| In vivo, ESC(Embryonic Stem Cell) could differentiate into any cell types. It can unlimitedly proliferate while maintaining its pluripotency. Thus its in vitro culture is very helpful for the platform construction of human disease model and the understanding the biological developments. In this thesis, we will discuss a conserve energy sensing pathway-m TOR signaling and a primodial organell-mitochondrion in the regulation of embryonic stem cell proliferation and pluripotency maintenance and we found that:1. In the m TOR aspect:(1)We compared the cell proliferation and size in two cultural conditions in E14 cell line, and after cultured 72 h in different condition, we found that cell cultured under Serum+Lif condition proliferate faster and has bigger cell size than cell cutured under 2i+Lif condition. Becaue cells cultured under both condtion are all naive pluripotent stem cell, and have the same level of pluripotency. We infer that the m TOR activity doesn't impact m ES pluripotency but affect its proliferation.(2)We use Si RNA to activate the m TOR signaling pathway and we found that cell proliferation reduced but it has no impact on the ESC pluripotency.2. In the mitochondria aspect:(1) We study the role of mitochondrial in mouse embryonic stem cell pluripotency maintenance. By manipulating the culture condition, we identified that the downregualtion of PGCa/b is able to prevent the m ESC loosing its pluripotency and also facilitate its pluripotency re-establishment. We also found that the down-regualtion of mitochondrial mass and ROS are the key factors that imfact m ESC pluripotent maintenance.(2) For further comfirming what we found above, we utilized Crispr/Cas9 technology and acquired three PGC1 a genetic knock out cell lines.We conclude that the manipulation of mTOR signaling can impact the m ESC proliferation, but it doesn't alterits pluripotency. The down-regulation of mitochondrial relavent genes might impact m ESC pluripotency. |
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