Establishment Of Canine Intestinal Epithelial Cell Line And Transcriptomic Study Of Echinococcus Granulosa Soluble Protein Induction

Cystic echinococcosis is caused by Echinococcus granulosus(E.granulosus)and is endemic all over the world.Dogs are the most important definitive host of the pathogen.Blocking dog infection is the core of controlling echinococcosis.The normal development of E.granulosus is the colonization of protoscolex in canine small intestinal epithelial cells.Blocking its colonization can prevent the occurrence of echinococcosis.At present,there is no canine small intestinal epithelial cell line that can be continuously subcultured,so the research on the early immune response and its regulatory mechanism of infection lags behind,which has become the bottleneck of the development of hydatid disease vaccine.Therefore,based on the establishment of immortalized canine small intestinal epithelial cell line,finding the target involved in e.g.protoscolex colonization may be the key to break through the bottleneck.Objective: This study intends to extend the life of canine small intestinal epithelial cells cultured in vitro by transfection of foreign genes,establish a canine small intestinal epithelial cell lines with normal epithelial cell morphology and function,and analyze the molecular events before and after E.granulosus protoscolex was colonized in canine small intestinal epithelial cells by using transcriptome sequencing technology,so as to provide candidate antigens for the development of effective protective vaccines suitable for the terminal host,It laid a foundation for the development of canine anti intestinal parasitic disease vaccine.Methods:(1)primary canine small intestinal epithelial cells were isolated and cultured by tissue block culture,and the characteristics of epithelial cells were identified by cell morphology and indirect immunofluorescence;After LPS induction,the transcription level of inflammatory factors was detected by real-time PCR to identify the function of primary canine small intestinal epithelial cells.(2)The canine small intestinal epithelial cell line stably transfected with h TERT gene based on lentivirus expression system was constructed.After continuous passage for more than 50 generations,the characteristics of its epithelial cells were identified by indirect immunofluorescence,the expression of hTERT protein was detected by Western blot,and the level of inflammatory factor transcription was detected by real time PCR after LPS induction.(3)The soluble extract of E.granulosus(PSA)was induced into canine small intestinal epithelial cells for 6h.After transcriptome sequencing,the differential genes before and after e.g.protoscolex was colonized in canine small intestinal epithelial cells were screened.The transcription results were identified by real time PCR,and the molecular events before and after E.granulosus protoscolex was colonized in canine small intestinal epithelial cells were analyzed,so as to provide a scientific basis for analyzing the response mechanism of E.granulosus infection.Results:(1)the primary canine small intestinal epithelial cells(p CIECs)obtained by tissue block culture can adhere to the wall within 24 hours,start cell proliferation on the 3rd day,reach the peak of proliferation on the 4th day,gradually reduce the cell activity on the 5th-7th day,and can be continuously cultured to the 10 th generation in vitro.After trypsin differential digestion and purification,paving stone like primary canine small intestinal epithelial cells were obtained.The growth curve was similar to "s" shape,and the identification of epithelial cell specific keratin 18 was positive.After LPS induced pciecs,the inflammatory factor IL-1β、 IL-6、IL-10、TNF-α The transcription level of was significantly improved.(2)A canine small intestinal epithelial cell line,named CIEC,was successfully established.CIECS express h TERT protein and have similar morphology and function to pciecs(3)the transcriptional profile of CIECS induced by PSA for 6 hours was analyzed by RNA SEQ technology,and 692 differentially expressed genes(difference > 1 time,P < 0.05)were selected for enrichment analysis of go and KEGG databases.Go analysis showed that the cell component(CC)of PSA induced 6h differential gene was extracellular region,and the main biological process(BP)was defense response;KEGG analysis showed that after 6 hours of PSA induction,26 genes significantly clustered in MAPK signaling pathway,including 17 up-regulated genes and 9 down-regulated genes.QRT PCR verified 10 differential genes,and the verification results were consistent with the results of omics screening.Conclusion:(1)primary canine small intestinal epithelial cells can be successfully isolated by tissue culture;The primary canine small intestinal epithelial cells have the morphological characteristics and biological functions of intestinal epithelial cells.(2)The foreign h TERT gene was successfully transferred into pciecs by lentivirus vector.There was no significant difference between CIECS in morphology,cell phenotype and pecs.(3)Transcriptomics analyzed that when canine small intestinal epithelial cells respond to the stimulation of CIECS by PSA,the main aggregation of differential genes is the extracellular region,and the main involved BP is the defense response,which is significantly concentrated in the MAPK signal pathway.