Experimental Study Of Calcium Phosphate Crystals Induced HMGB1Expression In Macrophages

Objective： Explore the calcium phosphate crystals after stimulatemacrophage U937High mobility group protein B1(HMGB1) release andexpression level.Methods: Microscopical observation of calcium phosphate crystals afterthe stimulation of macrophage U937people form change. Stimulation ofcalcium phosphate crystals with100ug/ml U937in macrophages, respectivelyafter the stimulus0,6,12,18,24,30,36h and48h, using Western blot test cellculture supernatant, cytoplasm and nuclei of HMGB1protein content. Use RT-PCR detection of HMGB1mRNA expression in cells. In calcium phosphatecrystals as after0,1,2h and4h, use Enzyme-linked immunosorbent assay(ELISA) determination of cell culture supernatant of TNF–α, IL-6, and thecontent of MCP-1.Results: The calcium phosphate crystals stimulate0to6h on cells afterclearly unable to clearly detect HMGB1,12h after cells began to increase inHMGB1protein,18hreached a peak, stable at a high level, after48h appearedobvious peak. Before calcium phosphate crystals stimulation, HMGB1is mainlydistributed in the nucleus, cytoplasm content is relatively small. In12-48h, cellplasma HMGB1content increased gradually, HMGB1in the nuclei in18to24h after stimulation content reduce,30-48h after HMGB1levels and increasegradually in the nuclei. RT-PCR results showed that calcium phosphate crystalsstimulus0-12h, HMGB1mRNA expression in cultured cells has no obviouschanges,18to24h of HMGB1mRNA expression in cultured cells increasedsignificantly. ELISA results showed that calcium phosphate crystals stimulus1h,the cell culture supernatant of TNF–α, IL-6and MCP-1expression and release,4h reach a peak.Conclusions： Calcium phosphate crystals can induce U937humanmacrophages produce HMGB1protein. Calcium phosphate crystals can inducehuman macrophage HMGB1, TNF-α, IL-6, MCP-1expression and releaseincreases, and HMGB1release time much later than TNF-a, IL-6and MCP-1inrelease time. Calcium phosphate crystals induce macrophage HMGB1from thenucleus to cytoplasm translocation, and released into the extracellular. Calciumphosphate crystal induced gene expression in human macrophages HMGB1mRNA up regulated. Calcium phosphate crystals can stimulate macrophageaccumulation, into the group.