| Objectives1. To evaluate the effect of cytokine combinations of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), recombinant human interleukin 4 (rhIL-4) and recombinant human tumor necrosis factor-α(rhTNF-α) on the cultivation of dendritic cell (DC) from healthy human peripheral blood mononuclear cell (PBMC).2. To evaluate the effect of calcium ionophore (CI) A23187 and rhGM-CSF on the cultivation of DC from healthy human PBMC.3. To explore the effect of DC stimulated with K562 cell lysate in inducing specific cytotoxic T lymphocyte (CTL) against K562 cell in vitro.MethodsPart 11. PBMC isolated from human peripheral blood by Ficoll-Hypaque density gradient separation were purified through adherence. rhGM-CSF and rhIL- 4 were added to induce the DC. After five days cultivation, rhTNF-αwere added to stimulate the mature of DC. Keep culturing for another two days.2. The morphology and quantity of DC were observed by inverted microscope all through the process. 3. The surface antigens of the induced cells on the 5th and 7th days were tested by flow cytometry (FCM).4. The proliferation of allogenetic T cell was examined by colorimetry.Part 21. PBMC isolated from peripheral blood of healthy human by Ficoll-Hypaque density gradient separation and purified through adherence were separated into two groups. The cells in group A were cultured with additional rhGM-CSF, rhIL-4 and rhTNF-αonly as control group. The cells in group B were cultured in the presence of rhGM-CSF and CI A23187.2. Preparation of K562 cell Lysate. The K562 cells were harvested in Logarithmic phase and washed twice in filtered phosphate-buffered saline. The cells were resuspended in phosphate-buffered saline and subjected to five cycles of freezing (-80℃) and thawing (37℃). The lysate was stored at -20℃.3. PBMC were incubated with K562 cell lysate for 30 minutes at 37℃before culturing. The cells in both groups were harvested in 96 hours'cultivation.4. The morphology and quantity of DC were observed by inverted microscope all through the process.5. The surface antigens of the induced cells after culturing for 96 hours were analyzed by FCM.6. The T cell was stimulated by DC loaded with K562 cell lysate. The proliferation of allogenetic T cell was examined by colorimetry.7. The nonspecific inhibition of DC loaded with K562 cell lysate against K562 cell was detected by colorimetry.8. The specific cytotoxicity of T cell primed with DC was examined by colorimetry.ResultsPart 11. After 5 days'incubation with rhGM-CSF and rhIL-4, most of immature DC formed clusters. Typical morphological features of mature DC could be observed on the 7th day in the presence of rhTNF-α.2. The expression of CD83, CD1a, CD86 and CD40 on the 7th day in the presence of rhTNF-αincreased significantly than those immature DC after 5 days'incubation (29.8% versus 14.3%, 18.2% versus 12.8%, 33.6% versus 20.1%, 28.1% versus 19.9%), respectively, P<0.05. The expression of CD14 also decreased significantly than that on the 5th day (8.0% versus 16.2%), P<0.05.3. Mixed lymphocyte reaction demonstrated that the capacity for stimulating T cell sharply increased when DC matured.( P<0.05)Part 21. Typical morphological features of DC could be observed in both groups.2. The expression of CD83, CD1a, CD86 and CD40 in group B (45.2%, 31.5%, 40.1%, 36.4%) are stronger than those in control group (16.9%, 20.4%, 26.5%, 22.3%), respectively, P<0.05. The expression of CD14 in group B is weaker than that in control group (5.7% vs 19.0%), P<0.05.3. As compared with the control group, DC in group B loaded with K562 cell lysate could evidently stimulate the proliferation of allogenetic T cell.(P<0.05, exclusion of the effector-to-target ratio of 1:40 )4. The DC in group B loaded with K562 cell lysate could inhibit the growth of K562 cells significantly. (P<0.05)5. In addition, both groups of the CTL stimulated by DC had specific cytotoxicity against K562 cell. At the effector-to-target ratios of 10:1 and 40:1, the CTL stimulated by DC of group B had stronger cytotoxicitiy against K562 cell. (P<0.05)Conclusions1. The cytokine combination of rhGM-CSF, rhIL-4 and rhTNF-αis beneficial to expanding and inducing PBMC into DC.2. rhGM-CSF and A23187 can induce PBMC into DC more effectively.3. DC loaded with K562 lysate can stimulate CTL and keep high immunocompetence with specific cytotoxicity against K562 cell.
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