The Study Of Th17 Cells Differentiation With Primary Biliary Cirrhosis Patients Induced In Vitro

BackgroundT helper 17 (Th17) cells belong to a recently identified T helper subset, in addition to the traditional Th1 and Th2 subsets. These cells are characterized as preferential producers of interleukin-17,IL-21 and IL-22. Th17 cells and their effectors cytokines mediate many autoimmune diseases. The effector of Th17 cells, therefore, mediate the crucial crosstalk between immune system and tissues, and play indispensable roles in tissue immunity. Primary biliarycirrhosis (PBC) is a organ specific autoimmune diseases, The pathogenesis and aetiology of PBC remain unclear. From the angle of Th17 to study PBC and seek the intervention measures to enhance the immune adjustment function are important to provide new way and experiment envidence for treating PBC.ObjectivesNaive CD4+ T cells were sorted by magnetic activated cell sorting technology(MACS), then cultered in vitro with various cytokines. The Th17 production was measured by flow cytometry(FCM), the IL-17 was measured by enzyme-linked immunosorbent assay (ELISA) and mRNA was measured by fluorescence quantitative PCR. The Th17 differentiation of naive CD4+T cells were compared between normal control and the primary biliary cirrhosis (PBC) patients. We hoped through the research, be able to explore the Th17 differentiation in vitro and reveal the correlation of Th17 with the pathogenesis of PBC.Method1. Naive CD4+ T cells were isolated from the peripheral blood of normal control and PBC patients by Ficoll-Hypaque density gradient centrifugation and MACS technology. Naive CD4+ T cells were cultured in 4 groups which with various cytokines, to induce the Th17 differentiated from Naive T cells. The 4 groups are:(1)control group:basal medium;(2)IL-1βgroup:IL-1β+basal medium;(3)IL-1β+IL-6 group:IL-1β+IL-6+basal medium;(4)IL-1β+IL-6+IL-23 group:IL-1β+IL-6+IL-23+basal medium(basal medium was anti-CD3,anti-CD28,anti-IL-4,anti-IFN-γ).The efficiency of Th17 differentiation was measured by flow cytometry (FCM) after 9 days cultured at 37℃,5% CO2.2 The differences of the Th17 differentiation between PBC patients and the normal group(1)Naive T cells were cultured with IL-1βand IL-6 and IL-23 combination. The supernatant of the cells were collected in 72 hours, the IL-17 levels was measured by enzyme-linked immunosorbent assay (ELISA).(2) Culture medium was changed once after an interval of 3 days, and replenish the corresponding cytokines for 9 days. Stimulated with PMA, Ionomycin and monensin cultured for 4 hours more, the cells were stained by surface and intracellular staining. At the same time the isotype were measured by FCM, CXP software was used to analyze frequency of Th17 cell differentiation.(3)the cells were collected after 9 days, then centrifugated, carefully discarded the supernatant, quickly added Trizol solution, mRNA was reversely transcribed into cDNA, IL-17 gene expression was measured by fluorescence quantitative PCR.ResultsFlow cytometry assay:Compared with the normal control, the frequency of Th17 cells differentiation of PBC patients was the highest in the IL-1β+IL-6+IL-23 group(P<0.01). To the PBC patients, IL-1βgroup;IL-1β+IL-6 group,IL-1β+IL-6+IL-23 group could induce Th17 cells differentiation significantly(P<0.01);the frequency of Th17 cells differentiation was the highest in the IL-1β+IL-6+IL-23 group comparing with other groups(P<0.01).ELISA results:For the PBC patients group, IL-1βand IL-6 and IL-23 combined,IL-17 levels significantly higher than the normal groups (P<0.05).Fluorescence quantitative PCR results:PBC patients with Naive CD4+T cells were cultured after 9 day were measured by fluorescence quantitative PCR, IL-17 gene expression rate is 3.73 times of the normal, significantly higher than the normal(P<0.01).Conclusion IL-1βalone and combinating with IL-6,IL-23 could induce Thl7 cells differentiation in PBC patients; IL-1βcombined with IL-6,IL-23 could induced Th17 differentiation significantly higher than normal control. Combined with IL-1β,IL-6,IL-23 could induce Naive CD4+ T cells of PBC patients Th17 cells differentiation in vitro to achieve the best.