Biological Effects Of Soluble CD40 Ligand On Non-hodgkin’s Lymphoma Raji Cells

Objective:To investigate the effect of soluble CD40 ligand(sCD40L)on proliferation and apoptosis of non-Hodgkin’s lymphoma Raji cells and the signal factor RAS/RAF/MEK/ERK on MAPK pathway,to provide more experimental and theoretical basis for the application of sCD40 L in the treatment of human Burkitt lymphoma.Method: 1.Raji cells were routinely cultured;cells in logarithmic growth phase were treated with concentration gradient of 0.25 μg/mL,0.5 μg/mL,1 μg/mL,2 μg/mL,and 4μg/mL for 24 h,48 h,and 72 h,and cells were added with CCK8 solution.Vitality,determine changes in cell proliferation to understand the biological effects of sCD40 L on Raji cells CCK-8 assay to detect the effect of sCD40 L on its proliferation.2.To detected the effect of sCD40 L on apoptosis of Raji cells by AnnexinV-FITC/PI double staining method.3.Flow cytometry was used to detect the effect of sCD40 L on the cell cycle of Raji cells.4.Used Real-time quantitative PCR to detect the effect of sCD40 L on the expression of ras,raf,MEK and ERK1/2 mRNA in Raji cells.Result: 1.Different concentrations of sCD40 L had different inhibitory effects on Raji cells at 24 h,48 h and 72 h,respectively,and there were significant differences compared with the control group(P<0.05).In this experiment,the maximum concentration of sCD40 L was 4 μg/mL,which was the most obvious inhibitory effect on Raji cells.At the same concentration but different time,the inhibition was most obvious at 72 h.2.AnnexinV-FITC/PI double staining showed that the apoptosis rate of Raji cells increased gradually with Raji cells treated with sCD40 L at 4 μg/mL for 24 h,48 h,and 72 h,and the apoptosis rate was the highest at 72 h.h and 48 h were statistically significant(P<0.05).It is suggested that sCD40 L can induce apoptosis of Raji cells,and the apoptosis rate of Raji cells increases with the prolongation of sCD40 L.3.Flow cytometry showed that the cell cycle showed that the proportion of cells in G0/G1 phase of Raji cells increased after treatment with sCD40 L at 4μg/mL for 24 h and48h,which was different from S and G2/M(P<0.05).It is suggested that the cells may be arrested in the G0/G1 phase.4.Real-time quantitative PCR showed that the expression of ras mRNA decreased at48 h,72 h after sCD40 L and Raji cells were cultured at 4 μg/mL for 24 h,48 h and 72 h,respectively.There was a difference in the control group(P<0.05).The expression of raf mRNA decreased at 72 h,and there was no significant difference between the two groups(P(29)0.05).The MEK mRNA decreased at 48 h and the expression was decreased.There was no statistical difference between the two groups(P(29)0.05);the expression of ERK mRNA decreased at 72 h,which was different from that of the control group at the same time(P<0.05),but the expression of each gene at other time points except the above time Both were higher than the corresponding control group,and the statistical comparison results were inconsistent.The above results show that,with the change of time,the expression results of each gene are different but not regular.Therefore,it is not possible to verify whether sCD40 L acts on Raji cells,and it is impossible to determine whether it is related to MAPK pathway.2.sCD40 L blocks Raji cells in G0/G1 phase in vitro.3.The expression levels of MAPK pathway-related factors ras,raf,MEK and ERK mRNA in Raji cells treated with sCD40 L were different,but it could not be explained by inhibiting MAPK pathway.