| Part ? Establishment of an animal model of ulcerative colitisObjectiveThe experimental animal model of UC mice was established.The changes of stool traits in the two groups of mice were observed every day.The DAI scores and colon histopathology(HE staining)were observed daily to evaluate whether the experimental model of UC mice was successful.MethodsTwenty BALB/c female mice at 6-8 weeks were selected and bred adaptively for one week.Twenty mice were randomly divided into two groups(10 mice/group)according to the random number table method:blank group(NC group)and model group(UC group).Mice in the UC group were given 3%DSS solution to drink freely for 7 days,and the UC experimental animal model was established.Mice in the NC group were given purified water for routine feeding.The fecal traits of the two groups of mice were observed daily,and the DAI scores were obtained for the two groups of mice.On the 8th day after modeling,all the mice were sacrificed by cervical dislocation,and the blood and colon samples of the two groups of mice were collected and stored for later use.One of the colon tissues of each mouse was subjected to HE staining to observe the pathological changes.ResultsThere was no difference in DAI scores between the two groups in the first two days after modeling.From the third day after modeling,the DAI score of the UC group modeled with 3%DSS was significantly higher than that of the control group(P<0.001).According to the pathological results,compared with the NC group,the mucosal structure of colon tissue in the UC group was changed,including the disappearance of normal glandular structure,the multifocal ulcers,destruction of crypt tissue,infiltration of neutrophil and other inflammatory cells,indicating the successful modeling of UC.ConclusionOn the fifth day during modeling,gross blood in stools were observed around the anus of mice in UC group.Compared to the NC group,the DAI score of the UC group was significantly higher,and the colon tissue structure was severely damaged,indicating that the UC mouse model was successful.Part ? To explore the mechanism of miR-155 in the pathogenesis of UC miee.ObjectiveTo compare the expression of miR-155,Ets-1,Thl7 cells,IL-23,IL-17 and IL-6 in plasma of two groups of mice,and to explore the mechanism how miR-155 induces UC.MethodsAfter one week adaptive feed,twenty BALB/c female mice aged 6-8 weeks were randomly divided into two groups(10 mice/group)according to random number table method:blank group(NC group)and model group(UC group).UC group mice were given 3%DSS solution to drink freely for 7 days for modeling.NC group mice were fed with pure water routinely.On the 8th day after modeling,all mice were killed by cervical dislocation.Blood and colon samples were collected.Real-time PCR,western blot and immunohistochemical staining(IHC)were used to detect the expression of miRNA-155,Ets-1,IL-23,IL-17,IL-6 in colon tissue and peripheral blood in both groups.Results1)Real-time PCR reSults showed that the expression level of miR-155 in UC group was significantly higher than that in NC group(P<0.05);the expression level of Ets-1 in UC group was significantly lower than that in NC group(P<0.001);the expression levels of IL-23,IL-17 and IL-6 in UC group were significantly higher than those in NC group(P<0.001).2)Western blot results:The results showed that the expression levels of IL-23 and IL-17 in peripheral blood leukocytes of UC group were higher than those of NC group(P<0.05);the expression level of IL-6 in UC group was higher than that in NC group(P<0.001);and the expression level of Ets-1 in UC group was lower than that in NC group(P<0.001).3)The results of IHC showed that the levels of IL-23 and IL-17 in UC group were significantly higher than those in NC group.ConclusionBased on the results,we confirmed that miR-155 induce UC by inhibiting the expression of Ets-1,accelerating the maturation of Th17 cells,and promoting the release of Th17 cells and their inflammatory factors IL-23,IL-17,IL-6,which reveals that microRNA-155 can induce UC through the IL-23/Thl7/IL-17/IL-6 pathway.Part ? Experimental study on the relationship between miR-155 and the pathogenesis of uleerative colitis in mieeObjectiveTo explore the relationship between miR-155 and UC,we up-regulated the expression of miR-155 based on a UC model,and observed the colonic inflammation degree in mice with UC and miR-155 high expression group.The expression of miR-155,IL-23,IL-17 and IL-6 in peripheral blood and colon tissue in two groups were compared to verify the relationship between miR-155 and UC mice.MethodsAfter one week adaptive feed,twenty female BALB/c mice aged 6-8 weeks were randomly divided into 2 groups(10/group)according to the random number table:the control group(UC+LV-Control group)and miR-155 high expression(UC+LV-miR-155)group.The UC+LV-Control group was given 3%DSS solution for free drinking for 7 days to establish a experimental UC animal model.In the UC+LV-miR-155 group,a fragment of about 100 bp containing miR-155 sequence was cloned into pCDH-CMV-MCS-EF1-copGFP lentivirus vector,which was packaged by t ransfected 4 plasmids system to strain 293T cells for 72 hours to generate lentiviral particles containing miR-155.The lentiviral particles were concentrated to about 109 TU/ml using a lentivirus concentration&puri fication kit.The prepared concentrated lentiviral particles were diluted and injected into the tail vein at a dose of 2 × 107 TU,and the molding was completed one week later.DAI scores were performed on both groups every day.On the 8th day after modeling,all mice were sacrificed by cervical dislocation and blood samples were collected.We observed the pathological changes in colon tissue through HE staining.Real-time PCR was used to detect the expression of miR-155,Ets-1 and Th17-associated inflammatory factors such as IL-23,IL-17 and IL-6 in peripheral blood and IHC was used for examination of' the expression of IL-23 and IL-17 in two groups.Results1)Real-time PCR:The results showed that the expression level of miR-155 in UC+LV-miR-155 group was significantly higher than in UC+LV-Control group(P<0.05),while the expression level of Ets-1 was lower(P<0.001).The expression of IL-23,IL-17 and IL-6 in UC+LV-miR-155 group was significantly higher than in UC+LV-Control group(P<0.05).2)DAI scores:The results showed that there was no difference in DAI scores between the UC+LV-miR-155 group and the UC+LV-Control group in the first two days during modeling.From day 3 to day 8,The DAI scores of UC+LV-miR-155 groups were significantly higher than those of UC+LV-Control group,and the DAI scores of the two groups were statistically significant(P<0.001).3)HE staining:Compared with the UC+LV-Control group,the erosion as well as increased number of inflammatory cells in the colonic mucosa and the disappearance of glandular epithelium of UC+LV-miR-155 group were more noticeable.4)IHC:In contrast to the UC+LV-Control group,a large number of inflammatory cytokines,IL-23 and IL-17,were observed in the colon tissue of mice in UC+LV-miR-155 group.ConclusionmiR-155 may have a correlation with UC and be one of the inducement.The mechani'sm may be that miR-155 accelerates Thl7 maturation and differentiation by inhibiting its target gene Ets-1,promotes the release of inflammatory factors associated with Th17 such as IL-17,IL-17,IL-6,and induces UC by the IL-23/Th17/IL-17/IL-6 pathway mediated by Th17 cells. |
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