| Objectives RNA lentivirus BCL6-shRNA and ICOS-shRNA were used to transfect TNBS induced ulcerative colitis(UC)mouse model,to observe the expression of follicular helper T cell(Tfh)/follicular regulatory T cells(T follicular regulatory cell)in UC mice,including their regulation of expression by their cytokines as BCL6 and ICOS,to study its effect in the pathogenesis of UC.Methods One hundred male C57 mice were randomly divided into normal control group,TNBS model group,model mice+BCL6-shRNA group,model mice+ICOS-shRNA group,and model mice+Ubi-EGFP(empty load body)group,each group was 20.The establishment of UC mouse model by skin sensitization combined with TNBS to enema.The normal group was given 0.9%Sodium Chloride Solution to enema with the same volume.Mice in model group+BCL6-shRNA,modelmice+ICOS-shRNAgroup,model mice+Ubi-EGFP(empty load body)group were injected respectively with BCL6-shRNA,ICOS-shRNA and Ubi-EGFP(empty load body)lentivirus vectors 100ul on the first,seventh,fourteenth and 21st day after first enemaing,all mice were killed 1 weeks after the last lentivirus vector injection.After establishing the model,the weight,hair,stool character and activity of all mice were observed and recorded daily.The degree of disease activity in mice,the gross morphological damage of the colonic mucosa of mice and the degree of histology injury were evaluated.Flow cytometry was used to detect the percentages of Tfh and Tfr cells and the proportion of Tfh/Tfr cells in the spleen of mice in each group,RT-PCR was used to detect CD4mRNA,CXCR5mRNA,BCL-6mRNA,FOXP3mRNA,ICOSmRNA and PD1mRNA levels in the colon of mice in each group,three color fluorescence labeling method was used to studyonthepercentagesofCD4~+CXCR5~+FOXP3~+Tfrand CD4~+CXCR5~+PD1~+Tfh cells the proportion of Tfh/Tfr cells in the lymphatic germinal center of mice colon mucosa in each group.Results The scores of DAI,CMDI and histology in the model group and the transfection group with the empty load body were significantly higher than those in the normal control group(p<0.01),the scores of DAI,CMDI and histology in the virus transfection group was between the normal control group and the model group.Compared with the normal control group,the Tfh cells in spleen and colonic mucosa of TNBS model group,model mice+BCL6-shRNA group,model mice+ICOS-shRNA group,model mice+Ubi-EGFP group(empty load group)increased significantly(p<0.01),the content of Tfr cells decreased significantly(p<0.01),the proportion of Tfh/Tfr cells increased significantly(p<0.01).Compared with the TNBS model group and the model mice+Ubi-EGFP(no-load)group,the model mice+BCL6-shRNA group and the model mice+ICOS-shRNA group after transfected with lentivirus,the content of Tfh cells in the spleen and colonic mucosa decreased(p<0.01),the content of Tfr cells increased(p<0.01),the proportion of Tfh/Tfr cells deCLined(p<0.01),but it is still higher than the normal control group(p<0.01).Compared with the normal control group,the expression of BCL6mRNA and ICOSmRNA in colon of TNBS model group,model mice+BCL6-shRNA group,model mice+ICOS-shRNA group,model mice+Ubi-EGFP group(empty load group)increased significantly(p<0.01).The model mice+BCL6-shRNA group and the model mice+ICOS-shRNA group after transfected by lentivirus,the expression of BCL6mRNA and ICOSmRNA in the colon was lower than that of the TNBS model group and the model mice+Ubi-EGFP(empty load group)group(p<0.01),but it is still higher than the normal control group(p<0.01).Conclusions There is abnormal regulation and differentiation of Tfh and Tfr two kinds of cells in TNBS induced colitis mice,which suggest that Tfh and Tfr may play an important role in the pathogenesis of UC.Tfh and Tfr cell content and Tfh/Tfr cell ratio were decreased by downing BCL6 and ICOS in UC mice,which can ameliorate the inflammatory response in the intestinal tract of UC mice.Tfh/Tfr and its related cytokines BCL6 and ICOS play an important role in promoting UC immune response,which is the key to UC’s disease. |
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