烟酰胺磷酸核糖转移酶基因编辑对人胚胎干细胞生长影响的研究

The effect of Nampt gene editing on the growth of human embryonic stem cells

目的研究烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyl transferase,Nampt)基因在人胚胎干细胞生长中的作用。方法采用CRISPR/Cas9技术对人胚胎干细胞H9细胞系的Nampt基因进行移码突变;随后,筛选Nampt基因突变细胞系进行基因测序,同时在显微镜下观察其生长状态。另外,给予H9细胞不同浓度的Nampt抑制剂FK866进行干预,采用CCK-8试剂盒检测细胞活力。结果 Nampt基因发生突变后,人胚胎干细胞会逐渐死亡。同样,Nampt抑制剂FK866也会抑制胚胎干细胞的活力,使细胞逐渐死亡。结论 Nampt基因对维持人胚胎干细胞生长具有不可或缺的重要作用。

Objective To study the role of nicotinamide phosphoribosyltransferase(Nampt)gene in the growth of human embryonic stem cells.Methods CRISPR/Cas9 technology was used for Nampt gene knockout in human embryonic stem cells by introducing frame-shift mutation to gene coding region.Then,the mutational cells were screened with gene sequencing and observed for the growth situation under the microscope.In addition,Nampt inhibitor FK866 was introduced at different concentrations to interfere the growth of human embryonic stem cells,followed by the cell viability test with CCK-8 kit.Results Human embryonic stem cells gradually died after the mutation of Nampt gene with CRISPR/Cas9 technology.Likewise,Nampt inhibitor FK866 significantly inhibited the cell viability and gradually led to the death of cells.Conclusion Nampt gene plays an indispensable role in maintaining the growth of human embryonic stem cells.