Regulatory T Cell Effects On Intestinal T-cell Immune Function And The Changes Of Expression Of T Helper 17 Cell And Regulatory T Cell And It Effects On Intestinal Barrier Injury In Severely Burned Rats

Objective: Regulatory T cells(Tregs) and CD3+T cells was isolated from intestinal lymph of burned rats and using differenet concentration Regulatory T cells to culture with CD3+T cells in vitro. To investigate the effect of differenet concentration Regulatory T cell to CD3+T cells on immunity.Methods: Ten SD male rats were burned with vapor under 3MPa pressure and 108℃ temperature for eight seconds to achieve III degree scalding,and a 30% total body surface area(TBSA) burn model was made. They were killed by cervical dislocation after 12 burn hour and their intestinal lymph nodes was extract to make single cell suspension. MACS techniques were used for the separating of the CD4+CD25+ Regulatory T cell and CD3+T cell was isolated by antigen-specical glass colums. Isolated CD3+T cells were randomly divided into blank control group, A group(1×104 Tregs), B group(2×104 Tregs), C group(5×104 Tregs)and D group(1×105 Tregs). Flow cytometry was used to detect the ratio of CD4+/CD8+cells,Th1/Th2 cells and T cell apoptosis. ELISA method was used for detection of IL-2 and IL-10 levels in the culture suspension.Results:(1) Compared with the blank control group(0.82±0.07), The proportion of CD4+/CD8+T cells had decreased with the increaseing of Treg concentration. The difference was statistically significant(P<0.05).(2) Compared with the blank control group Th1(8.62±0.21)、Th2(5.2±0.67), The proportion of Th1 had decreased with the increaseing of Treg concentration,The Th2 cells were just the opposite. The difference was statistically significant(P<0.05).(3) Compared with the blank control group, The T cell apoptosis had increased with the increaseing of Treg concentration. The difference was statistically significant(P<0.05).(4) Compared with the blank control group, The expression of IL-2 wassignificantly increased with the increaseing of Treg concentration.(P<0.05). The expression of IL-10 was significantly decreased with the increaseing of Treg concentration.(P<0.05).Conclusion: The expression of the Treg cells in the intestinal lymph nodes was increased in severely burned rats. With the increaseing of Treg concentration, the ratio of CD4+/CD8+cells and Th1/Th2 cells had decreased, what’s more,the T cell apoptosis had increased. The expression of IL-2 was significantly increased and IL-10 was significantly decreased.The changes may be responsible for the intestinal immunsuppression response. To inhibit the Treg cells,Can be used as a new target for reversing immunosuppression after burned.Objective: To observe the change of intestinal T Helper 17 cell(Th17) and Treg expression, the levels of IL-17 A,IL-6 in the portal vein blood(PV) and intestinal mucosa(IM)in severely burned rats,To investigate the changes of Th17/ Treg and its cytokines effect on intestinal barrier’s injury.Methods: Fifty SD male rats were randomly divided into normal control group(n=10)and burn model groups(n=40).Rats were burned with vapor under 3m Pa pressure and 108℃ temperature for eight seconds to achieve III degree scalding,and a 30% total body surface area(TBSA) burn model was made. Rats were sacrificed before(normal control group) and after 12, 24, 36,48 burn hour(PBH,marked T1-T4).Flow cytometry techniques were used for the detection of the expressions of T Helper 17 cell and regulatory T cells in intestinal lymph nodes which were separated. ELISA method was used for detection of IL-17 A and IL-6 levels in the PV and IM.One 1cm small intestine was observed with HE staining to invaule the gut injury score by chiu’s method.Results:(1) The percentage of Help T 17 cells in T1, T4 group rats were(1.54±0.07, 3.03±0.08), and that in normal control group is 0.82±0.07, There was a significant difference between control group and PBH groups(P<0.01).(2) The percentage of regulatory T cells in T1 groups(12PBH), T4 groups(48 PBH groups)rats were(17.42±1.17, 40.66±0.93), and that in normal control group is 8.07 ±0.82, There was a significant difference between control group and PBH groups(P<0.01).(3) Compared with the normal control group(36.3±2.83、16.3±1.25), The IL-6、 IL-17 A levels in T1 groups were(105.7±5.89 、 31.3±4.77), T4 groups were(263.9±5.30、107±3.43) in PV.The difference was statistically significant(P<0.01).(4) Compared with the normal control group(5.1±0.74、18.1±1.60), The IL-6、 IL-17 A levels in T1 groups were(16.3±1.16 、 29.2±2.49), T4 groups were(53.4±2.99、61.3±3.47)in IM. The difference was statistically significant(P<0.01).(5) Compared with the normal control group(0), The small intestine injury scores(chiu’s method)were(2.7±0.67,5.16±0.25)in T1 and T4 groups,The difference was statistically significant(P<0.01).(6) The expression of Th17 and Treg were positively correlated with small intestine injury scores( r=0.972,0.969.P<0.01). The expression of IL-6 and IL-17 A were positively correlated with small intestine injury scores in PV and IM( r=0.973,0.962,0.970,0.961..P<0.01),The rate of Th17 /Treg were negatively correlated with small intestine injury scores( r=-0.814,P<0.01). Multiple linear regression analysis shows that their expression were positively correlated with small intestine injury scores(R=0.978;p<0.01).Conclusion:The ratio of the intestinal Th17/Treg cells in the in severely burned rats was decreased. With the increaseing of IL-17 A and IL-6 levels in the PV and IM, The small intestine injury scores had increased. the Th17/Treg cells have portal effects on intestinal immunity barrier and results in spesis.