Analysis Of Differentially Expressed Genes At Different Time Intervals In RAW264.7 Cells Infected With Echinococcus Granulosus

Cystic echinococcosis(CE)is a zoonoses parasitic diseases caused by Echinococcus granulosus(E.g),which was widespread and complex,The pathogenic mechanism and immune response and other issues of CE is still to be solved.Protoscoleces(PSC)is a form of larval stage of E.g,and which can generated immunologically response with intermediate hosts.How the PSC destroying the host's immune response and producing lesions,It is remain dimness.This study were based on the in vitro observation of PSC infected mice macrophage RAW264.7,the host-parasite interaction mechanism was revealed by the cell level and the transcriptome level.1.In vitro culture and observation of PSC: The in vitro culture system was established to observe the growth and development characteristics of the PSC.The study showed that the PSC changed into capsules at 10 th day,and 50% of the PSC had fully developed into capsules at 25 th day.90% of the PSC developed into microcapsules and vesicles at 35 th day,and the thickness of stratum corneum was thickened,and the head hooks at the crest were disappeared completely.The microcapsules gradually increased and the wall gradually thickened at 50 th day.The microcapsules ruptured and the stratum corneum invaded the invader at 70 th day.This experiment successfully established the in vitro culture system of PSC,and the growth and development were observed.2.Effect of PSC on in vitro production of NO from RAW264.7: In this paper,mouse macrophage RAW264.7 was cultured with protoscolices of PSC,Hydatid cyst wall(HC)and Hydatid cyst fluid(HF),respectively,and detected the production of Nitric Oxide with Griess reagent in different infected time.The results showed that a large amount of nitric oxide was produced with PSC induced macrophages in a short time;HC also could induce macrophages to produce nitric oxide,which was significantly different from with the control group;HF induced a small amount of nitric oxide and which was not significantly different with the control group.In this study,we investigated the effect of NO on macrophage resistance to PSC,which indicated that macrophages could resist the infection of protopods by continuously releasing large amount of NO.The hydatid cyst wall could provide a favorable environment for the survival of PSC,and could prevent the further infection of parasite to the host.The effect of hydatid cyst fluid on macrophages release NO was not significant.The experiment laid a foundation for the further study of the mechanism of action of NO on resisting parasite infection.3.Effect of cytokines in RAW264.7 Cells Infected by PSC: By studying the changes of the four cytokines IL-2,IL-4,IL-5 and IFN-?were produced by Th1 and Th2 cells after PSC-infected mouse macrophage RAW264.7,for discuss the significance of Th1 and Th2 cells in re-immunization after parasites direct contact with host cells.The results showed that,at the initial stage of infection,the parasites the cells damage were most serious,and the immune protection and the immunological damage were simultaneously performed during the interaction between the parasites and host cells,and the secretion of Th2 cytokines was mainly,the secretion of Th1 cytokines was auxiliary.4.Analysis of gene difference expression of PSC infected RAW264.7: Using Illumina sequencing technology and bioinformatics analysis,we sequenced at the transcriptome level protoscoleces of E.g and investigated the transcriptome changes during host-parasite interaction.We obtained identified a large number of genes associated with immune.Changes analysis results suggest in gene expression profile among the two groups.The up-regulated DEGs in PSC include Proteinase inhibitor ? Serine proteases ? Glycoside hydrolase ?Phospholipase A2?MHC class I?Immunoglobulin?EF-Hand 1?Tumour necrosis factor?p53-like transcription factor and so on.The GO analysis showed that were mainly involved in oxidation,metabolism and other related processes;analysis of the KEGG indicated that the PSC genes were significantly enriched in multiple pathways mainly related to environmental information processing,metabolic pathways and genetic information processing.The up-regulated DEGs in RAW 264.7 include Antigen B ? HSP70 ? FABP1 ? Proteinase inhibitor 12?BMTI6 ?Glutamate dehydrogenase and so on.GO enrichment were mainly involved in biological processes and molecular functions such as oxidation,metabolism,and immunity.KEGG analysis shows that genes of DEGs were involved in processes such as metabolism.By analyzing the immune factors and biological enzyme functions of parasites and cells,this survey constitutes a valuable resource for gene prediction on the parasite genome and for further genomic analyses focused on cestodes and host-parasite interactions and new targets are available for insect-resistant vaccines.