ATM基因在体细胞重编程过程中的作用及机制研究

发布时间：2018-08-09 15:19

【摘要】：ATM(mutated in Ataxia-Telangiectasia)是一个重要的表达丝氨酸-苏氨酸蛋白激酶的基因,在DNA损伤与修复中发挥重要作用。DNA双链断裂会引起ATM的激活,从而引发下游一系列损伤修复反应。此外,染色体的高度动态变化也会激活ATM,iPS细胞诱导过程中染色体会发生巨大变化和高度修饰,但很少有研究报道ATM基因在重编程过程中的作用。本研究利用基因敲减技术分析了一些参与细胞代谢与生命活动的蛋白酶与蛋白激酶对于体细胞重编程的影响。结果显示,除chuk基因外,敲减其他磷酸化相关基因后iPS细胞的诱导效率均有所提高。其中,敲减ATM不但提高iPS诱导效率,同时获得的iPS克隆折光性很好,边界清楚,且形成的转分化细胞很少,iPS克隆非常均一。进一步分析显示,随着敲减ATM比例的增高,重编程的效率降低,且生成的克隆内源性Oct4未被激活,不能继续培养传代,而低敲减ATM的克隆经检测重编程完整,多能性基因表达正常。此外,敲减ATM后,抑制P53和H2AX的磷酸化,同时发现敲减ATM后,细胞自噬相关蛋白LC3被激活,自噬在重编程过程中也发挥着重要作用。最终我们得出结论,适当敲减ATM会通过降低P53-pi和1γH2AX的水平而提高重编程的效率。
[Abstract]:ATM (mutated in Ataxia-Telangiectasia) is an important gene expressing serine-threonine protein kinase. It plays an important role in DNA damage and repair. DNA double strand breaks can activate ATM and induce a series of downstream damage and repair reactions. In addition, the highly dynamic changes of chromosomes also activate the induction of ATMI-PS cells. However, there are few studies on the role of ATM gene in the process of reprogramming. In this study, the effects of protease and protein kinase involved in cell metabolism and life activity on somatic cell reprogramming were analyzed by gene knockout technique. The results showed that the induction efficiency of iPS cells was increased after knockout of other phosphorylation related genes except chuk gene. Knockout ATM not only improved the induction efficiency of iPS, but also obtained iPS clones with good refractive index and clear boundary. Further analysis showed that with the increase of the proportion of knockout ATM, the efficiency of reprogramming was decreased, and the cloned endogenous Oct4 was not activated and could not be cultured and subcultured, while the clone of low knock down ATM was tested and reprogrammed completely. The expression of pluripotent genes is normal. In addition, the phosphorylation of p53 and H2AX was inhibited after knockout of ATM. It was also found that after knockout ATM, the autophagy protein LC3 was activated, and autophagy also played an important role in the process of reprogramming. Finally, we conclude that proper tapping of ATM can improve the efficiency of reprogramming by lowering the levels of P53-pi and 1 纬 H2AX.
【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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