| Objective To analyse the level of HLA-DR in peripheral blood monocyte cells of patients with gastric cancer by FCS,and detected the relative level PI3K,PTEN,AKT,mTOR from protein and gene field,make insight into the PI3K signaling pathway how to exert regulation on HLA-DR positive cells.Methods 5ml EDTA anticoagulated Peripheral blood samples were collected from 37 cases of gastric cancer patients and 33 healthy persons.Flow cytometry was used to detect the relative expression of HLA-DR.PBMC was isolated through density gradient centrifugation method.MACS Sorting: blood HLA-DR cells were isolated from peripheral blood monocyte cell by immune magnetic beads.Furmore,HLA-DR cells were divided into two parts,one section was used for nucleic acids extraction and another for proteins extraction,we detected the m RNA and protein levels of PTEN,PI3K,Akt,and mTOR expression by RT-PCR and Westernblot technique,respectively.Searching mechanism of how PI3K/AKT/mTOR signaling pathway make effect on the HLA-DR cell with gastric cancer.Experimental data was repeated three times,and results descripted in the form of Mean±standard deviation,statistical analysis was performed by SPSS18.0 statistical software,groups significant differences analysis use chi square test in the condition of homogeneity of variance.P < 0.05 was treated significantly different.Results Flow cytometry results showed that the percentage of HLA-DR in gastric cancer patients and healthy human peripheral blood is(13.11 + 3.01)% and(9.02 + 1.42)%,respectively.And Variance analysis showed that,P < 0.05,HLA-DR expression in gastric cancer peripheral blood were significantly higher than these in healthy control group.Realtime PCR results showed that gene PI3K and mTOR expression in healthy human HLA-DR positive cells is higher than that in HLA-DR negative cells,nevertheless,on the contrary,the expression of PTEN in HLA-DR positive cells is lower than that in negative cell,suggesting that low expression of PTEN in positive cells is favor for activation of the PI3K signaling pathway in HLA-DR cell;Identically,patients with gastric cancer,gene PI3K, mTOR in HLA-DR positive cells is still higher than that in negative cells,but the expression of PTEN in positive cells were significantly higher than the negative cells,therefore,we speculated that under the state of gastric cancer,high expression of PTEN in tumor patients HLA-DR positive cells may be the negative regulator in PI3K signal pathway;furthermore,we compared gene expression in gastric cancer patients and healthy people.Find out PI3K signaling pathway gene expression were not significant in both group HLA-DR negative cell,however,the HLA-DR positive cells have obvious difference,expression of PI3K and mTOR in gastric cancer patients was significantly lower than the control.Immunoblotting results showed that expression of PTEN in healthy peripheral blood HLA-DR negative and positive cells was 1.27± 0.52,0.78± 0.24,respectively,PTEN in gastric cancer peripheral blood HLA-DR negative and positive cells was 2.55±1.20,3.17±1.39,respectively,results of variance analysis indicated that the higher expression of PTEN in gastric cancer HLA-DR positive cells has significant difference(F = 15.07,P =0.000).PI3K expression in healthy human peripheral blood HLA-DR negative and positive cells was 0.58±0.18,1.51±0.47,respectively,and in gastric cancer HLA-DR negative and positive cells was 1.38±0.61,2.12±0.54,respectively,according to the results of variance analysis(F = 10.21,P = 0.000),the expression of PI3K in the above groups was statistically significant.AKT expression in healthy human peripheral blood HLA-DR negative and positive cells was 0.77±0.24,0.64±0.16,respectively,and in gastric cancer HLA-DR negative and positive cells was 0.94±0.19,1.02±0.24,respectively,the variance analysis results(F =5.41,P =0.007)showed that the expression of AKT also has significant difference.Conclusion After the occurrence of gastric carcinoma,the expression of HLA-DR increased,suggesting the ability of tumor present by antigen-presenting cells enhanced.However,there is a limit to the expression of HLA – DR.Gene and protein expression of PTEN in gastric cancer HLA-DR positive cells was higher when contrast to other groups.To some certain extent,PTEN can inhibit the PI3K/AKT/mTOR pathway downstream signal activation,furthermore,inhibit the excessive proliferation of HLA-DR cells,decrease the double damage by body excessive immune response at the same time,provide limited immune response to gastric cancer. |
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