The Effects Of Lentivirus-mediated Dectin-1 Over-expression And Curdlan On The Defense Of Raw264.7 Against Talaromyces Marneffei Infection

Objective:To investigate the effects of over-expressed Dectin-1 and Curdlan on the defense of macrophage RAW264.7 against Talaromyces marneffei(TM)infection via the constructions of over-expressed Dectin-1 lentivirus and Dectin-1 over-expression cell co-culutred with TM model.Methods:(1)Primers were designed according to the sequence of objective gene Dectin-1.Objective fragments were amplified by PCR.Obtained the recombinants by the enzyme digestion and ligation of the objective fragments and lentiviral vector.The recombinant lentivirus was harvested from 293T cells after the recombints co-transfected with lentiviral packing materials into them for 72 h.Titer of virus was determined by hole dilution method.The positive and the negative control lentivirus were transfected into RAW264.7 in vitro(named RAW-Posi and RAW-Neg separately,the name RAW-Ctrl was given to the cells without treatments).The rate of tansfection was detected by the expression of green fluorescence protein observed under the fluorescence microscope.Relative expression of Dectin-1 mRNA was determined by real-time PCR.(2)Establish the model of co-culture macrophage and Talaromyces Marneffei conidia.The ability of RAW264.7 killing Talaromyces marneffei conidia was measured by serial dilution plating method.The concentration of Reactive Oxygen Species(ROS),Nitric Oxide(NO),TNF-α,IL-1β,IL-4 and IL-10 were detected by ELISA.The morphological changes of conidia phagocytosed by RAW264.7 were observed via calcium fluorescent white staining.The relative expression of iNOS and Arg-1 were detected by real-time PCR.Results:(1)Dectin-1 gene was cloned into lentivirus correctly verified by enzyme digestion and PCR.Titer of the acquired lentivirus was 3.12*109 Tu/ml.The highest intensity of fluoresence was observed under the fluoresence microscope after 72 hours transfection.The relative expression of Dectin-1 mRNA in RAW-Posi was increased significantly compared to RAW-Ctrl and RAW-Neg(P<0.05).(2)① The killing ability was revealed by serial dilution plating method:The killing ability of the cell co-cultured with TM for 48 hours was stronger than co-cultured for 24 hours(P<0.05);The killing ability of RAW-Posi was stronger than that of RAW-Ctrl and RAW-Neg(P<0.05);The killing ability of the cell pretreated with Curdlan was stronger than the cell without pretreatment of Curdlan(P<0.05).② The concentrations of cytokines in the supernatant detected by ELISA:The concentration of ROS in co-culture for 48 h was higher compared to 24 h(P<0.05),and the phenomenon was not observed among NO,TNF-α,IL-1β,IL-4 and IL-10.The concentrations of ROS,NO and TNF-α in the co-culture supernatant of infected RAW-Posi were higher compared to that of infected RAW-Ctrl and RAW-Neg group(P<0.05),and the phenomenon was not observed among IL-1β,IL-4 and IL-10.The concentrations of ROS、NO、TNF-α and IL-1β in the co-culture supernatant with the pretreatment of Curdlan were higher compared to that without pretreatment of Curdlan(P<0.05),and the phenomenon was not observed between IL-4 and IL-10.③ The relative expressions of iNOS and Arg-1 mRNA detected by real-time PCR:The relative expressions of iNOS and Arg-1 mRNA of the co-culture with TM for 48 h were higher than that of the co-culture for 24 h(P<0.05).The relative expressions of iNOS and Arg-1 mRNA of infected RAW-Posi were higher compared to that of infected RAW-Ctrl and RAW-Neg group(P<0.05).The relative expressions of iNOS mRNA of the co-culture with pretreatment of Curdlan was higher compared to that of co-culture without pretreatment of Curdlan(P<0.05).The difference of the relative expressions of Arg-1 mRNA between the co-cultured with pretreatment of Curdlan and the co-culture without pretreatment of Curdlan showed no statistic significance(P>0.05),except that between the RAW-Posi with and without pretreatment of Curdlan co-cultured with TM for 48 hours has statistic significance(P<0.05).Conclusion:(1)Lentivirus could mediate the over-expression of Decitn-1 in macrophage RAW264.7.(2)The over-expressing Dectin-1 and Curdlan helped increase the production of ROS、NO、TNF-α and IL-1β,and made the killing ability of macrophage RAW264.7 stronger.(3)Over-expressing Dectin-1 promoted both M1 and M2 polarization of RAW264.7 denfensing TM.Curdlan might get the ability of promoting M1 polarization of RAW264.7.