| Background:Biological therapy is an novel method in addition to the traditional method to treated cancer such as surgery?radiotherapy and chemotherapy.CIK cell therapy as one kind of so many biological therapies is widely used in the treatment of malignant tumor,especially in hematologic malignant deasese.The side effect of CIK therapy is seldom,CIK therapy is effective against refractory and recurrent malignant deasese.Survival time were prolonged in patients with CIK cell therapy,It is worthy to research forther.The traditional method to culture CIK is that collection and separation of peripheral blood mononuclear cell(PBMC)from healthy donors or patient,Stimulate the PBMC by CD3 monoclone antibody(CD3mAb),cytokines and culture the cells in vitro,and get a group of cells construct by many kinds of cells such as CD3+CD56+ cells?CD3+CD8+ cells and NK cells?CIK kill the tumer cells mainly by the CD3+CD56+ cells.Bonanno G found that when Thymoglobulin(TG)used instead of CD3 mAb to stimulate the cells can also obtained CIK cells,and has better performance than that CD3 mAb stimulated.I.Popow and J.Leitner found that TG contained little anti-CD3 antibody titer.That can not stimulate leukomonocyte to proliferated.TG contain high does of CD2 antibody.CD2 can stimulate the lymphocyte and lead lymphocyte to proliferation.Use to culture better performance CIK cells induced by TG may rely on CD2 stimulation.ATG-Fresenius is an Polyclonal antibody used in clinic,ATG-Fresenius is an antibody made by anti Jurkat T cells line.It has CD2 antibody titer and CD28 antibody titer,almost not CD3 antibody titer.In this study ATG-F instead od CD3-mAb to culture the CIK cells were observed.CIK cells stimulated by ATG-F or other antibodies were compared in proliferation ability,phenotype,secretion ability and the differences of tumor cell killing ability preliminary.Objective: 1.Culture CIK cells using ATG-F instead of anti-CD3 mAb,find the suitable concentrate of ATG-F.2.To compare the effect of ATG-Fresenius with other antibodies on the ability of proliferation,phenotype,secretion,and tumor cell killing of CIK cells.Methods:Collection and separation of peripheral blood mononuclear cell(PBMC)from healthy donors and culture CIK cells,CIK cells were divided into six groups accordeding to the different antibody treatment on day 1 : CD3 group,TG group,CD3 + CD28 group,ATG-F high-dose group,ATG-F middle dose group and ATG-F low dose group,Then,the CIK cells were supplied with culture medium which containing IL-2 every 3 days.Collect the cultured cells to count total cell number,and analyze the phenotype of the cells and the percentage of active and inhibitory receptor by flow cytometry on day 7 and day 14.On day 14 some CIK cells were washed and re-cultured,then measured the concentrations of IFN-? in the culture supernatant by ELISA after 24 hours.The cytotoxicity of CIK cells against NK-sensitive chronic myeloid leukaemia K562 cells were determined by Calcein-AM/PI staining and analyzed by flow cytometry in day 14.Results:1.The ATG-F group has a lower proliferation rate than CD3 group.The CD3 + CD28 group had the highest proliferation rate on day 7.The ATG-F high does group had the highest proliferation rate compared with the other ATG-F treated group on day 14,which was significantly higher than the ATG-F middle does group and the ATG-F low dose group(P< 0.05).Among the groups,the ATG-F low dose group has the lowest proliferation rate and the CD3 + CD28 group has the highest proliferation rate on day 14.The proferation rate of ATG-F high dose group has no statistical difference compare with the CD3 group(P> 0.05),but less than the CD3 + CD28 group(P< 0.05).2.On day 14,The ATG-F Low doses group has the highest percentage of CD3 + CD56 + cells among ATG-F treated group,but there is no statistical difference compared with high dose ATG-F treated group(P> 0.05).The percentage of CD3 + CD56 + cells in ATG-F high dose group and CD3 group has no statistical difference.The ATG-F high does group has the highest percentage of NK cells on day 14,higher than CD3 group(P< 0.05).The percentage of activated receptor CD314 expression highger in the ATG-F high does group than CD3 group(P< 0.05)on day 14.3.The level of IFN-? in the supernatant of CIK cells in the ATG-F high does group is higher than that in CD3 group on day 14(P<0.05).TG group has stronger IFN-? secretes ability than CD3 group on day 14 as well(P<0.05).The ATG-F high does group has higher cytotoxicity ability to lyse K562 cells than CD3 group(P<0.05).Conclusions:1.ATG-F could active lymphocytes proliferation through CD2 channels.and could be used to cultive CIK cells.The suitable concentrate was 500ng/ml.2.Instead of CD3 monoclonal antibody,ATG-F treatment can improve the percentage of NK cells in CIK cells,the ability of IFN-?secretion and the cytotoxicity ability to lyse K562 cells of CIK cells.3.ATG-F treated CIK has better performance.ATG-fresenius has the potential to replace of anti-CD3 mAb to culture the CIK and use to clinic in the future. |
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