| Objective: By conducting cell experiments in vitro,to investigate the effects of Pemetrexed on proliferation inhibition of lung adenocarcinoma cell line HCC827 and the effects of PD-L1(programmed cell death-ligand1)and PI3K(phosphatidylinositol 3 kinase)/ AKT(protein kinase B)pathway related proteins AKT and p-AKT,and further explore its possible mechanism.Methods:(1)Culture of human lung adenocarcinoma hcc827 cells: hcc827 cells were cultured in 1640 medium containing 10% fetal bovine serum,and placed in 5% CO2,37 ℃ constant temperature and saturated humidity incubator,and then logarithmic growth cells were used in subsequent experiments.(2)Hcc827 cells were treated with different concentrations of pemetrexed(0,0.01,0.1,1,10,100 μ mol / L)at different times(12,24,48h).The proliferation inhibition of hcc827 cells was detected by CCK-8 method and the IC50 of pemetrexed was determined.At the same time,the optimal action time(48h)was selected as the intervention time of the follow-up experiment The IC50 of mettrexate was used as the drug concentration in the follow-up experiment.(3)Flow cytometry was used to detect the expression of PD-L1 after pemetrexed at different concentrations(0,0.01,0.1,1,10,100 μ mol / L)was used to compare the differences among the groups.(4)Experimental grouping and treatment: the experiment was divided into four groups: control group(adding culture medium),IC50 PEM group,PI3 K / Akt pathway activator 740y-p group,IC50 PEM + 740y-p group.(5)Western blot was used to detect the protein expression of Akt and p-Akt in each group: according to the above experimental grouping and treatment,the protein expression of Akt and p-Akt in each group was detected by Western blot after 48 hours of treatment,and the differences among the groups were compared.(6)Flow cytometry was used to detect the expression level of PD-L1 in each group:according to the above grouping situation,the expression level of PD-L1 in each group was detected by flow cytometry after 48 hours of treatment,and the difference of PD-L1 expression in each group was compared.Results:(1)CCK-8 method was used to detect the inhibitory effect of pemetrexed on the proliferation of tumor cells: compared with the control group,the inhibitory rate increased gradually with the increase of pemetrexed concentration(P<0.05),and the difference was statistically significant(P<0.05);The inhibitory effect of pemetrexed 48 h group was stronger than that of pemetrexed 12 h group(P<0.05),and there was no significant difference between pemetrexed 48 h group and 24 h group(P>0.05);(2)Flow cytometry was used to detect the effect of different concentrations of pemetrexed on the expression of PD-L1 in tumor cells: with the increase of pemetrexed concentration,the expression of PD-L1 in the experimental groups decreased gradually(55.07 ± 1.81,44.83 ± 1.74,38.3 ± 1.73,27.13 ± 0.78,23.4 ± 1.21,16.73 ± 0.76),compared with the control group,the difference was statistically significant(P<0.05);(3)Western blot was used to detect the expression of p-Akt and Akt protein.Results: after 48 hours,the expression levels of p-Akt protein in control group,IC50 PEM group,740y-p group and IC50 PEM +740y-p group were 0.24 ± 0.01,0.05 ± 0.01,0.54 ± 0.03 and 0.13 ± 0.01,and the difference was statistically significant(P<0.05);Compared with the control group,the expression of p-Akt protein increased in 740y-p group(P<0.05),and decreased in IC50 PEM group and IC50 PEM + 740y-p group(P<0.05),and the expression of p-Akt protein in IC50 PEM group was lower than that in IC50 PEM + 740y-p group(P< 0.05).The expression levels of Akt protein in each experimental group were 0.63 ± 0.03,0.64 ± 0.04,0.63 ± 0.03 and 0.65 ± 0.02,respectively,and there was no significant difference among the groups(P>0.05);(4)The expression of PD-L1 was detected by flow cytometry: the expression of PD-L1 in each experimental group was 59.83 ± 2.58,32.5 ± 1.04,71.63 ± 2.82,41.1 ± 2.48 after 48 hours of treatment,and the difference was statistically significant(P<0.05);compared with the control group,the expression of PD-L1 in 740y-p group was up-regulated(P<0.05),The expression of PD-L1 in IC50 PEM group and IC50 PEM + 740y-p group decreased(P<0.05),and the expression of PD-L1 in pemetrexed group was lower than that in pemetrexed +740y-p group(P<0.05).Conclusions:(1)Pemetrexed could inhibit the activity and proliferation of hcc827 cells.With the increase of pemetrexed concentration,the proliferation ability of hcc827 cells gradually decreased,and had a certain concentration dependence.(2)PEM could down regulate the expression of p-Akt and PD-L1.(3)PEM down regulates the expression of PD-L1 in hcc827 cells,which may be related to the inhibition of PI3 K / Akt signaling pathway. |
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