树突状细胞CD80和CD86表达及胞外白细胞介素12分泌与不同剂量青藤碱干预的影响

背景:青藤碱是从抗风湿中药青风藤中分离提取的单体生物碱,具有抗炎、镇痛、免疫抑制等药理作用,该药用于治疗类风湿性关节炎等风湿性疾病具有较明确的疗效,在临床上得到了广泛的应用。树突状细胞是目前所知的机体内功能最强的抗原提呈细胞。目的:观察不同剂量青藤碱对树突状细胞CD80和CD86表达及胞外白细胞介素12分泌的影响。设计:重复测量实验。单位:广东医学院药理教研室。材料:实验于2004-09/2005-05在解放军第三军医大学全军免疫学研究所完成。健康志愿者外周血由西南医院血库提供。青藤碱由湖南正清药业有限公司提供。RPMI1640培养基购于Hyclone公司,FITC标记的单克隆抗体CD80、CD86、鼠免疫球蛋白亚型IgG1均购于eBioscience公司,重组人粒细胞巨噬细胞集落刺激因子,重组人白细胞介素4,肿瘤坏死因子α购于PE公司。人白细胞介素12ELISA试剂盒购于法国Diaclone公司。方法:取健康志愿者外周血稀释,培养,按重组人粒细胞巨噬细胞集落刺激因子1×106U/L、重组人白细胞介素45×105U/L终浓度加入细胞因子,培养7d后可获得树突状细胞。①将常规培养7d后的树突状细胞调整细胞浓度为2×108L-1,加入6孔培养板,分别加入青藤碱使其终浓度为3,10,30mg/L以及肿瘤坏死因子α使其终浓度为10μg/L,同时设对照组,于第9天分别收集悬浮细胞,每管取2×105细胞,用PBS洗涤2次,加入20μLFITC标记的CD80或CD86,对照管则加入鼠免疫球蛋白亚型IgG1,4℃避光孵育30min,充分洗涤后,10g/L多聚甲醛固定,将细胞悬液在流式细胞仪上进行分析。②将常规培养7d后的树突状细胞调整细胞浓度为2×108L-1,加入6孔培养版,分别加入青藤碱使其终浓度为3,10,30mg/L以及肿瘤坏死因子α使其终浓度为10μg/L,同时设对照组,继续培养48h后,收集上清进行白细胞介素12测定。白细胞介素12的检测采用夹心ELISA法检测胞外的白细胞介素12分泌量,按试剂盒说明操作。主要观察指标:①不同剂量青藤碱对树突状细胞表面CD80及CD86表达的影响。②不同剂量青藤碱对树突状细胞分泌白细胞介素12的影响。结果:①树突状细胞表面CD80及CD86表达:不同剂量青藤碱组与对照组树突状细胞表面CD80分子阳性表达率及平均荧光强度差异无统计学意义(P>0.05),各组细胞表面CD86分子阳性表达率及平均荧光强度差异亦无统计学意义(P>0.05)。②各组树突状细胞分泌白细胞介素12水平检测结果:由ELISA测量值可见,青藤碱小、中、大剂量组树突状细胞白细胞介素12水平分别为(863.1±33.7),(668.8±31.9),(512.6±29.6)ng/L,高于对照组(922.2±36.6),P<0.05-0.01],随青藤碱剂量增大,抑制树突状细胞分泌白细胞介素12水平呈依赖性减少。结论:青藤碱可剂量依赖性地抑制树突状细胞分泌白细胞介素12,而对树突状细胞表面CD80和CD83表达无明显影响;青藤碱的抗类风湿作用可能与其抑制树突状细胞分泌白细胞介素12有关。

Effect of different doses of sinomenine on CD80 and CD86 expressions and extracellular interleukin-12 secretion in dendritic cells

BACKGROUND: Sinomenine, an alkaloid monomer extracted from Chinese medicinal herb sinomenium acutum,possesses potent anti-inflammatory, analgesic and immunoinhibitory pharmacological activities. Sinomenine has certain therapeutic effect on rheumatoid arthritis and has been widely applied in the clinic. Dendritic cell (DC) is the known antigen presenting cell with the strongest functions in the body.OBJECTIVE: To observe the effect of different doses of sinomenine on CD80 and CD 86 expression and extracellular interleukin-12 secretion in DCs.DESIGN: A controlled repeated measuring study based on the cells.SETTTNG: Department of Pharmacology, Guangdong Medical College.MATERIALS: The experiment was carried out in the Institute of Immunology, Third Military Medical University of Chinese PLA between September 2004 and May 2005. The peripheral blood of healthy donors was supplied by the blood bank of the Southwest Hospital. RPMI1640 medium was purchased from Hyclone Company. Fluorescein isothiocyanate(FITC)-conjugated mouse monoclonal antibodies against CD80 and CD86 and FITC-conjugated mouse IgG1 were purchased from Bioscience Company. Recombinant human interleukin-4 (rhlL-4), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and tumor necrosis factor-α (TNF-α) were purchased from PharMingen Company. Sinomenine was supplied by Zhengqing Pharmacy Company in Hunan. ELISA kit specific for IL-12 was purchased from Diaclone Company.METHODS: Peripheral blood was collected from healthy volunteers. 1 ×106 U/L recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and 5×105 U/Lrecombinant human interleukin-4 (rhlL-4)were added to the peripheral blood. After 7 days, DCs were harvested. ①The DCs were counted and cultured at 2×108 L 1 in fresh medium supplemented with TNF-α and different doses of sinomenine(3, 10, 30 mg/L). After 48 hours, 2×105 cells were collected and washed twice with phosphate buffer solution (PBS). Then the expressions of CD80 and CD86 were analyzed using 20 μL FITC-conjugated mouse monoclone antibodies against CD80 or CD86.FITC-conjugated mouse IgG1 was used as control at 4 ℃ away from light for 30 minutes. After staining, cells were washed twice with PBS and fixed in 10 g/L paraformaldehyde. The samples were analyzed on a flow cytometry. ②The DCs were counted and cultured at 2 ×108 L-1 in fresh medium supplemented with TNF-α and different doses of sinomenine (3, 10, 30 mg/L). After 48 hours, the supernatants were collected and the concentration of interleukin-12 (IL-12) was determined in a sandwich ELISA using kit specific for IL-12 according to the manufacture's instruction.MAIN OUTCOME MEASURES: ①Effect of different doses of sinomenine on the expressions of CD80 and CD86 on DCs. ②Effect of different doses of sinomenine on the level of IL-12 secreted by DCs.RESULTS : ① Expressions of CD80 and CD86 on DCs: There were no significant differences in the percentage of cells positive and fluorescence intensity for CD80 and CD86 on DC between different doses of sinomenine groups and control groups (P ＞ 0.05). ② IL-12 level secreted by DCs: From ELISA measurement, we found that IL-12 level of the sinomenine 3 mg/L, 10 mg/L and 30 mg/L groups was (863.1±33.7), (668.8±31.9), (512.6±29.6) ng/L, respectively,which was higher than that of control group (922.2±36.6),P ＜ 0.05-0.01]. The level of IL-12 in the supernatants of DCs co-cultured with sinomenine was significantly lower compared with control, which was dose-dependent.CONCLUSTON: Sinomenine can inhibit the level of IL-12 secreted by DCs in dose-dependent manner, but do not influence the expressions of CD80 and CD86 in DCs; The therapeutic effect of sinomenine on rheumatoid arthritis may be related to its inhibition to secretion of IL-12 in DCs.