The Preliminary Study About The Expression Of CD19⁺CD24^hiCD38^hi Breg And Its Effects On The Differentiation Of Th1 In The Peripheral Blood Of PBC Patients

Primary biliary cirrhosis (PBC) is an autoimmune disease of slowly progress which is characterized by chronic cholestasis, anti-mitochondrial antibodies (AMA) and nonsuppurative destructive cholangitis. The condition of patients with PBC generally slows development, in which middle and advanced stage there are no effective clinical treatments. Moreover the therapeutic approaches are single. Consequently the early diagnosis and therapy are essential for PBC. Currently, it is a hot point to look for a new therapeutic.Abnormal activation of B lymphocytes, autoantibody production are the main factor that induced the pathogenesis of PBC. B cell not only possess the role of producing antibody、presenting antigen etc, but have the role of regulating the immune response. In the early 1990s, researchers have found the mice deterioration degree is higher after excluding B cells in encephalomyelitis mouse model experiment, and put forward the regulatory B cells for the first time. Then subsequently, series of studies further confirmed the existence of Bregs, and pointed out the importance of the immune regulation. In recent years, some reports indicated the biological nature and role of CD19+CD24hiCD38hi Breg in some autoimmune diseases such as system of lupus erythematosus> pemphigus、vasculitis. However, there are no relevant documents about the CD19+CD24hiCD38hiBreg in the PBC patients.Objectives:Detected the proportion of CD19+CD24hiCD38hiBreg in the peripheral blood of PBC patients. And analyze the correlation between CD19+CD24hiCD38hiBreg and the diseases. Then, we preliminarily explored the role of CD19+CD24hiCD38hiBreg in the differentiation of Thl、Th17. Thereby we can provide new experimental basis for immune mechanism research and treatment strategies development in PBC.Methods:1. Gathered the peripheral blood of EDTA anticoagulant from PBC patients and healthy controls (HC).We extracted the Peripheral blood mononuclear cell by density gradient centrifugation. Then the proportion of CD19+B cells and CD19+CD24hiCD38hiBreg cells in the PBC patients and healthy controls were assayed with flow cytometry. Then we compared the phenotypic expression changes of CD19+B cells and CD19+CD24hiCD38hiBreg between HC and PBC.2. We collected the serum of PBC patients, and, detected the ADA. ALT、 AST、GGT and ALP about patients, then analysed the correction between CD19+CD24hiCD38hiBreg and the above laboratory dates.3. Detected the proportion of Thl、Th17 in HC with Flow cytometry. Then we analyzed the correlation between the CD19+CD24hiCD38hi Breg and Th1、h17 in HC.4. Detected the proportion of Th1、Th17 in PBC with Flow cytometry and analyzed the correlation between the CD19+CD24hiCD38hi Breg and Th1、Th17 in PBC.5. CD4+T cells and CD19+CD24hiCD38hi Breg were isolated by Flow cytometry sorting from HC and PBC patients. The CD4+T cells from healthy controls were cultured alone as a control. In addition, CD19+CD24hiCD38hi Breg isolated from the healthy individuals and PBC patients respectively Co-culture with CD4+T cells of healthy controls. And, then we detected the differentiation of Thl with Flow cytometry.Results:l.The proportion of CD19*B cells in the peripheral blood:the group of HC was (8.469±3.399)%, the group of PBC was (12.210±6.204)%. Compared with the HC group, the proportion of CD19+B cells in PBC showed statistically significant increases (P=0.009). The proportion of CD19+CD24hiCD38hi Breg PBC group (12.379±6.417)%increases compared with that in HC (4.225±3.318)%, and, there was statistical significance (P=0.000).2. CD19+CD24hiCD38hi Breg in HC showed positive correlation with TBIL (P=0.049, r=0.535). DBIL (P=0.035, r=0.566)、IBIL(P=0.046, r=0.521) and TBA ((P=0.014, r=0.619)3. The proportion of Th1、Th17 in HC are respectively (11.807±5.144)%、 (0.846±0.904)%. And, CD19+CD24hiCD38hi Breg in HC showed negative correlation with Thl (P=0.017,r=-0.426).But there were no correlation between CD19+CD24hiCD38hi Breg and Thl7 in HC (P=0.941, r=0.014)4. The proportion of Th1, Th17 in PBC are respectively (17.689±7.444)%、 (1.797±2.784)%. There is a positive correlation between CD19+CD24hiCD38hi Breg and Thl in PBC (P=0.003, r=0.677). No correlation between CD19+CD24hiCD38hi Breg and Th17 in PBC (P=0.171, r=0.348)5. Taking the CD4+T from HC culturing alone as a control, the CD19+CD24hiCD38hi Breg from HC and PBC respectively co-culture with CD4+T cells of HC. The differentiation of Thl are as follows:(17.367±2.747)%、(4.173±1.878)%、 (48.567±7.522)%。The differentiation of Thl which detect from the CD19+CD24hiCD38hi Breg of PBC co-culturing with CD4+T cells of HC increased among with that in the CD4+T from HC culturing alone and the CD19+CD24hiCD38hi Breg of HC co-culturing with CD4+T cells of HC, there was statistical significance (P=0.001)、(P=0.000). The differentiation of Thl which detect form the CD19+CD24hiCD38hi Breg of HC co-culturing with CD4+T cells of HC decreased among with that in the CD4+T from HC culturing alone and the CD19+CD24hiCD38hi Breg of PBCC co-culturing with CD4+T cells of HC, there was statistical significance (P=0.043)(P=0.000).Conelusions:The express of CD19+CD24hiCD38hi in the peripheral blood of PBC patients increased. Moreover, there is a closely related to the degree of liver injury. In addition, The CD19+CD24hiCD38hi promoted the differentiation of Thl in PBC.