Effect Of Per2 Gene Overexpression On Radiosensitivity Of U87 Glioma Stem Cells

Objective:Study on transfection of U87 glioma stem cells with Per2 gene overexpression lentivirus,Then,the expression of Per2 gene in U87 glioma stem cells was identified,To investigate the effect of Per2 gene overexpression on the sensitivity of U87 glioma stem cells to radiotherapy? Methods:1.U87 glioma stem cells were induced by serum-free medium(with growth factor bFGF,growth factor rhEGF,B27 reagent and N2 additive)and suspension culture,and then transfected with lentivirus to transfect glioma stem cells in fluorescence Transfection efficiency under microscope?2.Real-time quantitative PCR and Western blotting showed that the expression of Per2 gene in the overexpressing group was significantly higher than that in the other two groups(P<0.05),while the expression of Per2 gene in empty vector group and normal cell group was detected.There was no significant difference(P>0.05).3.Three groups of experimental cells were irradiated with 8mV 100 cGy X-ray,and the expression of ?H2AX protein in each group was detected by Western blotting at 24 h and 48 h respectively.4.The apoptosis rate of the stem cells in the overexpressed group was(45.3±0.75)% and(51±0.82)% at 24 h and 48 h,respectively,and the cells in the empty vector group were at 24 h and 48 h.The apoptotic rates were(25±0.54)% and(29.8±0.49)%,respectively.The apoptosis rate of blank cells at 24 h and 48 h was(22.1±0.48)% and(26.9±0.50)at 24 h and 48 h.%,the apoptotic rate of stem cells in overexpressing group was significantly higher than that in empty vector group and blank group(P<0.05),but there was no significant difference in apoptosis rate between empty vector group and blank group(P>0.05).And with the prolongation of time after radiotherapy,the apoptosis of the three groups of cells increased with time.Results:1.Under the condition that the MOI value is 17,The fluorescence of U87 glioma stem cells observed under the fluorescence microscope is the most Strong,With the MOI worth increasing,Cell brightness is not significantly enhanced?2.Detection of Per2 gene expression in three groups of cells by real-time fluorescent quantitative PCR and Western blotting,The expression of Per2 gene in stem cells of overexpressing group was significantly higher than that of other two groups,There was no significant difference in the expression of Per2 gene between empty vector group and normal cell group.3.The expression of ?H2AX protein in the overexpressed cells was higher than that in the other two groups(P<0.01),and the expression of ?H2AX protein in empty vector group and blank group was not observed.Significant difference(P>0.05),and with the increase of time,the expression of ?H2AX protein in each group increased.4.X-ray irradiation for 24 h,48h,The apoptosis of stem cells in overexpressing cells was higher than that in the other two groups,There was no significant difference in apoptosis between empty vector group and normal group.Conclusion:1.The U87 glioma stem cell cell overexpressing the Per2 gene was successfully obtained by transfecting U87 glioma stem cells with Per2 gene overexpressing lentivirus.2.Overexpression of Per2 gene increased the expression of DNA damage marker ?H2AX protein in U87 glioma stem cells after receiving radiotherapy,which significantly promoted cell DNA damage,resulting in increased cell apoptosis after radiotherapy and prolonged time.The DNA damage and apoptosis of the cells also rise.3.When the Per2 gene is overexpressed,the sensitivity of U87 glioma stem cells to radiotherapy can be significantly enhanced.