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Objective To establish an in vitro model of mycobacterial granuloma.Methods Mononuclear cells were isolated from peripheral blood of healthy human subjects,and stimulated to differentiate into macrophages,which were then classified into four groups to be cocultured with Mycobacterium marinum,Mycobacterium tuberculosis,Bacillus Calmette-Guérin,and Mycobacterium leprae,respectively,for five days followed by incubation with peripheral blood mononuclear cells (PBMCs) from the corresponding donors to establish an in vitro model of mycobacterial granuloma.The macrophages cocultured with PBMCs or mycobacteria alone served as the control.Microscopy was performed to dynamically visualize the formation of granuloma in vitro,flow cytometry to detect the expressions of cell surface antigens at different stages,real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA) to determine the mRNA expressions of important cytokines and their protein levels in the supernatant of macrophages,respectively.Results After 7-9 days of coculture with mycobacteria and PBMCs,the macrophages aggregated to form granuloma-like clumps,and some cells fused to form multinuclear giant cells,along with the expressions of some surface antigens such as CD14,CD68 and CD86 on these macrophages.The mRNA expressions of some important cytokines,including tumor necrosis factor-a,interferon-γ interleukin (IL)-1 β and IL-10,were detectable in the macrophages cocultured with mycobacteria and PBMCs,and the secretion of these cytokines was confirmed by ELISA in the supernatant of these cells.Conclusions An in vitro model of mycobacterial granuloma is basically established,which may facilitate the investigation into the formation of granuloma caused by and immune response to mycobacterial infection.
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Objective To investigate the effects of keratinocyte growth factor (KGF) and KGF receptor (KGFR) antisense oligonucleotide (ASODN) on cell cycle and apoptosis of HaCat cells. Methods HaCaT cell, an immortalized keratinocyte cell strain, was cultured in vitro. Flow cytometry was used to measure the cell cycle and apoptosis mediated by KGF and ASODN. Results The rates of S phase and apoptosis in the group treated with KGF increased significantly than those in the control group (both P
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Objective To investigate the effects of the compound T0 extracted from Tripterygium wilfordii, a traditional Chinese herb, in comparison with those of cyclosporin A(CyA), on down－ regulating the expression and activities of IL－ 1 and IL－ 2. Methods The cellular reactive systems of peritoneal macrophage (rat)－ thymocytes (rat), and spleen cells (rat)－ CTLL－ 2 (mouse) were set up. The technique of 3H－ TdR incorporation was applied. Results The production of IL－ 1 and IL－ 2 were significantly inhibited by T0 and CyA. The activities of IL－ 1 and IL－ 2 were down－ regulated by T0,rather than by CyA. Conclusion T0 and CyA are potent inhibitors of the production of IL－ 1 and IL－ 2. Effects of T0 on the activities of IL－ 1 and IL－ 2 are different from those of CyA.