Stemness And Inducing Differentiation Of Hunman Glioma Cells

Objective:By studying the stem cell characteristics of human glioma cell line U251 and its potential of differentiating into adipocytes and osteoblast, to reveal the biological characteristics of glioma cells and explore the conditions and mechanisms of inducing differentiations.Inducing differentiation may be a novel method in treatments of glioma in further.Methods:For detecting expression of stem cell markers,the glioma cell line U251 were conventional cultured Then the U251 cells were seeded in 24 well plates which preset advanced sterile round slide.After fixed using 4% paraformaldehyde immunofluorescence staining was used to detct the expression of stem cell marker Nestin,sal-like4(Sall4),neural cell adhesionmolecule(NCAM),Octamer-binding transcription factor-4(Oct4),SRY-related high-mobility-group(HMG)-box protein-2(Sox2) and S100β in U251 cells.These U251 cells were observed with immunofluorescence microscope.The U251 cells were treated with adipogenic induction medium(containing 2μmol/L insulin,500μmol/L(IBMX),1μmol/L dexamethasone,200μmol/L indomethacin) and osteogenic induce medium(containing100mmol/L dexamethasone,10mmol/Lβ-glycerophosphate and 0.05mmol/L vitamin C),At the end of Inducing differentiation, adipocyte-like lipid droplet were detected by staining with Oil Red O in these cells and the ossification of differentiatedcancer cells were detected by staining Alizarin Red S. The induced differentiation growth curve of U251 cells was detected by CCK8 method.To explore the mechansims of aipogenic and osteogenic differentiation,the expression of adipogenic regulatory proteins,osteogenic regulatory proteins and apoptosis/authophagy associated proteins during inducing differentiation were detected by Western blotting.Result:1.The human glioma cell line U251 can stably express a variety of stem cellmarkers. Immunofluorescence staining showed that neural cell adhesion molecule NCAM, neural crest stem cell markers Nestin, glial cell markers S100β, pluripotent stem cell markers Sall4, Oct4 and neural stem cell markers Sox2 were positive expression.in U251 cells.2.U251 cells can be successfully induced into adipocytes. After inducing differentiation with adipogenic induction medium, the Oil Red O staining showed that lipid droplets were produced in cancer cells; and with the extension of the induction time, the lipid droplets gradually increased; The CCK8 results showed a slowing in the cancer cell proliferation after induction differentiation; Western blotting results showed that expression of adipocyte associated proteins increased in cancer cells after induction differentiation.3.U251 cells can be successfully induced into osteoblasts. After inducing differentiation with osteogenic induction medium, the Alizarin Red S staining showed that a large number of cell calcium nodules were produced in cancer cells; The CCK8 results showed a slowing in the cancer cell proliferation after induction differentiation;Western blot results showed that expression of osteoblastrelated proteins and apoptosis/autophagy/proliferation related proteins increased in cancer cells after induction differentiation.Conclusion:1.Most cancer cells in U251 cell line possessed high stem cell properties and plasticity.The U251 cells can be induced into adipocytes and osteoblasts.2.The proliferate of U251 can be inhibited cells by inducing differentiation,and inducing differentiation can promote terminal differentiation,autophagy and apoptosis of U251 cells.3.The experimental results show that inducing differentiation can be used as a potentially effective method of human glioma therapies.