| Objective Observing hypoxic regulate Osteopontin (OPN) secreting by mature dendritic cells (mDC). To Investigate regulation mechanism of OPN secreting by mDC.Method CD14+ cells were enriched using anti-CD 14 immunomagnetic beads, for induce to mature DCs, CD14+ cells were cultured with GM-CSF and IL-4 in hypoxia or normoxia in vitro. Concentration of OPN and TGF-β1 in supernatant were detected by sandwich enzymehnked inununosorbent assay (ELISA), OPN mRNA detected by RT-PCR. Intracellulra OPN were determined by FACS. Approach regulating function of A2R in expressing of OPN by mDCs by using NECA (surrogate of Adenosine). A2R agonist (CGS21680),A2R antagonist (SCH 58261) and investigate role of TGF-β1 in this process by using rhTGF-β1 and anti- TGF-β1 Ab. SPSS13.0 for Windows was utilized for statistical analysis.Results①Whether hypoxia or nomoxia, CD14+ monocytes could differentiate to mature dendritic cells.②Hypoxia inreased the level of OPN and OPN mRNA in mDCs, and this effect could be reversed by A2R antagonist.③Under nomoxia, both surrogate of Adenosine (NECA) and A2R agonist (CGS21680) could upregulate the level of OPN and OPN mRNA in mDCs significantly, but this positive effect could be reversed by A2R antagonist.④Hypoxic condition could not change the i-OPN generated by mDCs.⑤No matter what state A2R in, mDCs would not change production of i-OPN ⑥A2R played a role in regulating TGF-β1, and confirmed TGF-β1 involved in regulation of OPN by using rhTGF-β1 and anti-TGF-β1 Ab. Conclusion High adenosine induce the generation of TGF-β1 through the A2R on mDCs, and then TGF-β1 raise the OPN secreting by mDCs. |
