| Macrophages as immune effector cells widely distributed in the micro environment of the body can mediate the defense mechanism of innate immunity.Kupffer cells(KCs),a specific type of macrophages settled in the liver,have the ability to devour invading pathogenic microorganisms and are the first barrier for liver immune protection system.After stripping in gastric juice,Fasciola gigantica newly excysted juveniles(NEJ)pass through the small intestine and then the abdominal cavity to reach the liver.During liver migration,F.gigantica juvenile flukes play the role of immune regulation by contacting with host immune cells such as KCs directly.However,few studies have explored the interaction between F.gigantica and KCs.The research regarding immunoregulatory effect of F.gigantica and the host KCs and other liver immune cells will help to better understand the underlying mechanism of the interaction between F.gigantica and the host immune system,especially potential mechanism of liver local immunity.This study focused on the in vitro effects of F.gigantica NEJ on KCs/macrophages.After characterizing isolated liver KCs from mouse,NEJ were co-cultured with KCs/macrophages for 24h.By measuring cell polarization and apoptosis,preliminary study was carried out to explore the interaction between F.gigantica NEJ and host KCs/macrophages.The following is a specific study.(1)To obtain sufficient KCs as an important test material in this experiment,the Hepatocyte suspension medium was obtained by"in situ perfusion+in vitro digestion"and KCs were isolated by differential centrifugation and selective attachment method based on characteristics of KCs.At last,obtained cells were identified through the swallowing experiment,Wright-Giemsa staining and F4/80 immunofluorescence staining.The results showed that a single mouse can obtain(8.43±1.78)×10~5KCs,the cell purity was(82.75±1.71)%and the cell survival rate was(89.5±7.77)%.The obtained cells showed the basic characteristics of KCs/macrophages with strong adherence in vitro culture,the ability to devour ink particles and positive F4/80 specific antibody staining,which was in line with,and the number met the needs of subsequent experiments.The obtained cells quantity met the needs of subsequent experiments.(2)To determine the polarized direction of KCs/macrophage induced by NEJ,F.gigantica NEJ were co-cultured with KCs and Raw264.7 for 24h in vitro separately.Molecular markers(CD86,CD206,i NOS,Arg-1)and cytokines(TGF-β,IL-10)of macrophages were detected by RT-q PCR.Intracellular expression levels of i NOS and Arg-1 proteins were tested by Western blot.Cellular NO and Arg-1 secretion were measured by Griess assay and Arg-1assay respectively.The data showed after stimulating by F.gigantica NEJ for24h NO and Arg-1 secretion from KCs and Raw264.7 did not change,significantly accompanied upregulated CD206 and Arg-1 m RNA level and Arg-1 protein expression level compared with PBS group.The expression of i NOS protein was downregulated in KCs after stimulation by F.gigantica NEJ,but there was no significant change in Raw264.7.The results showed that F.gigantica NEJ can induce mouse KCs/macrophages to polarize to M2 phenotype in vitro.(3)To find out whether F.gigantica NEJ can induce KCs/macrophage apoptosis,NEJ were co-cultured with KCs and Raw264.7 for 24h in vitro separately.Apoptosis related gene(Bcl-2 and Bax)were detected by RT-q PCR,Caspase-3 activity was test by kit,nuclear apoptosis was observed by Hoechst33342 staining and cell morphology was checked by TEM.The data showed that there was significantly up-regulated(P<0.05)for the pro-apoptotic gene Bax m RNA level,and no significant difference for Bcl-2 in mouse KCs after 24hours of co-cultured by NEJ.No significant difference for Bax and Bcl-2 m RNA level was observed in Raw264.7.There was no significant change in intracellular Caspase-3 activity of these two types of cells after 24h of stimulation with F.gigantica NEJ.Hoechst 33342 fluorescence-labeled nuclei did not show dense staining and no obvious apoptotic features were observed in TEM.The results suggested that the 24 h interaction of KCs/macrophages and F.gigantica NEJ in vitro could not cause apoptosis.To summarize,the study that primary KCs of mice were successfully isolated and the number of cells can meet the research requirements accumulated experimental and technical basis for the researched based on KCs.In this study,we found F.gigantica NEJ can induce mouse KCs/macrophages to polarize to M2 phenotype in vitro after 24h co-culture.It indicated NEJ can regulate host immune cells by mediating anti-inflammatory effects.However,NEJ did not induce apoptosis within 24 hours.These research conclusions will provide basic theoretical research for the in-depth study of the mechanism of the F.gigantica juvenile flukes regulating the immunological function of macrophages to achieve immune escape. |
