| BackgroundPrimary immune thrombocytopenia(ITP) was a kind of hemorrhagic disease which was more common in children. 4-5 children with this disease would be found among 100,000,,and they always had skin, mucous membranes, internal bleeding more or less because of decreased production of platelets. ITP had no clear etiology at present, and if we wanted to diagnosis it,we must eliminate other diseases. Most children with ITP had good prognosis, while about 10%-20% were still likely to turn into chronic or refractory ITP despite regular treatment.It was very necessary to investigate the etiology of the disease for us.Th17 cells was a new subset of CD4+ T helper cells. It could secrete IL-17 and other pro-inflammatory cytokines, leading to consequent inflammation cytokines recruitment and tissue injury.More researchs about Th17 cells could be found in diseases, such as asthma, ankylosing spondylitis, systemic sclerosis and so on. Treg cells including natual and induced ones,which adopted different mechanisms to suppress immune responses: directly via cell contact and indirectly via reducing the capacity of antigen presentation on antigen presenting cells or via anti-inflammatory cytokines.Recent studies had demonstrated that Th17/Treg played an important role in ITP,While another studies were not. It was still a controversial topic,and it awlays focued on adults with ITP.Flow cytometry(FCM) was a kind of technology which can fluorescent signal detectable label to suspens of single cells or other biological particles, do cell quantitative analysis and sorting speedly and precisly.Enzyme linked immunosorbent assay(ELISA) was one of the three classic Labeled Immunoassays technology. the specificity, sensitivity and the degree of automation of the analysis and the measurement method were significantly improved. So the enzyme immunoassay constantly updated, range of applications is continuing to broaden.and it became useful in clinical. ObjectiveTo investigate the ratio of Th17/Treg cells in children with ITP newly diagnosed and the healthy ones by using ELISA to test the IL-17 and IL-23 levels in plasma and Flow cytometry to detect the frequency of Th17 cells and Treg cells,so did the children treated with globulin. Resultly could we find a new therapy for these children and a criterion during treatment. MethodsForm May 2015 to Augest 2015, 32 cases children with primary immune thrombocytopenia newly diagnosed in the First Affiliated Hospital of Zhengzhou University pediatric were enrolled in this research,including 18 males and 14 females, the median age of which was 2 years(2month-12 years old). The children with ITP who had been treated with hormone or globulin were not be in study.While 22 children were enrolled(the control group)at the same time which had been ruled out other diseases, such as cardiovascular diseases, active infection, autoimmune diseases and so on. and lost visitor’s follow-up time was the date of lost to the follow-up. The age, sex and living area had no significant difference(P> 0.05).Children with ITP were phlebotomized 4ml fasting peripheral venous blood before treatment, one week after treatment by globulin. Normal control group were phlebotomized 4ml fasting peripheral venous blood at the initial visit. 1 m L fasting peripheral venous blood were placed in the general drying tube and allowed to stand at room temperature for two hours and centrifuge in 1500 r/min for 5 minutes to do ELISA. The supernatant specimens were packed in non-pyrogenic and ono-ndotoxin EP tubes and placed in-80 ℃ refrigerator. Another 3 ml fasting peripheral venous blood wouldl be placed in the general drying tube to do FCM.(1)We used ELISA to test the level of IL-17 and IL-23 levels in plasma and Flow cytometry to detect the frequency of Th17 cells and Treg cells respectively. The correlation between plasma IL-23 and IL-17 levels, Th17 cells, Treg were analyzed at the same time.(2)After treated by globulin(400mg/kg.d, 3-5d or 1g/kg.d 1-2d),children with ITP were devided into complete response group, the effective and the ineffective; Comparison of the proportion of Th17 cells in CD4 + T cells、Treg cells in CD4 + T cells and the proportion of Th17 / Treg cells between the effcctive(include complete response group, and effective group) and the ineffective ones were compared. Results1.The percentage of Th17 cells of peripheral blood and Th17/Treg were increased significantly in children with ITP compared with the healthy[(2.03 ±0.55)%vs(0.54 ±0.21)%;(0.99±0.58)% vs(0.71±0.35)%](P<0.05);The percentage of Treg cells was on the contrary[(2.49±0.86)% vs(8.14±0.86)%)](P<0.05);2.The level of IL-17、IL-23 in children with ITP were significantly increased compared with controls[(80.75±12.98)ng/L vs( 21.64±4.20) ng/L;( 13987.26± 2200.18)ng/L vs(9143.31±3219.17)ng/L]( P<0.05). They were positively correlated with the percentage of Th17 cells(r=0.54,P<0.01;r=0.46,P<0.05),while both of them were negativly correlated with the percentage of Treg cells(r=-0.60, P<0.01;r=-0.52, P<0.01).3.Compared with the datas after treated, the percentage of Th17 cells and Th17/Treg were increased significantly[( 2.03±0.55) % vs( 0.96±0.45) %;(0.99±0.58)% vs(0.71±0.35)%],and the percentage of Treg cells was not [(2.49±0.86)% vs(4.54±0.95)%)](P<0.05).Children treated were divided into two groups, the effective and the ineffective.Compared with the effctive ones, the percentage of Th17 and Th17/Treg were increased significantly[(0.69±0.36)%)vs(1.51±0.45)%(P<0.05);(0.14±0.07)%)vs(0.52±0.27)%(P<0.05],and the percentage of Treg reduced[(3.59±0.92)% vs(5.21±0.78)%)](P<0.05). Conclusions1. From our study,we can get a idea that the imbalance of Th17/Tregs cells in children with Primary immune thrombocytopenia exist in blood.The level of IL-17 and IL-23 levels in plasma are important in the imbalance of Th17/Tregs cells in children with Primary immune thrombocytopenia,and it may be target threapy for children with ITP in the future.2. We may suppose that Globulin(400mg/kg.d, 3-5d or 1g/kg.d 1-2d) can treat children with ITP through improving the cellular immune function by changing the imbalance of Th17/Tregs cells. |
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