Studies On The Modification And Optimization Of Culture Methods Of Mature DC And On Relativities Of Susceptibility Test Between DC And Tumor Cells

Objectives: 1) To explore the method of culturing DC to maturity by two different methods; 2) to investigate the relationship of the drug sensitivity between T-DC conventionally cultured and tumor cells of isoplastic patients; 3) to transfect DC by using CEA recombinant vaccine virus for better preparation of tumor Ag and curative effects. Methods: 1) Pretreatment by TNF- a ,T-DC were generated by culturing in medium containing GM-CSF and LL-4 cellular factors. 2) Freshly isolated blood mononuclear cells were reversely penetrated by unstimulated human umbilical vein endothelial cells and cultured in medium containing GM-CSF, IL-4 and TNF- a .By this method E-DC can be obtained in 105 hours. 3) The morphology of DC obtained by the above two methods was compared with the DC generated by common culture ways and analyzed by scanning electron microscopy. In addition, bioactivity of the cells was measured by specific killing experiment with MTT tests .And T cell stimulating activity was determined by allogeneic and autogeneic mixed lymphocyte reaction ,auto-MLR and allo-MLR. 4) Human CEA vaccine virus was transfected into the conventionally cultured DC by Lipofectamine. The expression of the CEA antigen in the supematant and the cell membrane was observed in 24h after transfection with AS 180 chemiluminescence test. 5) T-DC and tumor cells of osteosarcoma patients were cultured and drug sensitivity experiments were conducted on the cultured cells with 12 chemotherapeutical drugs. The results were compared. Results: 1) The T-DC5 cultured by the way of TNF?a pretreatment for 8d and of reversely tran-endotbelial cells for 105h, which possessed typical Morphological characters and expressed CD8, CD864 , CD834 , CDI~4 CD544 , CDiia4 and HAC-DR4 , were similar to the DCs cultured by routine method. Whereas the expression of T-DC was higher than that of the other two groups. T-DC, E-DC and DC had strong allo-MLR and auto-MLR and strong killing tumor cells capacity. 2) The results were astonishingly similar between experiments on the sensitivity of T-DC and tumor cells of patients with osteosarcoma to the chemotherapeutical drugs. 3) Using vaccine virus as carrier, Lipofectamen could transfect DNA of CEA into DC and CEA could be expressed in DC. Conclusion: Both T-DC and E-DC were typical DCs and could present antigens. T-DC of osteosarcoma patients derived from human peripheral blood could take the place of tumor cells for drug sensitivity experiments. Vaccine virus could transfect DNA of CEA into DC.