| Objective: To investigate the expression of related factors CD47,adenosine triphosphate,and high mobility group protein-1 after thrombospondin-1 induces immunogenic cell death in poorly differentiated mucoepidermoid carcinoma cell lines.Methods:Select MC-3 cell line and divide the cells into blank control group,TSP-1group,paclitaxel and carboplatin positive drug control group,and drug combination group.CCK-8 and flow cytometry were used to detect the proliferation and apoptosis of MC-3cells in the above groups;immunofluorescence staining,ELISA and other methods were used to detect the levels of CD47,ATP,and HMG1 in the MC-3 cell lines in the above groups.Expression;3.All experiments are done 3 times independently,using Graph Pad prism 8.0 statistical analysis software,the results are expressed in the form of mean±standard deviation,the comparison between the two groups adopts the t test or the comparison between multiple groups adopts one-way analysis of variance(one-way ANOVA),P<0.05 was statistically significant.Results:1.When the concentration is constant(0.05 μmol/L,0.1 μmol/L,0.2 μmol/L,0.4μmol/L,0.8 μmol/L,1.6 μmol/L),the inhibitory effect of TSP-1 on MC-3 increases with time(24h,48 h,72h)Increased inhibitory effect on cells.According to statistical analysis,TSP-1 at each concentration has a strong inhibitory effect on MC-3 cells at 72 h,so in the follow-up experiments we use 72 h IC50,the concentration of which is 0.09027μmol/L(about 0.1 μmol/L).2.At the same time(72h),the drug experimental group has an inhibitory effect on MC-3 cell activity.Except for the cell inhibition rate of the CBP group and TSP-1+CBP group,there is no statistical difference compared with the control group.The other drug experiments Compared with the control group,the cell inhibition rate is statistically different(P < 0.05),so this experiment uses the TSP-1,PTX,TSP-1+PTX group as the drug experimental group for subsequent experiments.3.TSP-1 group,PTX group,TSP-1+PTX group can induce MC-3 cell apoptosis after 72 hours of treatment,and the apoptosis rate gradually increases.Among them,the dual combination drug has the highest rate of apoptosis,and the results are statistically significant.Difference(P <0.05).4.In the control group,CD47 was highly expressed in clusters on the cell membrane,while in the drug experimental group,CD47 was scattered in a mist and low expression,and the nuclei of the PTX and TSP-1+PTX groups were split and nucleolytic.The fluorescence intensity of CD47 in the experimental drug group was lower than that in the control group.5.The drug group was treated for 12 hours.Compared with the control group,the ATP content increased.Among them,the TSP-1+PTX group induced the most ATP secreted by MC-3 cells,which was statistically different(P<0.05).6.The drug group was treated for 24 hours.Compared with the control group,the content of HMGB1 increased.Except for the TSP-1 group,which had a statistical difference(P < 0.05)and induced MC-3 cells to secrete the most HMGB1 content,the other drug groups were compared with the control group.There was no statistical difference in comparison.Conclusion:(1)0.1 μmol/L TSP-1 has the best proliferation inhibitory effect on MC-3cells and can induce MC-3 cell apoptosis.(2)The combined effect of TSP-1 and PTX on inhibiting the proliferation of MC-3 cells and inducing their apoptosis is stronger than that of the single drug group.(3)TSP-1 can induce the down-regulation of CD47 protein expression in MC-3 cells,and change CD47 from dot clusters to scattered clusters.(4)TSP-1 can induce MC-3 cells to release ICD-related factors ATP and HMGB1. |
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