Abstract:

Objective Mouse bone marrow-derived dendritic cells were isolated and cultured in vitro and co-incubated with Candida albicans,in order to study the phenotype,phagocytosis and cytotoxicity of which against Candida albicans under different concentration and time. Methods Dendritic cells from C57 mouse bone marrow were isolated and cultured under aseptic conditions.The morphological and the growth curve and cell diameter of DC were observed by optical microscope and Cellometer Mini cell counter.The expression levels of CD11c,MHCII,CD40,CD80 and CD86 of DC were measured by flow cytometer.The phagocytosis and cytotoxicity of DC against Candida albicans were detected by microscope,Laser Scanning Confocal Microscope(LSCM) and flow cytometry. Results Typical morphology of DC cells was observed under inverted microscope 10 days after culture.10.2 μm was the most widely distributed diameter of DC as illustrated and the size of DC increased with the extension of incubation time.The growth curve showed that DC increased multiply.After inoculation,2-9 days were logarithmic phase and stagnate phase was from 9 days.The result of immunological phenotype detected by FCM showed that the CD11c and CD80 were highly expressed and MHCII,CD40,CD86 were lower expressed in control group.After stimulated by Candida albicans,the DC were appeared mature phenotype and CD11c,CD80,CD40,CD86 and MHCⅡ were all highly expressed.As the ratio(DC:Candida albicans) reduced(from 2:1 to 1:4),the phagocytosis effects of DC enhanced and the strongest effect appeared when the ratio was 1:4.When the ratio was 1:1,the phagocytosis effects of DC was no significant change(P=0.063) with the extension of co-incubation time(30 min to 1.5 h).But when the ratio were 2:1,1:2 and 1:4,the phagocytosis effects of DC were obviously rised with the extension of co-incubation time(30 min to 1.5 h),a statistically significant difference(P=0.003,P=0.002,P=0.002).When co-incubation time was prolonged,the cytotoxicity of DC was dramatic descend(P=0.000) detected by microscope.When the ratio(DC:Candida albicans) was 1:1,compared with the 2:1 group,the cytotoxicity effects of DC was dramatic declined.But when the ratio of Candida albicans increased within limits(from 1:1 to 1:4),the cytotoxicity was significantly increased(P=0.000),with statistical significance.Conclusion Dendritic cells isolated from mouse bone marrow had high purity and immature cell phenotype.We could obtain 3×10⁷ cells per C57 mouse DC which could meet the requirements of experiments very well.After stimulated by Candida albicans,mature DCs were obvious increased.DC had strong phagocytosis and cytotoxicity against Candida albicans,and the effects of phagocytosis and cytotoxicity enhanced with the ratio(DC:Candida albicans) reduced.When co-incubation time was prolonged,the phagocytosis of DC against Candida albicans was increased and the cytotoxicity was decreased.

Key words: bone marrow, dendritic cells, Candida albicans, phagocytosis, cytotoxicity