Research On Circadian Rhythms Of The Per2 Gene In Human Glioblastoma Stem Cells

ObjectiveTo explore whether there are stem cells in human U87 glioma cell lines,and to observe their growth and morphological changes,and explore the circadian rhythm of Period2.Methods(1)Isolation,extraction and identification of cells(1)Glioma U87 cells were cultured by suspension culture and adding bFGF,rhEGF,B27,and N2 into serum-free medium.The morphology and growth traits of the tumor spheres isolated and cultured were observed by inverted phase contrast microscopy;Identification of surface protein of stem cell tumor cells by immunofluorescence staining;Detection of mRNA expression of surface protein of stem cell tumor by RT-qPCR;(2)The stem cell tumor spheres were differentiated,induced and cultured by using a full medium containing serum(DMEM:fetal bovine serum: streptomycin =1: 10%: 1%).Immunofluorescence staining was used to identify the phenotype of differentiated cells and further determine the multidifferentiation ability of stem cell spheroids.(2)Rhythmicity of the biological clock gene per2DMEM containing 10% serum and serum-free stem cell culture medium supplemented with trophic factors were used to culture U87 cells and U87 stem cells respectively,Then PMA inducer was added to the medium to induce the expression of the circadian clock gene,Then the cells with different time points at 4 h,8 h,12 h,16 h,20 h and 24 h were selected,and the rhythms of per2 gene in two kinds of cells were detected by real-time fluorescence quantitative PCR.Results:1.In the complete culture medium with 10% serum,human U87 glioma cells show a state of adherent growth and a synapse protruding,and the shape of the cells is varied.2.The cells were cultured with bFGF,rhEGF,B27 and N2 in the DMEM/F12 without serum,and the cells were suspended in the form of spheric growth,and they could propagate continuously.3.The detection of cell immunofluorescence staining showed that The surface protein CD133,Nestin and Sox2 of the cultured tumor cells were positive.4.Real-time fluorescence quantitative PCR assay revealed that the expression of surface proteins CD133,Nestin,and Sox2 in tumor spheres was significantly higher than U87 cells.5.The U87 tumor spheres were induced and differentiated with a complete medium containing serum,and can be redifferentiated into parietal cells.Subsequently,immunofluorescence staining of cells was performed: GFAP-positive cells,β-tubllin-positive cells,and MBP-positive cells.6.The biological clock gene Per2 was detected by RT-qPCR method in U87 cells and U87 tumor spheres.The peak expression of per2 in U87 glioma cells is in 12 h and 24 h,and the valley value is 8h and 20 h,and the highest point is in 24 h,and the lowest is 20 h.The peak expression of Per2 in U87 tumor spheres was at 4h and 16 h,and the valley values were at 12 h and 24 h.The highest point was at 4h and the lowest point was at 24 h.Conclusion:1.U87 cells can isolate tumor stem cells from DMEM/F12 medium containing nutrient factors and serum-free,and this stem cell has strong ability of self propagation and generation and differentiation into multiple cells.2.The expression of Per2 gene in glioma U87 cells and U87 glioma stem cells is 12 hours,but there is a difference between the peak and the valley value.