| Objective:To optimize culture system of CIK cell to improve proliferation of the CIK cell, to enhance the secretion of cytokine such as IL-2、IFN-yand TNF-a, to promote the killing fuction of CIK cell to target cell and cost-effect of in clinic practice.Methods:The peripheral blood of32patients who were diagnosed as malignancy by histopathology and accepted biotherapy in biotherapy center of the First Affiliated Hospital of Zhengzhou University during August1,2011and January1,2012were gotten as the object of the study. The monocytes were extracted and devided into four groups after suspended in RPMI1640culture medium. The cells were cultured according to CIK cell culture technological process. The group1monocytes was added into LacNAc, the group2was added into LacNAc and anti-CD28mAb, the group3was added into anti-CD28mAb and the group4was added into double distilled water. The proliferation ability of cells was measured via cytometry on the4th,8th,10th,12th,14th. we also detect the proliferation by CFSE staining. Their ability to secrete cell cytokine such as IL-2、IL-10、IFN-γ and TNF-α. was measured in1×106cells were from each group on the12th day, and then Phenocyte of CIK cell and killer ability of CIK cell to target cell K562were tested on the14th day.Results:(1)Morphological changes and proliferation of CIK cells in each group: Peripheral monocyte morphology was observed under inverted microscopy(4X20) after adding kinds of cytokines, monoclonal antibodies into peripheral monocyte, the size, cytoplasm, nucleus of the CIK cells gradually changed Cells began to gather and clonely grow on the2-4day, cell morphological changes were no difference among4goups under the microscopy. The cell proliferation in each group accelerates noticeably the proliferation of the cells in group1,2,3were faster than that of the group4, there waere statistically significant difference among them (F=3.945, P＜0.05; F=2.341, P>0.05; F=72.121,P<0.05; F=121.118, P＜0.05; F=32.973, P＜0.05) (2)Quantity of secreted cell cytokine in each group:The quantity of IL-2、IFN-γ and TNF-a in group1、2、3was higher than group4, difference has statistically significant (F=5.87, P<0.05;F=4.79, P<0.05;F=5.26, P＜0.05), and there was no statistical significance in the three experiment groups while they compared with each other (P>0.05)(3)Cell percentage in each group:the percentage of CD8+cell was statistically higher in group1,2,3than in group4,(F=5.03, P<0.05), the percentage of CD4+T lymphocyte was statistically lower in group1,2,3than in group4(F=4.63, P<0.05) the ratio of CD8+/CD4+in group2was statistically higher than the forth group (F=5.72, P＜0.05). Difference of the ratio of CD8+/CD4+in group1and group3with the ratio of the group4was not statistically significant (P>0.05), there was no significance difference about the percentage of CD3+CD56+cell in every group (P>0.05)(4)Killing efficency to target K562cell in each group:the killing efficency to target cell(K562) in controlled group was statistically lower than the group of LacNAc、the group of LacNAc and anti-CD28mAb and the group of anti-CD28mAb (F=6.68,P<0.05)Conclusions:(1)LacNAc and anti-CD28mAb can increase proliforation rate of CIK cell population effectively. LacNAc and anti-CD28antibodies can activate CD8+T lymphocyte but not CD4+T lymphocyte.(2) LacNAc and anti-CD28mAb can promote CIK cell to secrete IL-2、IFN-γand TNF-a, and there was synergistic effect of LacNAc and anti-CD28monoclonal antibodies.(3)LacNAc and anti-CD28mAb advanced the killing ability of CIK cell to leukemia clone K562. This culture systerm is better than primary culture systerm. |
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