Effect Of EGFRv? Extracellular Modified Dendritic Cells On The Induction Of CTL Cells To Gastric Cancer Cells

Objective The epidermal growth factor receptor type ? mutant(EGFR v?)extracellular modified dendritic cells inducing CTL cells of EGFR v? positive role of 7901 gastric cancer cells,a preliminary explore its action mechanism.Methods To construct the lentivirus expressing EGFR v?,the titer was determined by fluorescence quantitative method,and the T lymphocyte and mononuclear cells were isolated from human peripheral blood by density gradient centrifugation,using cytokines rh IL-4(100ng/ml),rh GM-CSF(100ng/ml)and rh TNF-a(50ng/ml)induce monocytes to differentiate into dendritic cells(DC),by flow cytometry instrument detection of dendritic cell surface antigen CD80,CD83,HLA-DR verify DC mature,using the lentivirus transfection DC with EGFRv?,the expression of EGFRv? in DC was determined by Western-blot.Blank DC,EGFRv? slow virus infection were dendritic cells and dendritic cells,T lymphocytes in 1:5,1:10,1:20,1:40 trained,after 72 h after joining CCK 8,Carbon dioxide incubator at 37 C was placed for 4 hours,then observed under the microscope.Then the supernatant absorbed by each group of mixed cells cultured at 450 nm after adding CCK-8 was detected by enzyme labeling instrument.,T lymphocytes cultured from adult peripheral blood were co-cultured with DC,EGFRv?-infected dendritic cells and negative control dendritic cells at 37 C(stimulated cells/effector cells = 1:20)to collect supernatants of cell culture.T lymphocytes in 1:5,1:10,1:20,three days after 1:40 trained to join CCK8,use Bio-Rad microplate reader record in the 450 nm wavelength absorption value,Their own T cells respectively with blank DC,EGFRV? slow virus infection of dendritic cells,negative control of dendritic cells in 37 ? trained(stimulate cells/effect = 1:20)collect cell culture supernatant,using ELISA method,detection of IL-10 in the supernatant and IFN-r production.Pick up after the wedding of GC7901 cells and GC7901-v? as target cells,according to the different proportions of effector cells and the proportion of the target cells(1:5,1:10,1:20,1:40)to join the different groups of T cell,continue to develop for 24 hours,determined by MTT method to kill your situation.All the above experiments were repeated three times.Results 1.The mature dendritic cells and T lymphocytes is cultured with density gradient centrifugation and cytokines.2.The expression rates of CD80,CD83 and HLA-DR in dendritic cells without TNF-a were 35.5%,25.1% and 33.2%.The expression rates of H'LA-DR in dendritic cells with TNF-a is 82.7%,69.6%.The expression rates of CD-80 and HLA-DR-APC in dendritic cells infected with EGFR v? is 86.3% and 76.0%.3.Lentivirus carries the target gene EGFRv? to infect dendritic cells.Western Blot assay of EGFRv? protein showes that the expression rate of EGFR v? in infected DC is 58%.4.Microscopically observes dendritic cells carrying the target gene EGFR v? can obtain a high infection rate of 60 %.5.Infection EGFR v? of DC and T lymphocytes mixed culture itself,can stimulate T cells to produce more with antitumor activity of cytokines IL-10 and INF-r,and a dose dependent.6.In the same condition,EGFRv? extracellular stage modified dendritic cells induced CTL cells to be more effective in the killing of 7901 cells of EGFRv? positive gastric cancer.Conclusions 1.High purity T lymphocytes and dendritic cells can be obtained from human peripheral blood through density gradient centrifugation and cytokine stimulation,and TNF-a is the key to promote the maturation of dendritic cells.2.Dendritic cells stimulate T lymphocytes to produce cytokines IL-10 and INF-r with killer activity,which can kill 7901 cells of gastric cancer.3.The study showes that in vitro EGFRv? positive dendritic cells significantly enhanced the immunity of CTL cells to 7901 cells of gastric cancer.