Three Strategies For Tumor Antigen Loading Of Cord Blood-derived DCs To Induce The Immunity Against Ovarian Cancer In Vitro

Objective: To observe the activation effect on T lymphocyte by cord blood-derived dendritic cells (DCs) loading with freeze thaw lysis, mild acid-eluted peptides and total RNA of HO-8910 tumor cells and the killing rates of tumor specific cytotoxic T lymphocyte (CTL) on ovarian cancer cells in vitro. To find the optimal means loading cord blood derived DCs for inducing high effective immunity against ovarian cancer, to set the base for future animal experiment and clinical trial, and to give an new approach in the biological treatment of ovarian carcinoma.Methods: Cord blood was collected from healthy term delivery mother. Cord blood mononuclear cells. (CBMNCs) were separated by density gradient centrifugatio.n(Ficoll-Hypaque). The adherent fraction of CBMNCs was cultured with the presence of relevant cytokines (rhGM-CSF2000u/ml, rhIL-4 500u/ml and rhTNF-a100u/ml) to generate DCs. The tumor cells of human ovarian cancer cell line HO-8910 were froze and thawed three times in rapid succession for freeze thaw lysis and treated with citrate-phosphate buffer(PH3.3) for mild acid eluted peptides. Total cellular RNA was extracted from tumor cells using the RNeasy kit following the manufacture's protocol. As tumor antigens, they primed DCs in vitro. The ability of DCs stimulating T lymphocyte proliferation was observed by mixed T lymphocyte reaction using 3H-TdR incorporation. MTT assay was employed to test the inhibition rates of tumor specific cytotoxic T lymphocyte on HO-8910 ovarian cancer cells.Results: DCs with typical morphology and phenotype were harvested from the adherent fraction of CBMNCs with the presence of rhGM-CSF2000u/ml, rhIL-4 500u/ml and rhTNF-a100u/ml in vitro. Freeze thaw lysis, mild acid-eluted peptides and total RNA were successfully get from HO-8910 tumor cells. Loading three kinds of tumor antigen, DCs were able to stimulate the amplification of allogeneic mixed T lymphocytes. The simulate index(SI) of the loading groups are higher than the unloading group at effectontarget ratios of 1:10,1:50 and 1:100. The killing rates ofthe loading groups are larger than the unloading group and inactivated T cells group at effectontarget ratios of 10:1, 50:1 and 100:1. The killing rates increased with the improving of effectontarget ratios and the killing rates of the loading groups to human nasopharyngeal carcinoma CNE tumor cells are low.Conclusion: CBMNC could be induced to DCs in the presentation GM-CSF, EL-4 and TNF-a. Cord blood-derived DCs loading with freeze thaw lysis, mild acid-eluted peptides or total RNA could activate CTL and induce specific immunity against ovarian cancer effectively in vitro.