A Study On The Relationship Between CD4~+CD25~+Treg Cell And CD40/CD40L In Lymph Fluid And Blood Of Asthmatic Rat

Object:To determine the effect of the CD40/CD40L signaling pathway to the number of the CD4+CD25+Foxp3+Treg and the secretion of TGF-β1,IL-10in lymph fluid and blood of the asthma rat.Method:Build the asthma model. After the last challenge, the percentage of the CD4+CD25+Foxp3+Treg from the lymph fluid and the blood were detected by FCM(flow cytometer), then the blood and lymph fluid were collected from the rats at0h、12h、24h after the last challenge. MNC were isolated and seeded in24well plate, after72h-cocultured with CD40L monoclonal antibody to block, the percentage of the CD4+CD25+Foxp3+Treg were detected by FCM(flow cytometer), the expression of PE-CY5-Foxp3was detected by laser scanning confocal microscope, the levels of the TGF-β1and IL-10in supernatant were detected by enzyme-linked immune-sorbent assay.Result:1. The asthma model was copied successfully and the lymph drainage patterns were established.2. The percentage of CD4CD25Foxp3+Treg in the lymph fluid and the blood of the asthma rats were significantly lower than the control group (P<0.05). The percentage of CD4+CD25+Foxp3Treg in the lymph fluid were higher than in the blood.3. In vitro culture system, the percentage of CD4+CD25+Foxp3+Treg in the lymph fluid were significantly higher than in the blood in each group. With the interval time which the sample collected from the last challenge, the percentage at the trend from rising to dropping in the lymph fluid and in the blood, and the percentage of CD4+CD25+Foxp3+Treg in the lymph fluid and in the blood in asthma group were significantly lower than the control group and the anti-CD40LMAb group(P<0.05).4. Cocultured with the anti-CD40L monoclonal antibody,72h later, the average optical density of the PE-CY5-Foxp3in the surface of MNC in anti-CD40LMAb group were higher than in asthma group in the blood and the lymph fluid.5. In vitro culture system, with the interval time from the last challenge, the levels of IL-10in the supernatant of the MNC from the lymph fluid and the blood were at the rising trend, the levels of IL-10in the supernatant of the MNC from the lymph fluid in the anti-CD40LMAb groupwas higer than the asthma group and the control, the significant differences was at24h(.P<0.05), there were no significant differences during the asthma group and the control group; and there were no significant differences in the supernatant of the mixed lymphocyte from the blood during each group.6. In vitro culture system, The levels of TGF-β1in the supernatant of the MNC from the lymph fluid in the asthma group was higer than the control and the anti-CD40LMAb group at Oh; The levels of TGF-β1in the supernatant of the MNC from the blood in the anti-CD40LMAb group were higher than the control group and the asthma group at Oh and12h (P<0.05); The control group and the asthma group had no significant difference.Conclud:1. Thenumber of CD4+CD25+Foxp3+Treg in asthma rats was lower than the control; In vitro culture system, with the interval time from the last challenge, the percentage of CD4+CD25-Foxp3+Treg at the trend from rising to dropping.2. Blocking the CD40/CD40L signaling way with anti-CD40LMAb can promote the proliferation of CD4+CD25+Foxp3+Treg in vitro culture system.3. Blocking the CD40/CD40L signaling way with anti-CD40LMAb in vitro culture system can rise the level of TGF-β1secreted by CD4+CD25+Foxp3+Treg from the lymph at Oh and12h after the asthma were excited, and can rise the level of IL-10secreted by CD4+CD25+Foxp3+Treg from the lymph at24h after the asthma were excited.