The Study Of Mechnisum Of Th17/Treg In The Lymph Fluid Of Asthmatic Rats And Effect Of Human Placenta Derived Mesenchymal Stem Cells Intervention

Object:In this experiment, we will investigate the proliferation、differentiation and characteristics of the relevant factors between Th17 and CD4+CD25+ Foxp3+ Treg and effect of human placenta derived mesenchymal stem cells transplantation in lymph fluid of asthmatic rat.Method:First, we establish the asthma model and induce lymph drainage. Human placenta mesenchymal stem cells are isolated by the enzyme digestion with a high proliferative ability. HPMSCs are cultured, passaged, counted, curve and identified by multipotent differentiation ability into neural cells, osteoblasts after three generations in vitro. Rats accept mesenchymal stem cells transplantation therapy. The 40 SD rats are randomly divided into four groups of 10 each:(1) normal control group (2) asthma model group (3) hPMSCs transplantation group (4) Asthma+PBS group; MNC which were collected from the asthma and hPMSCs rats from lymph and blood at 24 h after the last challenge were cultured in 24 well plate. When cultured for 18 h, the percentage of the Th17 and CD4+CD25+Foxp3+Treg were examined by FCM and FOXP3 mRNA、RORγt mRNA were examined by qRT-PCR. Lung tissue sections were collected at 24 h after the last challenge, IL-17 mRNA and IL-10 mRNA were checked by qRT-PCR; Western Blot detected FOXP3, RORγt protein expression in rats of lung tissue; IL-17 and IL-10 levels in the blood and lymph supernatant were determined by ELISA.Result:1. The asthma model was copied successfully and the lymph drainage patterns were established.2. The mult idiferention of hPMSCs, The reproductive activity of hpMSCs was attenuated after passages. The in vitro inductive culture demonstrates cartilage and neural differentiation.3. MNC were collected at 24 h after the last challenge and cultured 18 h, the percent of CD4+CD25+Foxp3+Treg in the MNC from lymph were obviously higher than that from blood in each group(P<0.05); The percent of CD4+CD25+Foxp3+Treg in the MNC from lymph and blood in asthma group and asthma with PBS group were obviously lower compare to the control group (P<0.05); The percent of CD4+CD25+Foxp3+Treg in the MNC from lymph and blood in the hPMSCs group obviously increased compare with the asthma group (P<0.05).4. MNC were collected at 24 h after the last challenge and cultured 18 h, the expression of FOXP3 mRNA in the MNC from lymph were obviously higher than that from blood in each group(P<0.05); The expression of FOXP3 mRNA in the MNC from lymph and blood in asthma group and asthma with PBS group were obviously lower compare to the control group (P<0.05); The expression of FOXP3 mRNA in the MNC from lymph and blood in the hPMSCs group obviously increased compare with the asthma group (P<0.05).5. MNC were collected at 24 h after the last challenge. The level of IL-10 in the MNC from lymph and blood in asthma group and asthma with PBS group were obviously lower compare to the control group (P<0.05); The level of IL-10 in the MNC from lymph and blood in the hPMSCs group obviously increased compare with the asthma group (P<0.05). there were no significant differences of IL-10 level in the supernatant of MNC from lymph and blood in each group.6. MNC were collected at 24 h after the last challenge and cultured 18 h, the percent of Th17 in the MNC from lymph were obviously higher than that from blood in each group(P<0.05); The percent of Th17 in the MNC from lymph and blood in asthma group and asthma with PBS group were obviously higher compare to the control group (P<0.05); The percent of Th17 in the MNC from lymph and blood in the hPMSCs group obviously decreased compare with the asthma group (P<0.05).7. MNC were collected at 24 h after the last challenge and cultured 18 h, the expression of RORyt mRNA in the MNC from lymph were obviously higher than that from blood in each group(P<0.05); The expression of RORyt mRNA in the MNC from lymph and blood in asthma group and asthma with PBS group were obviously higher compare to the control group (P<0.05); The expression of RORyt mRNA in the MNC from lymph and blood in the hPMSCs group obviously decreased compare with the asthma group (P<0.05).8. MNC were collected at 24 h after the last challenge. The level of IL-17 in the MNC from lymph and blood in asthma group and asthma with PBS group were obviously higher compare to the control group (P<0.05); The level of IL-17 in the MNC from lymph and blood in the hPMSCs group obviously decreased compare with the asthma group (P<0.05). there were no significant differences of IL-17 level in the supernatant of MNC from lymph and blood in each group.9. The expression of IL-10 mRNA in the lung tissue was obviously lower in asthma group and asthma with PBS group than that in the control group (P<0.05); The expression of IL-10 mRNA in the hPMSCs group obviously increased compare with the asthma group (P<0.05). The expression of IL-17 mRNA in the lung tissue was obviously higher in asthma group and asthma with PBS group than that in the control group (P<0.05); The expression of IL-10 mRNA in the hPMSCs group obviously decreased compare with the asthma group (P<0.05).10. FOXP3 protein expression in asthma group and asthma with PBS group in lung tissue was significantly lower than the control group (P<0.05); FOXP3 protein expression in hPMSCs treatment group was significantly higher than the asthma group (P<0.05). ROR gamma protein expression in asthma group and asthma with PBS group in lung tissue was significantly higher than the control group (P<0.05); ROR gamma protein expression in hPMSCs treatment group was significantly decreased than the asthma group (P<0.05).Conclusion:1. The hPMSCs isolation and purification were successful by collagenase Ⅳ digestion and stem cells obtained were multipotency.2. There was imbalance between Th17 and Treg in lymph fluid and blood of asthmatic rat. MNC which were collected at 24 h after the last challenge from the lymph and blood indicated the percent of CD4+CD25+Foxp3+Treg were significantly reduced, and the percent of Th17 were significantly increased。Th17 promote the secretion of IL-17 in lymph and blood from asthma, Treg inhibit the secretion of IL-10 in lymph and blood from asthma.3. There was imbalance between FOXP3 and RORyt, manifested as low FOXP3 gene expression, RORyt gene high expression.4. Human placenta-derived mesenchymal stem cells may suppress airway inflammation in asthmatic rats by regulating the expression of related factors related Th17 and Treg.5. In each group, the levels of Th17 and CD4+CD25+Foxp3+Treg in lymph were significantly higher than those in blood, which suggested the immune function of lymph circulation may be a more sensitive indicator reflecting the change of immune status in the body.