| Objective:To study the anti-lung cancer effect of ARS on lymphocyte,and to observe whether the expression of CD3 9 and PD-1 molecules on lymphocyte surface after ARS induction has changed,especially the expression level of CD39 and PD-1 molecules on CTLs' surface,and it has statistical significance or not.ARS-treated lymphocytes were co-cultured with lung cancer cells to observe whether the proliferation of lung cancer cells was significantly inhibited or increased,and whether the apoptotic level of lung cancer cells was significantly increased or decreased.Methods:FCM was used to detect the inhibition rate of different concentrations of ARS on lymphocyte,and obtained the optimal concentration of ARS-induced lymphocyte.FCM was used to detect the expression levels of CD39 and PD-1 molecules on the surface of CTLs in healthy donor and lung cancer patients,and to compare the effects of ARS induction on its expression level.FCM was used to detect lymphocyte and lung cancer before and after ARS induction.Proliferation and apoptosis of lung cancer cells after co-culture.FCM was used to detect the expression of GrzB and PerF,Fas and FasL in CTL of lung cancer patients induced by ARS.Results:1.The time-dependent and drug concentration-dependent inhibition rates of ARS-treated cells were plotted based on the data obtained by FCM.According to the drug concentration screening experiment,the induction concentration of ARS was determined to be 200?M.The experiment was divided into drug treatment group with ARS induction concentration of 200?M and negative control group with ARS induction concentration of 0?M..2.According to the flow dot plot,the lymphocyte groups of normal donors were gated.Then the CD3/CD8 axis and the CD3+CD8+T cell group,the positive rate of CD39/PD-1 axis was compared between the experimental group and the control group.After Two-way paired T test,the expression level of CD39 molecule in the experimental group of normal donors was significantly higher than that in the control group.The P value of the two groups was 0.002.The results has statistically significant.The positive expression rate of PD-1 molecule in the two groups was 0.4107,with no statistical significance.According to the flow dot plot,the lymphocyte groups of lung cancer patients were circled and selected.Then the CD3/CD8 axis and the CD3+CD8+cell group,namely CTL cell group,were gated.The positive rate of CD39/PD-1 axis was compared between the experimental group and the control group.After Two-way paired T test,the expression level of CD39 molecule in the experimental group of lung cancer patients increased significantly compared with the control group,the P value of the two groups was 0.0004,the results has statistically significant;the expression level of PD-1 molecule was significantly increased compared with the control group,the positive expression rate of PD-1 molecule in the two groups was 0.0002,the results has statistically significant.Compared with CD3+CD4+cells group,also named Th cells,there was no significant difference in P value,suggesting that ARS had no significant effect on the expression of CD39 and PD-1 molecules on Th cells.On this basis,the clinical epidemiological information of experimental specimens was classified according to different conditions(such as sex,age,type of lung cancer,history of disease,history of treatment and etc.).The results showed that the factors influencing the expression of CD39 molecules on CTL cells induced by ARS might be the history of radiotherapy and absolute value counting of monocytes,and the factors influencing the expression of PD-1 molecules on CTL cells induced by ARS might be the following.The P values of lung cancer type,radiotherapy,sex and absolute value of monocytes were statistically significant.3.According to the flow chart of co-culture,the number of strong positive CFSE cells in lung cancer cells of the experimental group was significantly higher than that of the control group,the number of weak positive CFSE cells was significantly lower than that of the control group,those results suggest that the antiproliferative activity of CTL induced by ARS is significantly increased,and the pathway for ARS to achieve this function may be paved by increasing the expression of CD39 and PD-1 molecules on the surface of CTL.And the Annexin V positive cell rate was higher than that of the control group,the P value are 0.0123 and 0.0182,all respectively with statistical significance,those results suggest that CTL induced by ARS can significantly increase the apoptotic ability of tumor cells,and the mechanism may include that ARS affects the expression levels of CD39 and PD-1 molecules on the surface of CTL.4.The experimental results of investigating CTL-induced apoptosis of lung cancer cells showed that GrzB expression in CTL of ARS-induced lung cancer patients was significantly up-regulated and statistically significant,P value was 0.0302,while the expression of PerF,Fas and FasL was not significantly affected,P value was 0.1549,0.3934 and 0.9273,respectively.Conclusion:This study found that ARS could induce significant positive changes in the expression of CD39 and PD-1 molecules on the surface of killer T lymphocyte.The analysis of epidemiological data showed that the factors influencing the expression of CD39 and PD-1 on CTLs induced by ARS might be the history of radiotherapy,absolute number of monocytes and types of lung cancer,radiotherapy,sex and absolute number of monocytes,which revealed that ARS was more suitable for the types of lung cancer induced by ARS In co-culture of induced lymphocyte and lung cancer cells,it was found that the proliferation of lung cancer cells was significantly inhibited,while the apoptosis of lung cancer cells was increased.It proved that ARS could indirectly induce apoptosis of lung cancer cells by changing the level of CD39 and PD-1 molecules on the surface of CTLs.At the same time,it blocked the proliferation of lung cancer cells.The mechanism of ARS has proved that ARS induces apoptosis of lung cancer cells by inducing the high expression of GrzB in CTL cells of lung cancer patients.Figure 49 table 2 reference 61... |
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