Study The Impact Of Intestinal Macrophages Of Acute Colitis Induced By DSS Using CD11c- DTR Mice

Objective:Ulcerative colitis(UC) is a kind of chronic intestinal inflammatory diseases with uncertain etiology and pathogenesis.It is found that the amount of macrophages increase obviously in the intestine inflammation of UC patients and secrete massive cytokine and bioactive substances,which play the important role in inflammatory response. Inject DT(Diphtheria Toxin)into abdominal cavity of CD11c-DTR mice can completely eliminate intestinal macrophages and dendritic cells while they will develop after window period.Dendritic cells will grow within three days after injection,however,the macrophages need two weeks.We use this method to analyse the influence on acute colitis induced by DSS(Dextran Sodium Sulfate)with the loss of macrophages.Methods:1 Using flow detection to observe dendritic cells and macrophages’ development in window period as soon as DT is injected into the abdominal cavity of CD11c-DTR mice.Using FCM(flow cytometry) to record the quantitive and proportional variation of macrophages(IA/IE+F4/80+)and dendritic cells(CD11c+CD11b+) at lamina propria of large intestine related to different time point(1d、3d、1w、2w) after DT injection.2 Establish the model of acute colitis induced by DSSC on 57BL/6 mice.According to the Cooper and other methods to establish the model of acute colitis induced by DSSC with 57BL/6 mice.The specific operations are as follows.First,make up DSS solution of five percent by dissolving themolecular weight of 5,000 DSS into distilled water.Then the TC57BL/6 mice drink the mixture for seven days to get the model of acute colitis on mice.3 Detect the impact of macrophages in acute colitis induced by DSS on C57BL/6 mice. Using FCM(flow cytometry) to record the quantitive and proportional variation of macrophages(IA/IE+F4/80+) at lamina propria of large intestine.Using immunohistochemical detection to record the quantitive and proportional variation of macrophages(IA/IE+F4/80+) at lamina propria of large intestine.Adopting Real-Time PCR,ELISA methods to detect the secretion situation of rectum tissue cytokine under acute colitis induced by DSS.4 Detect the immunopathogenesis variation of acute colitis induced by DSS on C57BL/6 mice during DT window period(lack of macrophage).Using five percent DSS solution to lure the mice in window period(about seven or fourteen days after DT injection on CD11c-DTR mice) to establish the model of acute colitis.Observe the situation of acute colitis induced by DSS without macrophages by FCM and HE stain methods.Using Real-Time PCR and ELISAas supplemental methods to inspect the secretion situation of rectum tissue cytokine with acute colitis induced by DSS without macrophages.Results:1 It exists window period for the growth of macrophages and dendritic cells after DT injection on CD11c-DTR mice:After DT(200 ng for each mouse)injection on CD11c-DTR mice, macrophages and dendritic cells eliminate completely within one day;After three days of DT(200 ng for each mouse)injection on CD11c-DTR mice, the percentage of dendritic cells increases but not to the normal level while the macrophages are still without any growth;After seven days of DT(200 ng for each mouse) injection on CD11 cDTR mice, the percentage of dendritic cells recovers to normal while the macrophages are still without any growth;After fourteen days of DT(200 ngfor each mouse) injection on CD11c-DTR mice, the percentage of dendritic cells recover to normal while the macrophages are still without any growth.2 Establish the model successfully of acute colitis induced by DSS:Lure the C57BL/6 mice to drink DSS solution of five percent,the mice start to appear the symptom of acute colitis,on the third day appear weight loss, faeces decompaction and occult blood tested positive;on the fourth day appear the loose and blood stools;the water content of faeces increase obviously and the weight decrease to fifty percent,comparing to matched groups shows clear statistical differences.On the eighth day, choose the whole color for comparison,there is statistical discrepancy between the mice in model and the matched group,the ones in model with shorter color. Choose the above whole color through HE stain under the light microscope, the mice in model appear exuviation in section, permeation of inflammatory cells and the decrease of goblet cell in epithelium. There is significant statistical discrepancy of pathological inflammation score with the matched groups.3 The quantities of macrophages increase obviously in the model of acute colitis induced by DSS:The flow result displays that on the seventh day the quantity and percentage of mcrphage at the lamina propria of large intestine improves obviously on theC57BL/6 mice with acute colitis induced by DSS,which shows greatly difference from the matched group.4 Lack of the gut macrophages reduces the symptom of the colitis: on the seventh day after DT injection on CD11c-DTR mice,establish the model of acute colitis induced by DSS. The clinical symptom of CD11c-DTR mice in window period reduces comparing with WT mice(wild type).There is significant statistical discrepancy at the aspects of pathological and HE inflammation score comparing the CD11c-DTR micein window period with WT mice(wild type).Conclusion:Successfully find out the window period for the growth ofmacrophages and dendritic cells with CD11c-DTR mice after DT injection,meanwhile,dring the window period, to observe the function of macrophages of acute colitis induced by DSS. It shows that the symptom of the mice of acute colitis would be lower without macrophages,so that the macrophages play important role in the growth of inflammation of the acute colitis model induced by DSS.