| Cytokine-induced killer (CIK) cells are the human body (autologous or allogeneic)peripheral blood mononuclear cells in vitro by a variety of cytokines to stimulate theactivation and proliferation of a group of heterogeneous cells. It has high proliferativeability, tumoricidal activity, broad spectrum anti-tumor and without MHC (majorhistocompatibility complex, MHC) restriction and so on. Especially for postoperativeclearing micrometastases, preventing the spread and recurrence, improving the patient’sown anti-tumor immunity plays an important role.CD28is expressed in human T cells, and some important molecules in the surfaceof the NK cells. CD28of T cells as receptor molecules and B7molecules on theexpression of antigen presenting cells (APC) bind together to mediate T cell activationrequired coordinatory signal, so as to stimulate the proliferation of T-cells and play animmune response. Agonist monoclonal antibody against human CD28and CD28molecule binded can directly transduce cell activation signal, then promote cellproliferation and immune effects. The purpose of this study was to base on preparingAgonist monoclonal antibody mouse against human CD28, inducedCIK cells combined with different cytokines, analyzed the method induced CIKproliferation, phenotypic characteristics and on human breast cancer cell lineMCF-7killing effect, in order to look for new scheme of safe and efficient CIKamplification.PartⅠ Preparation and identification ofagonist monoclonal antibody mouseagainst human CD28Objective：To prepare mouse anti human CD28agonist monoclonal antibody andidentify its biological function. Methods：The strain of hybridism cells lines built was amplified in vitro bylarge,which would stimulate the secretion of mouse anti-human CD28agonistmonoclonal antibody. Using in vivo induced ascites, monoclonal antibody was preparedand puried by Protein A affinity chromatography.The titer of monoclonal antibody wasidentified by indirect immunofluorescence method, and the binding capacity ofmonoclonal antibody and membrane-type CD28molecule was analyzed through flowcytometry.Results：Ascites formation rate was about80%, yield of ascites of2ml/only. Therate of ascites antibody protein was1.5mg/ml by means of purification. Purifiedantibody was used in indirect immunofluorescence assay in an amount of0.5ug/5×105cells.Conclusions：Preparation of agonist monoclonal antibody mouse against humanCD28can be well combined with antigen molecules.Part Ⅱ Agonistmonoclonal antibody against human CD28involved in theinduction of CIK cells and its phenotypic analysisObjective： To research agonist monoclonal antibody against human CD28involved in inducing proliferation ability and cell phenotype characteristics of CIK.Methods：The Ficoll separation method to obtain healthy human peripheral bloodmononuclear cells (PBMC), adjusted the concentration of2×106/ml, then inoculated in24well culture medium,1ml/hole. According to the following two groups addinginducer: A, anti-CD3+IL-2+IFN-γ B, anti-CD3+anti-CD28+IL-2+IFN-γ. The finalconcentration of inducer anti-CD3was1μ g/ml, anti-CD280.5μ g/ml, IL-21000units/ml, IFN-γ100ng/ml. Every2-3day liquid changing or expanding bottle. On theseventh day calculating the number of the cells in each well, the expressions of surfacemolecule CD8, CD4and CD56of CIK were analyzed via using flow cytometry singlemarker analysis, and flow cytometric double labeling analysis for surface moleculeexpression CD4/CD25, CD8/CD25, CD4/FasL, CD8/FasL of CIK at the same time.Results：Cultured for up to seventh days, agonist monoclonal antibody againsthuman CD28group participating in the total CIK cells with an average of8.2×106/hole, significantly higher than that of the control group of2.8×106/hole. Thedifference was statistically significant (P<0.05). The results of single marker methodshowed, CD4+T cells, CD8+T cells and CD56+cells of CIK involved in agonist monoclonal antibody against human CD28were68.4±6.1%,34.6±7.2%and15.1±3.9%, and the control group52.5±3.6%,26.9±2.7%and7.7±1.2%. The differencehad statistical significance (P<0.05). The results of double labeling analysis showed,agonist monoclonal antibody against human CD28involved in the induction of CIKcells in CD4+FasL+T cells, CD8+FasL+T cells, CD4+CD25+T cells and CD8+CD25+Tcells were36.6±4.7%,40.7±3.2%,60.1±5.3%and58.5±4.1%. And the controlgroup21.9±3.9%,24.3±5.1%,45.6±4.6%and44.6±3.4%were statisticallysignificant (P<0.05).Conclusions：Agonist monoclonal antibody against human CD28can enhancecapability of CIK amplification and increase activation of membrane-type moleculesexpression.Part Ⅲ Study on cytotoxicity of agonist monoclonal antibodyagainst humanCD28involved in the induction of CIK cells to human breast cancer cell lineMCF-7Objective：To study on killing effect of agonist monoclonal antibody againsthuman CD28involved in the induction of CIK cells to human breast cancer cell lineMCF-7Methods： According to the above method induced CIK, with its as effector cells,human breast cancer cell line MCF-7as target cells, effector to target ratio of20:1byadding culture plates, and cultured for3days. With CCK-8incorporation assay forcytotoxicity, while using Annexin V/PI method analyzed CIK on apoptosis andmortality of MCF-7.Results：CCK-8incorporation assay showed that the killing rate of CIK for MCF-7cells which was involved in being induced by agonist monoclonal antibody againsthuman CD28was24.3%, higher than that of the control group9.68%. The differencewas statistically significant (P<0.05). Annexin V/PI analysis showed that agonistmonoclonal antibody against human CD28involved in the induction of CIK, theinduction of apoptosis on MCF-7cells was13.7%, and mortality was13.9%, while thecontrol group, apoptosis rate and mortality rate were10.1%and9.88%, respectively.Thedifference between the two groups had statistical significance (P <0.05). Conclusions： Agonist monoclonal antibody against human CD28can strengthenthe killing effect of CIK on human breast cancer cell line MCF-7. The antibody can beused as new inducers of CIK to enhance cell proliferation and killing ability. |
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