来氟米特抑制狼疮肾炎患者树突状细胞成熟的实验研究

Leflunomide Inhibit Peripheral Blood Dendritic Cells Maturation of Lupus Nephritis

目的:观察来氟米特活性代谢产物A771726处理前后狼疮肾炎(lupus nephritis,LN)患者树突状细胞(DC)表面标志及功能的改变,探索来氟米特治疗LN的新途径。方法:分离LN患者外周血单个核细胞,用粒细胞巨噬细胞集落刺激因子(granulocyte macrophage colony stimulating factor,GM-CSF)、白细胞介素-4(interleukin-4,IL-4)等细胞因子诱导DC成熟,来氟米特组再加入A771726培养。培养第9天收集DC细胞,流式细胞仪检测CD80、CD86和HLA-DR的表达。MTT法检测DC刺激淋巴细胞增殖的能力,ELISA法检测混和淋巴细胞反应培养上清IL-10和IFN-γ水平。结果:A771726处理后的DC表达CD80、CD86和HLA-DR百分数较对照组均明显降低[(62.80±1.67)vs(78.35±4.50),(64.50±13.06)vs(84.91±9.80),(71.44±11.56)vs(91.51±7.63)],P均<0.01;A771726处理后的DC与T细胞混合培养,其刺激T细胞增殖相应的OD值较对照组明显降低[DC∶TC=1∶10时,(0.295±0.069)vs(0.468±0.100);DC∶TC=1∶50时,(0.196±0.044)vs(0.391±0.023)],P均<0.01。其混合培养的上清液中IL-10水平较无A771726处理的DC与T细胞的混合培养上清液明显降低[(205.0±35.8)pg/mlvs(443.6±93.2)pg/ml,P<0.01],而IFN-γ两者间无统计学差异[(89.3±11.9)pg/mlvs(99.9±15.7)pg/ml,P>0.05]。结论:A771726在体外可抑制LN患者外周血DC的成熟,未成熟DC能抑制T细胞增殖及T细胞向Th2细胞转化。

Objective：To detect the effects of leflunomide active metabolites （A771726） on dendritic cells （DCs） phenotype and function in lupus nephritis （LN） patients. Methods： The mononuclear cells were isolated from peripheral blood in LN patients and developed into DCs by adding GM - CSF and IL - 4. In leflunomide group, A771726 was then added after adding the above cytokines, DCs were harvested after 9 days culture. CD80 ,CD86 and HLA- DR surface markers on DCs were detected by flow cytometry. MTT assay detect mixed lymphocyte reaction. IL- 10 and IFN - γin the supernatant of MLR were detected by ELISA. Results：The percentage of CD80 ,CD86 and HLA- DR expression on the DCs treated with A771726 were lower than them on the DCs without A771726 - treated[ （62.80 ± 1.67） vs （78.35 ± 4.50）, （64.50 ± 13.06） vs （84.91 ± 9.80）, （71.44 ± 11.56） vs （91.51 ± 7.63）], all P〈 0.01. The OD value related with T lymphocytes proliferation were lower in the MLR with the DCs treated with A771726 than in the MLR with the DCs without A771726- treated （at DC：TC= 1 ： 10, （0. 295 ± 0. 069） vs （0. 468 ± 0. 100） ;at DC： TC = 1：50, （0. 196 ± 0. 044） vs （0. 391 ± 0. 023）], both P〈 0.01. IL- 10 level in the MLR supernatant of the group treated with A771726 was lower than in the control group [ （205.0 ± 35.8）pg/ml vs （443.6 ± 93.2）pg/ml, P〈 0.01 ]. but IFN - γ level in both MLR supernatant have no significant difference [ （89.3 ± 11. 9） pg/ml vs （99.9 ± 15.7） pg/ml, P 〉 0.05 ） ]. Conclusion： A771726 can inhibit DCs maturation, and then the immature DCs can also inhibit T cells proliferation and refrain T cells from dividing into Th2 subtype in LN patients.