摘要
目的建立小鼠Thl7细胞体外培养方法并研究姜黄素对Thl7细胞分化、功能的影响及机制。方法分离、纯化小鼠脾脏CD4^＋CD25-T细胞，培养体系内加入抗CD3、抗CD28抗体活化，并加入转化生长因子（TGF）-β、白细胞介素（IL）-6、IL-23、抗干扰素-1抗体、抗IL-2抗体等促进Thl7细胞分化。培养的Thl7细胞分为对照组、姜黄素低剂量（5pμmol／L）及高剂量（25μmol／L）组、西罗莫司（100ng／m1）组。流式细胞术检测Thl7细胞比例，荧光定量聚合酶链反应（PCR）及蛋白印迹法检测视黄酸相关孤儿受体（ROR）γt表达水平及信号转导和转录活化因子（STAT）3磷酸化水平，最后检测细胞IL-17A、IL-21mRNA相对表达量。采用t检验、单因素方差分析对实验结果进行统计学处理。结果本方案可明显促进Thl7细胞分化。与对照组相比，姜黄素低组、高剂量组及西罗莫司组Thl7细胞比例明显降低[分别为（54．1±3．4）％、（40．3±2．8）％、（25．8±2．3）％、（25．0±2．0）％，t值为6．15，12．63，12．97，P值均〈0．01]。姜黄素低剂量组、高剂量组及西罗奠司组RORγt的mRNA水平、蛋白水平、STAT3磷酸化水平以及IL-17A、IL-21mRNA水平均较对照组明显降低[（IL-17AmRNA分别为0．81±0．05，0．61±0．05，0．58±0．05，1．01±0．11，t值为4．81，8．52，8．89，；IL-21mRNA分别为0．73±0．06，0．49±0．03，0．59±0．03，1｜12±0．11，t值为5．98，9．22，7．95；P值均〈0．01]，且姜黄素高剂量组IL-21mRNA水平较西罗莫司组降低（P〈0．05）。结论姜黄素体外可以抑制小鼠脾脏CD4^＋CD25^-T细胞向Thl7细胞分化，并抑制IL-17A、IL-21mRNA合成，其机制可能与抑制细胞内孤儿受体RORγt表达及STAT3磷酸化有关。
Objective To establish a culture protocol for Thl7 cells in vitro and evaluate the effect of eurcumin on the differentiation and functions of Thl7 cells and explore the related mechanisms. Methods Splenic CD4^＋CD25^- T ceils of C57BL/6 mice were isolated and purified with magnetic bead methods, and were co-cultured with plate-bound anti-CD3 and anti-CD28 antibodies and with transforming growth factor （TGF）-β, interleukin （IL）-6, IL-23, anti-interferon-γ antibody and anti-IL-2 antibody for Th17-polarization. The cuhured Thl7 cells were allocated into four groups： the control group, in which T cells were cultured on the basis of the above protocol; the low-concentration curcumin （CM-L, 5 p.mol/L） and high-concentration （CM-H, 25 p^mol/L） cureumin groups, and sirolimus （SRL, 100 ng/m]） group. The prop^a＇tion of Thl7 ceils was detected with flow cytometry, and the m RNA expression of retinoic acid-related orphan receptor （ROR）Tt, IL-17A, IL-21 was examined with real-time quantitative polymerase chain reaction. Finally, the protein expression of RORγt and the phosphorylation levels of signal transducer and activator of transcription （STAT） 3 were measured using western blotting analysis. Results The proportion of Thl7 cells in the freshly isolated CD4^＋CD25^- T cells was （3.1±0.4）%, whereas the proportion of the cells cultured with the protocol could get [（54.1±3.4）%, P〈0.01 ]. As compared with the control group, the Thl7 cells proportion in CM-L, CM-H and SRL groups was significantly decreased [CM-L （40.3±2.8）%, CM-H （25.8±2.3）%, SRL （25.0±2.0）% versus the control （54.1±3.4）%, t=6.15, 12.63, 12.97, P〈0.01], and no marked difference was seen between CM-H group and SRL group and（P〉0.05）. Moreover, the mRNA levels of RORγt, IL-17A and IL-21, the protein expression of RORγt and the phosphorylation levels of STAT 3 in CM-L, CM-H and SRL groups were also lower than those in the control group （IL-17A mRNA： CM-L 0.81±0.05, CM-H 0.61±0.05, SRL 0.58±0.05, Control 1.01±0.11, t=4.81, 8.52, 8.89; IL-21 mRNA： CM-L 0.73±0.06, CM-H 0.49±0.03,SRL 0.59±0.03, Control 1.12±0.11, t=5.98, 9.22, 7.95, P〈0.01）. The mRNA level of IL-21 in the CM-H group was lower than that in the SRL group （P〈0.05）. ConcLusion In vitro, curcumin can inhibit the differentiaton of splenic CD4^＋CD25^- T cells into Thl7 cells in the setting of Thl7-polarization and inhibit the expression of IL-17A and IL-21 mRNA, which is associated with the inhibitive effect of curcumin on RORγt expression and STAT 3 phosphorylation.
出处
《中华风湿病学杂志》
CAS
CSCD
北大核心
2011年第7期476-480,共5页
Chinese Journal of Rheumatology
