自噬相关蛋白ATG5和ATG7在神经干细胞体外扩增中的作用

Roles of autophagy-related proteins ATG5 and ATG7 in the expansion of neural stem cells in vitro

目的探讨自噬及自噬相关蛋白5(ATG5)和ATG7在神经干细胞(NSC)体外扩增中的作用。方法从SD胚胎大鼠脑组织中分离NSC在体外进行培养,在NSC体外扩增过程中测量神经球直径大小变化,并用CCK-8试剂盒检测细胞增殖率,通过Western印迹检测ATG5、ATG7、微管相关蛋白1轻链3(LC3)及增殖细胞核抗原(PCNA)的蛋白表达水平,利用透射电子显微镜观察自噬泡形成情况,免疫荧光分析LC3及Ki67的表达,EdU掺入法检测细胞增殖能力。进一步经过自噬抑制剂Baf A1干预处理,或用siRNA敲低Atg5/Atg7表达后评价自噬及自噬蛋白ATG5/ATG7在NSC体外扩增中的作用。结果在NSC体外扩增过程中自噬活性及增殖能力逐渐增强;用Baf A1抑制自噬后NSC的增殖受到明显抑制,表明自噬活性增加能促进NSC增殖。此外,与对照组相比,敲低Atg5或Atg7后,NSC的自噬活性和增殖能力均明显下降。结论自噬能促进NSC在体外扩增中的增殖能力,在这一过程中ATG5和ATG7对于维持NSC自噬活性是必不可少的。

Objective To investigate the roles of autophagy and autophagy-related proteins ATG5/ATG7 in the expansion of neural stem cells(NSCs)in vitro.Methods NSCs were isolated from the brains of embryonic SD rats and cultured in vitro.During the expansion of NSCs in vitro,the changes in the diameter of neurospheres were observed,the cell viability was detected by CCK-8 kit,the protein expression levels of ATG5,ATG7,LC3 and PCNA were analyzed by Western blotting assay,the formation of autophagosomes was observed under a transmission electron microscope(TEM),the expressions of LC3 and Ki67 were analyzed by immunofluorescence,and the cell proliferation ability was tested by EdU assay.The role of autophagy and ATG5/ATG7 in the expansion of NSCs was evaluated after Baf A1 treatment or knockdown of Atg5/Atg7 with siRNA.Results Autophagy and proliferation were gradually enhanced with the expansion of NSCs in vitro.The proliferation of NSCs was significantly inhibited after autophagy was blocked with Baf A1,indicating that the increase in autophagy could promote the proliferation of NSCs.In addition,compared with the control group,autophagy and proliferation of NSCs decreased significantly after knockdown of Atg5 or Atg7.Conclusion The expansion of NSCs in vitro can be promoted by autophagy,in which ATG5 and ATG7 are critical to autophagy of NSCs.