| BackgroundMacrophages are a kind of immune cells which local lives in cornea,in the process of fungal keratitis,As the lesion development,the number of macrophages increase gradually,Suggesting the macrophages play an important role in the development of the fungal keratitis.However,the interaction between macrophages and fungushas not been well studied at present.ObjectiveTo investigate whether the mouse macrophageshave phagocytosis of fungi to fungus(fusarium solani),and whether the macrophages can kill(or inhibit)fungi,and whether the fungus(Fusarium oxysporum)has a toxic(or proliferative)effect to macrophages.Materials and methodsThe macrophages(RAW 264.7)were co-cultured with fungal spores(Fusarium oxysporum),Constantly take pictures and Observe the phagocytosis of macrophages to fungal spores and the number of spores thathave been swallowed.To divide the fungal spores into two parts,one part was cultured separately,and the other part was co-cultured with mouse macrophage(RAW 264.7),then to randomly selected the number of the count spores and the number of budding spores in visual fields at different time.To calculate the germination rate of fungal spores,that is: the sprouting rate of fungal = the number of sprouting spore / total spore number×100%.To divide the spore suspension into two parts,one part was cultured separately,and the other part was co-cultured with mouse macrophage(RAW 264.7),After diluting the spore suspension 1000 times at different timepoints,Tile in Potato Dextrose Agar Petri dish and count CFU after 48h.The macrophages(RAW 264.7)were co-cultured with different concentrations of fungal spores,then to add the CCK 8 reagent at different time,4h later,using microplate reader to determine the optical density value.ResultsAccording to the observation of macrophages and fungal spores in the same visual field we can see that macrophages can capture and swallow fungal spores,but the fungal spores that are swallowed can continue growing in the cell.All the spores of the fungus that are swallowed are 100% budding.The macrophages were co-cultured with fungal spores,the rate of co culture group at 1h,2h,3h,4h,5h,6h,7h,8h were:(0.24 ±1.06)%,(1.15 ± 2.33)%,(10.99 ± 5.70)%,(34.31 ± 6.95)%,(46.79 ± 8.29)%,(59.42 ± 12.88)%,(65.77 ± 9.63)%,(73.03 ± 6.15)%.The rate of pure spore group were(0.38 ± 1.67)%,(2.76 ± 1.06)%,(22.4 ± 10.49)%,(54.92 ± 5.85)%,(71.36± 6.94)%,(80.12 ± 7.24)%,(91.62 ± 6.16)%,(97.25 ± 8.20)%.The differences were statistically significant at 3h,4h,5h,6h,7h,8h time point(P < 0.05).The differences were statistically insignificant at 1h,2h time point(P > 0.05).The macrophages were co-cultured with fungal spores,the number of co culture group at 2h,4h,6h,8h were: 44.46 ± 0.68×10~4,41.74 ± 0.67×10~4,38.63 ± 0.52×10~4,37.57 ± 0.70×10~4,37.57 ± 0.70×10~4,36.59 ± 0.50×10~4,35.72 ± 0.66 ×10~4.Compared with the pure spore group,the number of colonies decreased significantly.The differences were statistically significant at each time point(P < 0.05).Inactivation of fungal spores can promote macrophage proliferation,but the degree of proliferation is independent of concentration and time.At 1h,2h,3h,4h,5h,6h,7h,8h,after comparing spores of different concentrations group with the PBS group,there is a significant difference between the two groups(P <0.05).At the same time point,after comparing spores of a same concentration group,there is no significant difference(P >0.05).ConclusionsAfter macrophages were co-cultured with fungal spores(Fusarium solani),the results showed that the macrophages were able to kill fungal spores,but did not destroy the fungi.However,the germination rate and CFU number of fungal spores decreased,and the proliferation of macrophages was significant,above all suggest that macrophages can kill the spores of the fungus and inhibit their growth,and fungal spores can promote macrophage proliferation in turn.Background In the inflammatory response of the body,macrophages,foreign bodies,neutrophils,and so on,often exist simultaneously.They interact with each other to produce complex inflammatory reactions.Therefore,the determination of ability of macrophage phagocytosis can indirectly reflect the ability of immune response.In the preliminary study,we found that the abilities of macrophages to different foreign bodies are different.At present,when evaluating the phagocytic capacity of macrophages,most studies often use latex beads as the experimental object.However,it is unknown whether the latex beads can represent a variety of foreign bodies to accurately evaluate the phagocytic capacity of macrophages.Objective To compare the similarities and differences between macrophage phagocytosis of latex beads and macrophage phagocytosis of specific fungal pathogens,and neutrophils,in order to accurately detect macrophage phagocytosis to provide a new method.To compare and evaluate the feasibility of latex beads method objectively.Materials and methods Mouse macrophages(RAW 264.7)were incubated with Latex beads,inactivated fungal spores and neutrophils respectively.The total number of macrophages in each visual field,the number of macrophages with foreign body and the number of foreign bodies swallowed in each visual field respectively were counted at 2h,4h,6h and 8h.The macrophage phagocytosis rate and phagocytic index were calculated and the results are expressed as median statistics.Results Macrophageshad thehighest phagocytosis Percentage of inactivated fungal spores,reaching 22.73 %(18.78 %,28.04 %)at 8h,neutrophils followed by 18.75 %(14.46 %,23.20 %),latex beads was the least and only 12.50%(10.71 %,13.79 %).There was significant difference between the three groups at 8h compared with the three groups 2h(P<0.05).Compared with latex beads group,phagocytosis percentage was significantly different at each time in each group(P <0.05).The phagocytic index of macrophages to inactivated fungal spores washighest at 8h,reaching 0.30(0.23,0.31),neutrophils followed by 0.21(0.16,0.28),latex beads was only 0.14(0.12,0.18).There was significant difference between the three groups at 8h compared with the three groups 2h(P <0.05).Compared with the latex beads group,There was significant difference between the three groups at 2h,6h and 8h(P <0.05),and there was no significant difference between the three groups at 4h(P > 0.05).Conclusions The phagocytosis of mouse macrophages on latex beads,inactivated fungal spores and neutrophils were significantly different.Therefore,using latex beads as an indicator to evaluate macrophage phagocytosis is inaccurate and incomprehensive. |
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