| Objective:Henoch Schonlein Purpura(HSP) is one of the most common childhood hemorrhagic diseases, which the multiple body organs getting involved in and the systemic vascular inflammation in small vascular participate into the pathological changes. The main clinical manifestations are: skin purpura, gastrointestinal bleeding, joint swelling and nephritis. The targets for this study are:(1) to detect the Treg/Tfh cell levels in children with HSP at acute and recovery phase compare with health control by flow cytometry, and to explore its role in immunonregulation among children with HSP;(2) to detect the differences of cytokines IL-10, IL-21 at the acute and recovery phase compare with health control by ELISA, to investigate its role in the pathogenesis of children HSP.Methods:1. We collected the 6ml fresh anticoagulant fasting blood from the acute Children HSP at the outpatient department or inpatient wards in the Affilated Hospital of Ning Xia Medical University from September, 2013 to September, 2014. After 2 weeks, when the rash, joint pain, abdominal pain and other clinical symptoms disappeared, similarly, collected the 6 ml fasting blood from those recovery patients at the impatient wards. The 6ml fasting blood from the children healthy control were collected from the children physical check at the health examination center of Ningxia Medical University General Hospital. 2. To collect the serum from the blood samples by centrifuge 1500r/min,20 minutes and aliquot to each tubes frozen storage in-80℃fridge and to liquid nitrogen until get enough samples to check the IL-10, IL-21 level by ELISA. 3. To isolated the peripheral blood mononuclear cells(PBMC) by Density gradientcentrifugation, and aliquot to each tubes and use the programmed- freezing box to storage the PBMCs in-80℃ fridge and to liquid nitrogen until get enough samples to run flow. The antibody we used to mark the PBMCs were: anti-human-CD4-V450,anti-human-CD127-PE,anti-human-CD25-APC-CY7, anti-human-CXCR5-Alexa and anti-human-CXCR5-Fluor®488, anti-human-CD279-Percp-5.5 fluorescent antibody, and then to check the numbers or percentages of Treg, Tfh cell by flow cytometry. 4. To compare the numbers or percentages of Treg/Tfh from the children HSP at acute and recovery phase with normal control group by different variance analysis. 5. To compare the expression of cytokines IL-10, IL21 from the children HSP at acute and recovery phase with normal control group by different variance analysis..Results:1. The percentage(3.284%±0.5897) of CD4+CD25+Treg cells from the Children with HSP at the acute phase compared with health control group(8.195%±1.504)significantly decreased, P<0.01; the percentage(3.996%±0.3052) of CD25+CD127LowT cells within CD4+ T cells compared with health control group(6.541%±0.6195) significantly decreased, P<0.01; the percentage(5.314%±0.6934) of CD4+CD25+Treg cells from the Children with HSP at the recovery phase compared with the acute phase(3.284%±0.5897) significantly increased, P<0.05; and the expression level almost getting close to the normal control group. The percentage(6.121%±0.5195) of CD25+CD127LowTregs within CD4+ T cells from the Children with HSP at the recovery phase compared with the acute phase(3.284%±0.5897) significantly increased, P<0.01; and the expression level almost get close to the normal control group. 2. The percentage(6.310%±1.073) of CD4+CXCR5+Tfh cells from the Children with HSP at the acute phase compared with health control group(3.626%±0.4419) significantly increased, P<0.05; The percentage(2.747%±0.4335) of CD279+Tfh cells within the CD4+T cells from the Children with HSP at the acute phase compared with health control group(1.425%±0.2035) significantly increased, P<0.05; The percentage(1.296%±0.1632) of CXCR5+CD279+Tfh cells within the CD4+T cells from the Children with HSP at the acute phase compared with health control group(0.6935%±0.1007) significantly increased, P<0.01;The percentage(3.67%±0.7241) of CD4+CXCR5+Tfh cells from the Children with HSP at the recovery phase compared with the acute phase(6.310%±1.073) significantly decreased, P<0.05; and tend to be normal. The percentage(1.644%±0.3091)of CD279+Tfh cells within the CD4+T cells from the Children with HSP at the recovery phase compared with the acute phase significantly decreased, P<0.05. The percentage(0.7788%±0.1409)of CXCR5+CD279+Tfh cells within the CD4+T cells from the Children with HSP at the recovery phase compared with the acute phase decreased, P<0.05. 3. The serum average expression level(29.82±3.89) pg/ml of IL-10 from children HSP at the acute phase compared with the healthy control group(62.03±4.16) pg/ml significantly decreased(P<0.01); the serum average expression level(49±4.99) pg/ml of IL-10 from children HSP at the recovery phase compared with the acute phase significantly increased(P<0.05), the expression level was almost similar to those from the normal control group. 4. The serum average expression level(2198±107.7) pg/ml of IL-21 from children HSP at the acute phase compared with the healthy control group(1492±181.9) pg/ml significantly increased(P<0.01); the serum average expression level(1473±74.4) pg/ml of IL-21 from children HSP at the recovery phase compared with the acute phase significantly decreased(P<0.01), the expression level was almost similar to those from the normal control group.Conclusions:The different subsets of Treg and Tfh cells from the PBMC are contributed to the occurrence and development of children HSP. The percentage of the different subsets of Treg cells from the Children HSP at the acute phase decreased, but the percentage of the different subsets of Tfh cells from the Children HSP at the acute phase increased. Similarly, the serum expression level of IL-10 reduced and the serum expression level of IL-21 increased at the acute phase of children HSP, while its expression level tend to be normal at the recovery phase, suggesting the secretion of IL-10, IL-21 are involved in the occurrence, development and prognosis of children’s Henoch Schonlein purpura. Conclusion: the subsets of Treg, Tfh cells and their relevant cytokines might be involved in the pathogenesis of children with Henoch Schonlein Purpura. |
PDF Full Text Request