The Regulation Of Gastric Cancer-secreted Exosomes To Dicer1?PTEN Gene In HLA-DR^neg Leukocytes

Objective 1.By detecting to investigate the correlation between gastric cancer-secreted exosomes and HLA-DR^neg leukocytes;2.To analyse the influence of gastric cancer-secreted exosomes to Dicer1?PTEN gene in HLA-DR^neg leukocytes from protein and gene field,then to discuss the mechanism that how tumor derived exosomes promote immunosuppression.Methods 1.Screening 15 patients who were confirmed by gastric cancer and 16 healthy examined persons of the first affiliated hospital,Henan university of Traditional Chinese medicine,collected the peripheral blood.Extraction of gastric cancer patients serum exosomes and identification by the Exosome Isolation kit;observe the exosomes under the transmission electron microscopy?tem?;Westernblot indicates the protein of exosomes;2.Paramagnetic particle method sorting of HLA-DR^neg leukocytes;Acridine orange staining exosomes and CFSE staining HLA-DR^neg leukocytes,at 37?,5%CO₂ co-cultured 12 h;3.Gastric cancer patients and healthy controls were detected the co-cultured in peripheral blood of HLA-DR^neg leukocytes,the expression of Dicer1?PTEN gene and protein by the QRT-PCR and Westernblot.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 1.Successful purified peripheral blood exosomes of gastric cancer patients;under the transmission electron microscopy?tem?shows that they are double membrane,their diameter ranged 30-100 nm;Westernblot indicate its rich in surface CD9 and CD63 protein molecules;2.Co-cultured after 12h,fluorescence microscope can significantly observe the exosomes were stained red and the HLA-DR^neg leukocytes were stained green,the HLA-DR^neg leukocytes effective absorbing exosomes.3.From the QRT-PCR detection results we found that the relative expression of Dicer1mRNA?PTEN mRNA is respectively under healthy controls in the HLA-DR^neg leukocytes?0.46±0.19?0.48±0.27 P<0.05?;Compared with healthy controls Westernblot shows Dicer1?PTEN is inferior to healthy controls in the HLA-DR^neg leukocytes?0.15±0.11?0.33±0.19P<0.05?.Conclusion 1.The exosomes outside body is rich in the surface of the membrane protein,and it can be mediated information transmission between cells;2.The HLA-DR^neg leukocytes can effective absorbing gastric cancer-secreted exosomes,show that the information in exosomes might affect the expression of the HLA-DR^neg leukocytes;3.Gastric cancer-secreted exosomes can inhibit the expression of D Dicer1?PTEN gene in gastric cancer HLA-DR^neg leukocytes.