The Paracellular Permeability Of T84Monolayer And Gal-9Express Influenced By IL-17

BackgroundInflammatory bowel disease(IBD)-ulcerative colitis and Crohn’s disease-isemerging as a Chronic nonspecific intestinal inflammatory, morbidity increaseobvious in recent years, its exact pathophysiological mechanism remains largelyunknown. It was shown recently that an impairment of intestinal barrier is associatedwith Crohn’s disease and ulcerative colitis. along with the research about Th17cellsand the cytokines IL-17it secretion, the role of IL-17in the disease graduallyattention. Galectin-9is a member of the galectin family, Exists in various organisms,akind of Polysaccharide connection protein. We in Previous studies found thatgalectin-9May be associated with the close connection between enterocyte.Galectin-9is likely to be involved in maintain enterocyte connected. However,therelationship between intestinal barrier and IL-17,and the expression of galectin-9relationship with the integrity of the mucosal barrier is less frequently studied.ObjectiveT84cell monolayers were cultured in Transwell Common culture systems acrosscell membrane for simulating intestinal epithelial barrier. Transepithelial electric resistance(TER) of the T84monolayers and flow of HRP was determined aftertreatment with IL-17, so that permeability of the T84monolayers was got, thereforeenabling us to better explore the relationship between intestinal barrier and IL-17.While T84cell monolayer permeability changed further explore galectin-9relationship with the integrity of the intestinal epithelium mucosa barrier.Methods1. T84monolayers were employed in this study. To evaluate the influence ofparacellular permeability of T84monolayer by IL-17and HRP were added to theapical chambers of transwell systems. HRP in samples was determined byenzyme-linked immune assay(ELISA). HRP flux and TER were performed toevaluate the permeability of T84monolayers.2. IL-17with different concentrations and IL-17were added to the transwellsystems. HRP flux and TER were performed2h after the addition of IL-17withdifferent concentrations to evaluate the permeability of T84monolayers.3. Transwell systems were randomly divided into groups: the naive controlgroup, the IL-17with different concentration groups. HRP flux and TER wereperformed2h after the addition of flagellin.Collectioning the basal chambers liquid,Gal-9levels in basolateral liquid were determined by ELISA. To evaluaterelationship between T84cell monolayer permeability and Gal-9expression.4. To observe T84cell monolayer permeability increased the cell Gal-9expresschange. Cells were collected from the plates2h after the addition of IL-17.ThenT84cell monolay erexpress Gal-9determined by immunocytochemistry.5. The data was recorded by statestecal software SPSS17.0.Measurement datawere expressed as means±standard diviation. Shapiro-Wilk of normality testwas used in the data of statistics analysis.Levene test of homogeneity of varianc wasused in the data of statistics analysis too.Three or more groups were analyzedwith the analysis of variance(ANOVA) and the comparison between two groupswas performed with LSD-t test. Differences between two groups wereanalyzed with the Student’s t-test.Then Spearman correlation was used to describe the relationship between the variables.Differences between means at a levelof P <0.05were considered significant.Result1. IL-17with different concentrations added to the apical chambers oftranswell. When IL-17concentration less than50ng/ml, to increase the flagellinconcentration with luminal side, does not cause a decline with TER and a increaseswith HRP flow. When IL-17concentration greater than50ng/ml, with theconcentration of flagellin increases, The transmembrane resistance gradually declineand HRP flow gradually increase, compare with naive control group and the smallconcentration flagellin group, the difference was statistically significant.2. Immunochemical results observed under an optical microscope cells: IL-17through the intervention of T84cells (IL-17group) cytoplasm, nucleus, cellmembrane was dyed brown, with respect to the normal growth of T84cells (negativecontrol group) cytoplasm, nucleus, cell membrane is dyed brown significantlyreduced extent. The results suggest that: cell monolayer permeability increased, Gal-9over the cell monolayer permeability of normal cells expressing the Gal-9reduced.Levels by ELISA in cell supernatants luminal side showed Gal-9: Highconcentrations of IL-17were significantly higher than the normal control group, thedifference was statistically significant (P <0.05), IL-17acts on tips T84cellmonolayer permeability caused by the change in the role of cell supernatantscorresponding expression of Gal-9in the intervention group was significantly lowerthan those without.3. HRP flow and correlation analysis of the results of Gal-9: between Gal-9andHRP flow r=-0.942, P <0.01, explain negative correlation between the concentrationof the supernatant Gal-9and HRP traffic.Conclusion1. IL-17increase human intestinal epithelial cell line T84monolayersparacellular permeability. 2. After T84cell monolayers increased permeability reduced the expression ofGal-9.