1对于hESCs多能性维持具有一定的作用。第二部分PHC1对于维持hESCs基因组稳定性的作用目的:建立诱导hESCs DNA损伤的模型,探索敲降PHC1后hESCs对DNA损伤修复的反应。方法:沉默PHC1基因表达后,分别采用紫外(UV)辐射,阿霉素(Dox)处理诱导hESCs DNA损伤模型,检测细胞凋亡水平变化细胞增殖情况。结果:对比载体对照组,敲降PHC1可以造成hESCs基因组稳定性下降,对DNA损伤敏感度增加。结论:PHC1对hESCs基因组稳定性维持具有一定作用。第三部分PHC1在人体细胞重编程中的作用目的:探讨PHC1在过表达转录因子介导的体细胞重编程中的作用。方法:建立利用慢病毒载体过表达OCT4,SOX2,KLF4,c-MYC重编程人成纤维细胞为iPSCs的体系,检测沉默PHC1表达后对重编程效率的影响。结果:重编程过程中沉默PHC1后引起细胞周期阻滞、细胞凋亡增加以及重编程效率降低。结论:PHC1参与人体细胞重编程过程中发生的DNA损伤反应,从而影响重编程效率。
[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.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

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