Study On The Expression Of MicroRNAs In Hbe Cells Infected With Chlamydia Psittaci And The Role Of TNF-α In Chlamydia Muridarum Reproductive Tract Infection In Mice

The effects of Chlamydia psittaci infection on the expression of host miRNAs was analyzed to predict the role of miRNAs and their target genes in C.psittaci infection.And to investigate the role of Tumor Necrosis Factorα(TNF-α)in the development of protective immunity and pathology during C.muridarum urogenital infection in mice.In chapter 1,the research status of miRNA and TNF-αin chlamydial infection were briefly introduced.In chapter 2,HBE cells were infected with C.psittaci with a multiplicity of infection(MOI)=3 for 44 hours.Total RNA was extracted and small RNA sequencing technology was used to detect miRNA expression in HBE cells after C.psittaci infection.Softwares such as TarBase,miRWalk,miRanda and Pictar were used to predict the target genes of the significantly changed miRNAs.DAVID online tools were used to perform Gene Ontology(GO)analysis and the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of target genes.Cytoscape software was used to construct the miRNA-gene regulatory network.qRT-PCR was used to verify the expression of 13 candidate miRNAs.Based on this,140 up-regulate miRNAs and 11 down-regulate miRNAs were found when HBE cells infected by C.psittaci.These miRNAs may play a role in C.psittaci infection by regulating the corresponding target genes.In chapter 3,fifteen female wild-type(WT)C57BL/6J mice and 15TNF-αreceptor knockout(TNF-αR KO)mice were inoculated intravaginally with 1×10~4 inclusion forming units(IFUs)of live C.muridarum.Eight of the mice in each group were similarly carried out on56 days after the initial infection.Vaginal swabs were taken every 3 to 4days to detect the number of inclusion bodies of chlamydia.On day 80after the primary infection,mice were sacrificed,then the peritoneal macrophages were collected,and the vaginal tract and spleen were isolated.The degrees of inflammation and lumen dilatation of hydrosalpinx and uterine horn were observed and semiquantitatively scored.The levels of IL-6,IL-8,IL-1α,IL-1βand TNF-αin the supernatant of macrophage were detected.The spleen cells were stimulated with chlamydia EB in vitro to determine the level of cytokines such as IL-4,IL-5,IL-17 and IFN-γ.The experimental results showed that TNF-αis not associated with protective immunity,but promotes the inflammatory pathological damage of vaginal tract caused by C.muridarum.