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Objective To investigate the in vitro hormone refractory prostate cancer cell lines cryoablation expression of the law of the high mobility group protein B1 (high mobility group protein B1, HMGB1), and in vitro induction of allogeneic peripheral blood-derived dendritic cells (dendritic cell DC) differentiation and maturation of the relationship, to explore the impact of HMGB1 frozen immune response. Materials and methods develop hormone-refractory prostate cancer cell line PC-3, cryosurgery (using Endocare company cryotherapy system diameter 1.7mm frozen) supernatants were collected after the cells were lysed by the enzyme-linked immunosorbent assay (ELISA) The method for quantitative analysis of HMGB1 in the supernatant before and after freezing. Then application westernblot to detect different number of PC-3 cells frozen necrosis the supernatant HMGB1 release. Acquisition of prostate cancer patients anticoagulated fresh peripheral blood adherent mononuclear cells (PBMC), using lymphocyte separation medium (Ficoll), recombinant human granulocyte - macrophage colony-stimulating factor (rhGM-CSF) Recombinant human interleukin-4 (rhIL-4), induced in vitro culture get immature DC (imDC). PC-3 cells are frozen necrosis supernatant directly or by adding a sufficient amount HMHB1 (?) (?) And antibodies, respectively, were mixed with imDC cultured under suitable culture conditions in vitro. Cell morphology was observed under an inverted microscope and further changes in DC skeleton structure is observed under a confocal microscope. Measured by flow cytometry DC table and mature costimulatory sub-sub-CD83, CD86, HLA-DR expression. Observed HMGB1 stimulation with DC surface of mature costimulatory molecules CD83, CD86 and HLA-DR expression when - effect relationship, and dose - effect relationship. 1 Flow cytometric analysis of cryoablation normal before PC-3 cells, apoptosis, necrosis of the proportion of the three states were (95.58 ± 1.23)% and (2.00 ± 1.61)% and (0.56 ± 1.09)%, PC-3 after cryoablation normal apoptosis, necrosis of the proportion of the three states were (12.22 ± 9.53)% and (5.46 ± 1.14)%, (82.70 ± 11.35)%, the difference was statistically significant (P lt; 0.05). ELISA results show that: 1 × 106/ml group, 1 × 107/ml group, 1 x 108/ml set of PC-3 cell supernatant HMGB1 concentration before freezing (4.59 ± 0.25) ng / ml, (6.04 ± 0.13) ng / ml, (7.39 ± 0.18) ng / ml increased to (14.28 ± 0.84) ng / ml (71.68 ± 4.62) ng / ml, (329.64 ± 32.89) ng / ml, the difference was statistically significance (P lt; 0.01). The western blot same confirmed: HMGB1 concentration in the supernatant frozen necrosis also increases with increasing number of PC-3 cells. Dendritic cells after frozen necrosis supernatant stimulus microfilament protein increase cell volume increases, touch mutation long thicker, stress fiber formation, cytoplasmic sol thickening, the cells increased toughness and strength. HMGB1 neutralizing antibodies frozen necrosis supernatant, visible touch mutation short, thinning around the cell membrane and the fluorescence intensity of synapses were reduced, stress fibers decreased production, cytoplasmic sol thinning. Frozen necrosis supernatant HMGB1 induced DC surface of mature costimulatory divided high expression: the the frozen stimulation group, DC surface of mature costimulatory molecules CD83, CD86, HLA-DR expression than the control group increased significantly (P lt; 0.01) ; antibody intervention group DC surface of mature costimulatory molecules CD83, CD86, HLA-DR expression than frozen stimulation group decreased significantly (P lt; 0.01), compared with the control group remained high (P lt; 0.05) 5. frozen necrosis supernatant HMGB1 induced fluid mature DC surface costimulatory molecule expression when - effect relationship: a concentration of 1 × 106/ml PC-3 cells frozen bad necrosis supernatants were co-cultured with iDC 24h, 48h and 72h, DC surface mature Total stimulate molecules CD83, CD86 and of HLA-DR expression, respectively, in 24 ~ 72h significantly raised (P lt; 0.05, P lt; 0.01), which to cultivate 48h group DC surface of the total stimulus molecule expression raised the most significant with (P lt; 0.01 ). 6. Frozen necrosis supernatant HMGB1 induced mature DC surface costimulatory sub-expression dose - effect relationship: compared with the blank control group, 1 × 106/ml, 1 × 107/ml group DC costimulatory molecules CD83 , CD86, HLA-DR, were significantly increased (P lt; 0.05, P lt; 0.01), the DC surface 1 x 108/ml group only CD83 expression were significantly increased (P lt; 0.05), CD86, HLA-DR, no significant change (P gt; 0.05) Conclusion frozen ablation leads to tumor cell necrosis, necrotic tumor cells release HMGB1 as an endogenous danger signals, DC cytoskeleton can change, and enhance their ability to walk migration, prompting the immune synapse formation, and to facilitate their access to lymph nodes, with the T cells and trigger an immune response, thereby starting the specific T cell immune response. Meanwhile, HMGB1 in the freeze-immune response having a dual regulatory role, the low concentration of HMGB1 may be as important immune stimulation signals that promote mature DC surface high expression of costimulatory molecules, and reach peak intensity within a certain time; high concentrations of the HMGB1 the DC surface of mature costimulatory molecule expression is suppressed immune function of DC activation play a certain extent.
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