Formation of Neutrophils extracellular traps induced by Echinococcosis multilocularis protoscolex
Objective Preliminary exploration of the formation of Neutrophils extracellular traps(NETs)in-duced by Echinococcosis multilocularis protoscolex by qualitative and quantitative methods.Methods 1.The Echi-nococcosis multilocularis protoscolex was isolated from a gerbil model to detection its activity rate.2.Neutrophils were extracted by Polymorph Prep gradient centrifugation.The isolated neutrophils were labeled with PE-CD66b,whose purity was identified by flow cytometry.3.Protoscolex was co-cultured with neutrophils in vitro,and the con-centration of dsDNA in the co-culture supernatant was detected by using a PicoGreen dsDNA quantification kit.4.The structure of the formed NETs was observed under laser confocal microscopy.5.The levels of myeloperoxidase(MPO)and citrullinated histone H3(citrulline H3,H3Cit),which were the markers of NETs formation after co-cul-ture,were detected by flow cytometry.6.PCR was adopted to identify the origin of DNA in NETs.Results 1.The activ-ity rate of the protoscolex isolated from gerbil can reach more than 90%.2.The neutrophils extracted from the pe-ripheral blood of healthy people were isolated and purified,and their purity reached more than 87%.3.PicoGreen ds-DNA quantification showed that the production of NETs increased with the number of protoscolex and co-culture time.4.The NETs produced after co-cultivation were observed under laser confocal microscopy,with a network-like distribution of DNA skeletons.The number of labeled MPO and H3Cit significantly increased compared to the con-trol group.5.The results of flow cytometry showed that the number of MPO+and H3Cit+neutrophils in the experi-mental group co-cultured with the protoscolex(14.33±4.27)%was significantly increased compared with the nega-tive control group(0.53±0.12)%(P<0.05),and the difference was significant compared with the positive control group(17.33±2.59)%(P<0.01).6.The DNA of NETs produced by co-cultivation came from both nuclear DNA and mitochondrial DNA.Conclusion The infection with alveolar echinococcus can induce the formation of NETs.
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