Effects Of Overexpression Of CD40L Gene On Biological Function Of Leukemia NB-4 Cell Line

Objective: Lentivirus transfection was used to construct NB-4 cell lines with overexpression of CD40L,and the effects of overexpression of CD40L gene on proliferation,apoptosis and cycle of leukemia NB-4 cells were further investigated.Methods:(1)CD40L was transfected into NB-4 cells by lentivirus.The stable transformation strain was screened by puromycin.The green fluorescence of lentivirus vector was observed under fluorescence microscope.The infection efficiency of cells was detected by flow cytometry.The overexpression of CD40L was verified by q PCR.(2)CCK-8 method was used to detect the effect of CD40L overexpression on NB-4 cell proliferation;YF647a annexin V / PI double staining was used to detect the effect of CD40L overexpression on cell apoptosis;PI staining was used to detect the effect of CD40L overexpression on cell cycle.Results:(1)The results of fluorescence microscopy showed that the lentivirus could be successfully transfected into NB-4 cells.After the stable transformation strains were screened by puromycin,the GFP fluorescence expression rate of three groups of cells was detected by flow cytometry,and the GFP fluorescence expression rates in the group transfected with empty vector and CD40L overexpression group were 96.66% and 95.92% respectively by flow cytometry;the relative expression folds of CD40L in NB-4,group transfected with empty vector and CD40L overexpression group were 1.00 ± 0.05,1.03 ± 0.19 and 813 ± 51.97 respectively by QRT PCR,The relative expression level of CD40L gene in overexpressed CD40L group was significantly higher than that in NB-4 group and idlling group(P<0.05).(2)Under the same culture conditions,CCK8 method was used to observe the effect of CD40L expression on NB-4 cell proliferation,The OD values were 0.426 ± 0.228,0.418 ±0.012 and 0.328 ± 0.003 in NB-4 group,group transfected with empty vector and CD40L overexpression group at 24 h,0.770 ± 0.034,0.715 ±0.034 and 0.567 ± 0.036 in 48 h,and 1.206 ± 0.070,1.180 ± 0.014 and 1.055 ± 0.009 in 72 h,respectively.The OD values in CD40L overexpression group were lower than those in NB-4group andgroup transfected with empty vector(P < 0.05).(3)Flow cytometry was used to detect the effect of CD40L overexpression on NB-4 apoptosis.The results showed that the apoptosis rates of NB-4 group,idling group and CD40L overexpression group were(2.92±0.51)%,(3.320.73)%,(11.03±1.61)%,respectively.The apoptosis rate of CD40L overexpression group was significantly higher than that of NB-4 group and idling group(P < 0.05).(4)The cell cycle of the three groups was detected by flow cytometry,The percentages of G0/G1 phase,S phase and G2/M phase were 40.6%±2.52%,41.1%±5.82% and 19.0%±4.09% in NB-4 group,40.1%±1.82%,38.6±3.51% and 22.4%±4.36% in group transfected with empty vector,and 62.0% ± 3.80%,21.8%±1.68% and 16.6%±1.70% in CD40L overexpression group.The proportion of G0/G1phase in CD40L overexpression group was significantly higher than that in NB-4 group and group transfected with empty vector(P<0.05),and the proportion of S phase in CD40L overexpression group was significantly lower than that in NB-4 group andgroup transfected with empty vector(P<0.05).Conclusion: CD40L can inhibit the proliferation of NB-4 cells,promote the apoptosis of NB-4 cells,and inhibit the cell cell cycle of NB-4 cells in G0 / G1 phase.Our results are helpful to find new targets for AML immunotherapy.