The Study Of The Influence Of Interferon-a On The Differentiation, Maturation And Functions Of Dendritic Cells In Vitro

Objectives To investigate the influence of different concentrations of IFN-a on differentiation, maturation and immune function of human cord blood derived dendritic cells in vitro. To evaluate DC with hepatoma cell lysates induced antit-hepatoma effect and the feasibility of experimental methods of cultivation DC and antitumor efficiency.Methods Isolated from umbilical cord blood mononuclear cells (CBMNC), in complete culture medium (RPMI-1640 +5% fetal calf serum) added GM-CSF (1000U/ml), IL-4 (500U/ml) and different concentrations of INF-a (A to E five concentration 0,100,500,700,1000 U/ml) to induced DC through CBMNC, on 7th day, DC Cell morphology was observed in microscope, flow cytometry detected DC surface markers CD14, CD83, CD86, CD40, CDla and HLA-DR expression, The methed of MTT determination the proliferation of allogeneic lymphocytes and the rate of cell killing about DC-mediated CTL, The methed of ELISA determination of DC supernatants of cytokine levels of IL-12, IFN-y and IL-10.Results 1.cell factor GM-CSF+IL-4 and INF-a with different concentrations to cultivate CBMNC, CBMNC cultured for 3 days, most of the cells still adhered to the wall and some cells forming small colonies, no dendritic-like processes,On the 7th day, the surface of cells significantly pseudopod-like protrusions, showing a typical dendritic morphology; 2.Cultured cells detectd surface markers by flow cytometry,we found that D groups cell surface expression of CD83, CD1a, HLA-DR, the expression rate (%) were CD86 (95.10±1.53), CD83 (75.11±1.63), CDla (38.75±1.93), HLA-DR (88.75±1.65);3.Within a certain range (0,100,500,700 U/ml), INF-a induced DC to stimulate allogeneic lymphocyte proliferation increased gradually (P <0.05),When the concentration of INF-a reach 700U/ml and the T: DC= 10:1, stimulating the proliferation of allogeneic lymphocytes about OD value (0.485±0.100)is highest, and obvious significant difference with other groups (P<0.05);4.In this study,when INF-a concentration reach 700U/ml, the load of the DC antigen SMMC-7721 liver cancer cells induce allogeneic CTL killing is the highest percentage (%), (59.0±2.0); and had significantly difference with other goups (P <0.05); 5.the methed of ELISA to detect A, B, C, D, E groups cytokines in DC supernatants, on 3th day cytokines in DC supernatants had no significant difference with other groups (P> 0.05); 7th day cytokines levels of each group were high, in which D group IL-12 (37.69±2.67 pg/ml), IFN-y (49.94±1.35 pg/ml) was highest, and had significant difference with other groups (P<0.05); and D group of IL-10 (32.34±2.49 pg/ml) were the lowest, and had significant difference with other groups (P<0.05).Conclusion CBMNC isolated from cord blood, GM-CSF, IL-4 and different concentrations of INF-a (0,100,500,700,1000 U/ml) can promote differentiation and maturation of DC in vitro; different concentrations of INF- a can enhance contention of cytokine IL-12 which DC secretion, IFN-y, DC loaded with tumor antigen can promote the induction of allogeneic lymphocyte proliferation and enhance DC-mediated CTL killing rate of hepatocellular carcinoma. In this experiment, INF-a concentration reaching 700U/ml is the optimal concentration. this experiment provide some experimental basis for clinical application.