Condition of human Th17 ceII differentiation induced in vitro / 中国药科大学学报

ABSTRACT

In this study,a series of related indicators were investigated via flow cytometry,enzyme-linked immu-no sorbent assay (ELISA)and quantitative real time PCR (Q-PCR)technology to assess the in vitro differentia-tion of human Th17 cells.Human peripheral blood mononuclear cells (PBMCs)were purified from fresh human blood using gradient centrifugation and then the Th17 cells were induced with different cytokines (IL-1β,IL-6, TGF-βand IL-23)at different induction time (1,2,3,4 d)to compare the effects on Th17 cell differentiation un-der these conditions.The experiment data showed that IL-1β,IL-6,TGF-βor IL-23 alone play a promotion role in the Th17 differentiation and combination of IL-1β,IL-6,TGF-βand IL-23 could induce efficient human Th17 cell differentiation in vitro to achieve the best.Further optimization of the induction time found that the Th17 cell dif-ferentiation efficiency gradually increased with the extension of the time;howerver,when culturing for 3 d,it reached the peak number and then decreased in regardless of the time increasion.Finally the optimal condition of in vitro polarization of human Th17 cells was established,and the purified PBMCs were cultured with anti-CD3 and anti-CD28 as the basal conditions,and co-culturing with IL-1β,IL-6,TGF-βand IL-23 for 3 d to effectively induce the differentiation of Th17 cells.The inducing efficiency is significantly higher than in normal control.At the optimal condition,we observed the Th17 cell differentiation frequency (CD4 +IL17A +)was increased to nearly 10% through flow cytometry analysis and the secretion level of IL-17A in cell supernatants was also detec-ted to reach 3 ng/mL using ELISA methods.In addition,gene expression of IL-17A were determined by quantitativereal-time PCR using pre-designed primers by the comparative method of relative quantitation (ΔΔCt)and β-actin gene was used as an internal control for sample normalization.The results showed that the expression of IL-17A mRNA could be increased about 15 times with IL-1β,IL-6,TGF-βand IL-23 co-culturing for 3 d.The protocolof efficient human Th17 cell differentiation we presented in this paper is simple,rapid and easy to be repeated.This study provides an effective detection platform for the research of Th17 cell function and development ofrelated drugs targeting Th17 cells for autoimmune disease treatment.