| Dendritic cells (DCs) are the most potent antigen-presenting cells (APC) in vivo that can active resting na?ve T lymphocytes, and play crucial roles in capturing, processing and presenting antigens, especially, the function of which in anti-tumor activities has been emphasized in recent years. Alpha-Fetoprotein (AFP) is a tumor-associated fetal protein, and its serum level is elevated in 80% patients with hepatocellular carcinoma (HCC). In clinic, AFP is mainly employed to diagnosis HCC, make census in high-risk population and evaluate the efficacy of operation and other therapies. However, with the further study, AFP is fund that has immunosuppressive effects, which can effectively curb maternal rejection of the fetus, inhibit the immune system of patients with HCC and promote lymphocyte apoptosis, thereby induce tumor to evade the immune surveillance. However, AFP is a protein with complicated biological function, the mechanism of immuno- suppressive effects is not clear yet.Therefore, on the basis of successfully preparing human peripheral blood DCs, we incubated DCs with different concentration AFP in vitro, and mainly researched the effects and mechanism of differentiation, maturation, function and apoptosis of DCs. They follow three aspects:1. Identification and culture of human peripheral blood DCs in vitro Human peripheral blood mononuclear cells was induced mature DCs.Because of adhesion properties, progenitor cells (mainly mononuclear cells) were purified from non-adherent cells (mainly mixed lymphocyte). After inducing five days, non-adherent DCs was collected in new plate for culture, in case of the effect of other cells (such as macrophages) on the growth of DCs. Under the role of cytokines(GM-CSF, TNF-αand IL-4), peripheral blood mononuclear cells gradually performed typical form of DCs. Nine-day training cells highly expressed phenotypes (HLA-DR, CD1a, CD11c), costimulatory molecules (CD80, CD86), specific markers of mature DCs(CD83), and had a strong ability to stimulate proliferation of allogeneic lymphocytes. With analyzing phenotypes, morphology and mixed lymphocyte reaction(MLR), we confirmed that the cells from the peripheral blood mononuclear cells were DCs.2. Effects of AFP on the phenotypes and functions of DCsTo explore the effects of AFP on the proliferation of DCs, five-day training DCs was incubated with AFP (1μg/ml, 5μg/ml, 10μg/ml,20μg/ml, 40μg/ml) for 72 hours. MTT assayed the proliferation of DCs. Results showed that the AFP inhibited the growth of DCs. With an increasing concentration of AFP (1μg/ml to 40μg/ml), the inhibitory effect becomes stronger.To research the effects on phenotypes of DCs, the AFP, seven-day DCs was cultured with AFP (20μg/ml) for 24 hours, then added six antibodies (anti-HLA-DR, anti-CD1a, anti-CD83, Anti-CD80, anti-CD86, anti-CD11c). The results were recorded by flow cytometry. Results showed that AFP treatment DCs significantly lower expressed HLA-DR, CD1a, CD11c, CD80, CD83 and CD86 than the control group. The difference was significant (P <0.05).To study the impacts on the function of DCs to activate lymphocytes, MLR was acted with AFP-treated DCs (stimulating cells) and allogeneic lymphocytes (reacting cells). MTT assayed OD value in the wavelength of 570nm. The results showed that the mature DCs had a strong power in activating lymphocytes proliferation than immature DCs. The proliferation of AFP-treated groups was significantly lower than the control group, and the higher of the concentration of AFP, the lower of the proliferation of lymphocytes.3. Study on AFP-induced apoptosis of DCs in vitroTo study the effects of AFP on apoptosis of DCs, AFP(20μg/ml), human serum albumin(HSA), Anti-AFP, SB203580 (p38-MAPK pathway inhibitor) and DEVD-fmk(Caspase inhibitor) were respectively added in 5 groups (DCs+AFP,DCs+HSA,DCs+AFP+Anti-AFP,DCs+AFP+SB203580,DCs+AFP+DEVD-fmk), and untreated DCs was taken as blank control. After culturing for 24 hours, Annexin V-FITC and PI were added and the apoptosis of DCs was measured by flow cytometry. The results showed that all AFP of different concentration could promote apoptosis of DCs. With the increasing concentration, the apoptosis index increased. SB203580 and DEVD-fmk blocked the apoptosis of DCs, however HSA and AFP antigen-antibody complex could not affect the apoptosis.To further study the mechanism of apoptosis of DCs induced by AFP, We used JC-1 staining method to test the mitochondrial membrane potential changes in AFP-treated DCs and Western Blot to test the expression of active caspase-3 and phospho-p38. The results showed that the apoptosis of DCs induced by AFP (20μg/ml) accompanied the decline in mitochondrial membrane potential, expression of active Caspase-3 and phospho-p38. Taken the HSA group and the antigen-antibody complex group as control, it is that AFP induces the decline of mitochondrial membrane potential, expression of active Caspase-3 and phospho-p38. Among them, DEVD-fmk blocked the apoptosis induced by AFP, inhibited the expression of active caspase-3, but had no impact on the decline in mitochondrial membrane potential. And SB203580 could significantly reduce apoptosis induced by AFP, partly stop the decline in mitochondrial membrane potential and blocked expression of active Caspase-3 and phospho-p38.To sum up: (1) AFP inhibits the proliferation and expression of surface molecules of DCs, inhibits their ability to stimulate lymphocytes proliferation; (2) AFP may promote apoptosis of DCs through mitochondrial pathway; (3) p38 MAPK signal transduction pathway may be involved in the AFP-mediated apoptosis of DCs; (4)AFP may induce apoptosis of DCs by "AFP-p38MAPK-mitochondrial- Caspase" signal transduction pathway.
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