| Objective:This paper mainly explores on the impact of human chemokine CCL17 and CCL22 on lymphocyte chemotactic ability and anti tumor function,so as to connect with the destruction of immune cells,the CIK cells with non restrictive cytotoxicity to tumor cells with high expression ligand CCR4 of CCL17 and CCL22 can be chemotactic to tumor immune to kill.Method: 1.Comparative study on chemotaxis of human chemokine CCL17 and CCL22 on CD8 cell and CD4 cell that is two kinds of T lymphocyte subsets.In this paper,we focus on the differences of chemotaxis of CCL17 and CCL22 on the CD4 and CD8 two kinds of T cells and their effects on the internalization of CCR4 molecules on the surface of T cells.We first analyze the distribution of CCR4+T cells in the peripheral blood of healthy adults PBMC in flow cytometry,then we use Mo Flo to select CD4 separation and CD8 T lymphocytes by flow cytometry to carry out cell chemotaxis assay.we detected the chemotactic effect on CD4 and CD8 T cells of two kinds of chemokines in different concentrations by Transwell.Chemokine binding to cell surface receptors may cause desensitization and internalization of receptor molecules,and the proportion of receptor internalization depends on the activation and function of receptor signaling.We further investigated the effects of CCL17 and CCL22 on the internalization of CCR4 receptor in CD4 and CD8 cells.2.The effect of CCL17 and CCL22 on receptor expression in CIK and DC-CIK In this section of the experiment,we first examined the expression of CCR4 in CIK and DC-CIK cells and the chemotaxis experiment of CCL17 and CCL22 on CIK and DC-CIK cells.Flow cytometry analysed the expression of CCR4 on CIK and DC-CIK,finding that they were almost all CD56+CCR4+.Finally,the CIK cells were stimulated to express CCR4 with CIK cells that were acted for a period of time with a certain concentration of two chemokines.3.The chemotaxis of CIK cells transfected with CCR4 and the killing effect on tumor cells In this section,we mainly done that the CCR4 gene was transfected into CIK cells to make CIK cells with high expression of CCR4 gene,and then CIK cells transfected CCR4 had stronger killing effect and chemotaxis than CIK cells non transfected.It is of great significance to tumor killing.Result: 1.The two kinds of concentration of chemokine acted on the same receptor molecule CCR4 on CD4 and CD8,the concentration of 100ng/ml had stronger chemokine ability than that of 10ng/ml,showing that in a certain concentration range,the higher concentration of chemokines on cell had stronger chemotactic ability.During the same time,the number of CD4 cells attracted through Transwell by CCL17 and CCL22 were more than CD8 cells,while the overall chemotaxis of CCL22 was stronger than that of CCL17.CCR4 receptor internalization experiments confirmed that CCL22 is more likely to cause CCR4 receptor internalization than CCL17.For the CD4 and CD8 cell,after the role of CCL22 and CCL17,the internalization of receptor CCR4 on CD4 cells is stronger than that on CD48 cellsr.2.During the 14 day of culture,the expression of CCR4 on CIK cells increased first and then decreased,the highest in fourth days.the longer the incubation time,the expression of CCR4 was more and more low,until the final recovery of CIK cells,CCR4 was almost no expression.DC-CIK cells with DC cells taken in tenth days until its recovery in the fourteenth day,the expression of CCR4 during this period did not decrease but increased,because the secretion of chemokines by DC increased expression of CCR4.In the treatment of immune cells,you could add the appropriate concentration of CCL17 and CCL22 to promote the expression of ligands CCR4 on cytokine induced killer CIK.The environment around the tumor cells often secrete these two chemokines,more CCR4 immune cell surface expression,tumor microenvironment can chemotaxis more immune cells expressed CCR4 ligand to kill tumor cells.3.Transfecting CCR4 gene into CIK cells,CIK cells were highly expressed CCR4 gene,and then CIK cells transfected CCR4 had a stronger chemotaxis and stronger killing effect than CIK cells non transfected.It is of great significance to kill the tumor.Because many tumor microenvironment had proved the high expression of the two kinds of chemokines CCL17 and CCL22,so the liquid environment surrounding tumor cells can chemotaxis more immune cells to kill the tumor.This was of great significance to kill the tumor.Conclusion: Within a certain range of concentration,the higher the concentration of chemokines,the stronger the chemotaxis of cells were.But CCL22 can better promote CCR4+T cell chemotaxis and receptor internalization phenomenon than CCL17.For the CD4 and CD8 cell,after the role of CCL22 and CCL17,the internalization of receptor CCR4 and chemotaxis on CD4 cells is stronger than that on CD8 cell.And the more CCR4 expression on the surface of immune cells,the tumor microenvironment can chemotaxis more and more immune cells expressed CCR4 ligand to kill tumor cells.Our study was to associate the killing immune cells and the CCL17 and CCL22,Our study is the CCL17 and CCL22 and the killing associated immune cells,so that these CIK cells with non restrictive cytotoxicity with high expression of CCL17 and CCL22 ligand CCR4 can be chemotactic to tumor cells to destruct rather than the Treg cells with high expression of CCR4 to inhibit tumor killing... |
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