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目的观察小鼠肾小管上皮细胞(TEC)在白介素4(IL-4)及CD40激活状态下钙调神经磷酸酶(CaN)活性,并进一步探讨其与趋化因子RANTES分泌的关系。方法取Ⅱ级6～7周的雌性DBA小鼠,分离肾小管上皮细胞进行培养、刺激。采用RT-PCR检测RANTESmRNA表达;ELISA检测培养上清中RANTES含量;流式细胞仪检测CD40表达。结果(1)常规培养TEC可表达一定量的CD40;用IL-4刺激TEC24h,细胞表面CD40平均荧光强度(MFI)显著高于对照组(7.92±0.56vs4.33±0.30,P<0.001)。(2)常规培养TEC中有一定量的CaN活化(8.98±0.56)nmol/mg.pro;用IL-4、CD40抗体(mAb)及IL-4+CD40mAb刺激细胞,3组CaN活性均显著高于对照组(P<0.05)。(3)常规培养TEC仅分泌极少量的RANTES;在IL-4刺激或CD40mAb激活后TECRANTES蛋白分泌增高,分别为(43.61±13.73)pg/ml和(73.77±4.28)pg/ml,显著高于对照组(14.78±2.20)pg/ml(P<0.001)。(4)常规培养TEC微量表达RANTESmRNA;用IL-4、CD40mAb及IL-4+CD40mAb刺激细胞24h,各刺激组RANTESmRNA表达显著高于对照组(P<0.05)。(5)在IL-4、CD40mAb及IL-4+CD40mAb刺激下,FK506对TEC分泌RANTES蛋白及mRNA有显著抑制作用(P均<0.05)。结论Th2细胞因子IL-4及CD40-CD40配体(CD40L)共刺激信号可通过活化TECCaN,调控RANTES基因表达及分泌、
Objective To observe the activity of calcineurin (CaN) in mouse renal tubular epithelial cells (TEC) under interleukin-4 (IL-4) and CD40-activated states, and to further explore its relationship with the chemokine RANTES secretion. Methods Female DBA mice of grade Ⅱ 6 to 7 weeks were harvested and cultured for renal tubular epithelial cells for stimulation. The expression of RANTES mRNA was detected by RT-PCR, the content of RANTES in culture supernatant was detected by ELISA, and the expression of CD40 was detected by flow cytometry. Results: (1) TEC could express a certain amount of CD40 in normal culture; meanwhile, the average fluorescence intensity of CD40 on the surface of cells was significantly higher than that of control (7.92 ± 0.56 vs 4.33 ± 0.30, P <0.001). (2) There was a certain amount of CaN activation (8.98 ± 0.56) nmol / mg.pro in conventional culture TEC; the activity of CaN was significantly higher than that of IL-4, CD40 (mAb) and IL-4 + Control group (P <0.05). (3) Conventional culture of TEC secreted only a very small amount of RANTES; the increase of TECRANTES protein secretion was (43.61 ± 13.73) pg / ml and (73.77 ± 4.28) pg / ml respectively after IL-4 stimulation or CD40 mAb activation, Control group (14.78 ± 2.20) pg / ml (P <0.001). (4) RANTES mRNA was routinely cultured in TEC. RANTES mRNA expression in each stimulated group was significantly higher than that in control group (P <0.05) after stimulation with IL-4, CD40mAb and IL-4 + CD40mAb for 24h. (5) Under the stimulation of IL-4, CD40mAb and IL-4 + CD40mAb, FK506 significantly inhibited the secretion of RANTES protein and mRNA by TEC (all P <0.05). Conclusions The costimulatory signals of Th2 cytokines IL-4 and CD40-CD40 ligand (CD40L) can regulate the expression and secretion of RANTES gene by activating TECCaN,