CAJ | 학술논문

Objective: To observe the changes of glioma stem/progenitor cells (GSPCs) before and after induction so as to explore the relationship between the differentiation and autophagy of GSPCs. <br> Methods: GSPCs were divided into 2 groups and cultured in stem cell culture medium and differentiation-inducing culture medium [DMEM/F12 containing 10% fetal calf serum (FCS)], respectively. The latter was divided into experimental group added with autophagic inhibitor 3-MA with terminal concentration of 10 mM/L, and control group was free of any autophagic inhibitor. The culture solutions of both groups were changed every two days. The culture was ended on d7, and cellular morphology was observed. Immunofluorescence staining against microtubule-associated protein light chain-3 (LC3), transmission electron microscopy (TEM) and Western-blot method were used to detect the changes of autophagic activity before and after differentiation of GSPCs. Autophagy inhibitor 3-methyladenine (3-MA) was added after differentiation was induced to observe its inlfuence on GSPCs differentiation and activator rapamycin (RPM) was applied to make further detection. <br> Results: Autophagic activity of GSPCs was relatively low before GSPCs differentiation, but was increased after differentiation. GSPCs cultured in serum-containing medium for 7 d showed adherent growth, star-like or fusiform protractile convex-like structure, vesicle-like structure in proximal cytoplasm of the “convex” under microscope and punctiform distribution of LC3 in corresponding sites by immunological staining. Under TEM, there were obvious and typical autophagosomes in GSPCs differentiated cells. 3-MA could inhibit the adherence differentiation of GSPCs, and Western blot analysis showed that after the differentiation of GSPCs using 3-MA, expression of glial fibrillary acidic protein (GFAP) and ammonium dihydrogen phosphate-2 (MAP-2) decreased signiifcantly than that in control group. In addition, as to GSPCs cultured in serum-free medium, the concentration and action time of RPM had no obvious impact on their morphology, however, the ratio of GSPCs with adherent differentiation increased apparently when RPM concentration was 12.5 ng/mL. <br> Conclusion: Autophogy is in close association with differentiation of GSPCs, which means that increasing autophagy can improve differentiation whereas inhibiting autophagy can suppress the differentiation of GSPCs, and the autophagic activity of GSPCs is improved after inducing differentiation.