| Objective:Study the different expression of the CD11c、CD80、CD86、 CD40、MHC Ⅱ on the DC surface before and after the H22mouse hapatoma cells whole-cell antigen loaded, and the anti-mouse hepatoma effect of tumor infiltrating lymphocytes (TILs) stimulated by dendritic cells (DCs) pulsed with the H22mouse tumor full-cell antigen in vitro, to discuss the significance of the phenotypic changes of DC pulsed with H22whole-cell antigen in improving the killing effects on liver-carcinoma of TIL activated by DCs against H22cell.Methods:①The the BALB/c mouses turned into Tumor-bearing mouses after12days injection of cell growth H22in the logarithm growth period in the annpits subcutaneous。②Separat out H22cells and TIL from the H22liver cancer tumors by the discontinued density gradient method. TIL was expand by culture with restructuring mouse interleukin2(rmIL-2) in vitro, and a part of the H22cells are preparated into the H22whole-cells antigen, the other part of H22cells are cultured in vitrol for detections of killing effects.③the H22whole-cells antigen were made by freeze-thaw the H22cells in the logarithm growth period after four freeze-thaw cycle.④Sterile take out mouse spleen, grinding, filter into cell suspension liquid, cracking the red blood cells, culture with restructuring mouse interleukin2(rmIL-2).⑤Bone marrow cell suspensions were seperated from bones of mouses, then the granulocytes, lymphocytes and NK cells were removed by growth characteristics of the stick. ImmatureDC were cultured with recombinant murine granulocyte marcophage-colony stimulating factor,(rm GM-CSF) and recombinant murine interleukin-4(IL-4).⑥In the fifth day of the culturing of the DC to hybrid the freeze-thaw H22whole-cell antigen, cultivate48hours to completed stimulation. Determination the expression rate of CD11c,CD80,CD86, CD40,MHC Ⅱ on DC before and after stimulation,﹕timulated the TIL and splenic lymphocytes With the DC loaded H22whole-cell antigens, non-sensitizated DC and H22whole-cell antigens.⑧Cytoxicity assays on the H22cells was test by the CCK-8, and divided the effector cells into eight groups:TIL stimulated by DC loaded H22whole-cell antigens; TIL stimulated by DC loaded non-antigen; TIL stimulated by H22whole-cell antigens; TIL without stimulation; splenic lymphocytes stimulated by DC loaded H22whole-cell antigens; splenic lymphocytes stimulated by DC loaded non-antigen; splenic lymphocytes stimulated by H22whole-cell antigens; splenic lymphocytes without stimulation. Effect than the target at10:1.Results:(1) Dendritic cells morphological observation:The progenitor cells of dendritic cells are stick wall cells, the film is smooth, without dendritic protrusions stabilized, stick on wall growth in small group of cells sample, cell clong stick on wall could be seen on the2days culture, and bigger cell clong, more dendritic protrusions could be seen on the5days culture, Antigens sensitization2days later (7days) dendritic cells clongs, was suspended growth, the body is big and round, raised much and dense, and have some cells peripheral bumps for fluffy samples.(2) dendritic cells phenotype:5days culturing, DC purity (CDllc+cells rate) of65%above; In H22whole-cell antigen after allergic sensitization, mice CD80CD86CD40MHC DC in the expression of a significantly higher rate.(3) cytotoxic activity test results:(1) killing activity at H22cells detection results indicate, the killer activity TIL [(47.35±3.40)%] is far higher than the killer activity splenic lymphocytes [(28.45±2.56)%], the difference was statistically significant (P<0.01); Different methods of activated to killing effect at H22, TIL activity are higher than the corresponding ways to activate splenic lymphocytes H22cells kill activity, the difference was statistically significant (P<0.01).(2) kill activity of TIL stimulated by DC loaded H22whole-cell antigens [(81.90±2.90)%] higher than any other way of handling TIL and inactive TIL, the difference was statistically significant (P <0.01); All the handled TIL effects are higher than the TIL without handled, the difference was statistically significant (P<0.01).(3) the killing activity of splenic lymphocytes stimulated by DC loaded H22whole-cell antigens [(62.64±3.94)%] are higher than the splenic lymphocytes without stimulated [(28.45±2.56)%], and the difference was statistically significant (P<0.01).Conclusion:the mouse DC could matured by the stimulated of H22whole-cell antigens. The expression rate of CD80, CD86, DC surface CD40, MHC Ⅱ on DC are highter after stimulated by the H22whole-cell antigens. The H22cell all cells antigen sensitization to DC can induce activation TIL, obviously improve the killer activity at the H22cells in vitro, the increasing express of phenotypes CD80、CD86、CD40、MHC Ⅱ on DC is a significant process in improving the killing effects of TIL stimulated by DC with the H22whole-cell antigen against H22cell. |
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