1,25-二羟维生素D_3对脐血单核细胞来源树突状细胞分化、成熟及凋亡的影响

Effects of 1α,25-dihydroxyvitamin D_3 on differentiation,maturation and apoptosis of cord blood monocyte-derived dendritic cells

目的探讨1,25-二羟维生素D3[1,25(OH)2D3]对脐血单核细胞来源树突状细胞(DCs)分化、成熟及凋亡的影响。方法体外分离获得脐血单核细胞,经重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF)、重组人白介素-4(rhIL-4)和重组人肿瘤坏死因子-α(rhTNF-α)诱导分化为未成熟树突状细胞(iDCs)以及成熟树突状细胞(mDCs),用1,25(OH)2D3(10 nmol/L)干预(干预组)。采用流式细胞仪检测单核细胞来源DCs分化、成熟各阶段细胞表面分子的表达;Real-Time PCR方法检测1,25(OH)2D3对DCs分化成熟过程中维生素D受体(VDR)和1α-羟化酶(CYP27B1)mRNA表达的影响;Annexin V-FITC和PI染色流式细胞术检测DCs的凋亡情况。另设不使用1,25(OH)2D3干预者为对照组。结果①干预组iDCs表面CD14分子表达显著高于对照组(40.05%vs 5.14%,P<0.001),而CD1a和CD11c分子表达则显著低于对照组(12.73%vs 30.07%,P<0.01;91.27%vs 95.94%,P<0.05),mDCs表面CD80、CD86和HLA-DR分子表达均显著低于对照组(3.52%vs 17.75%,P<0.01;51.10%vs 69.76%,P<0.01;69.38%vs 92.35%,P<0.01)。②随着DCs的分化成熟,iDCs和mDCs的CYP27B1表达均显著高于单核细胞(P=0.005,P=0.002),而且mDCs CYP27B1的表达显著高于iDCs(P=0.01)。iDCs和mDCs的VDR表达显著低于单核细胞(P=0.01,P=0.003),但iDCs和mDCs的VDR表达差异无统计学意义(P=0.43)。③干预组iDCs的CYP27B1表达显著高于对照组(P<0.01),而VDR的表达显著低于对照组(P<0.01),但1,25(OH)2D3对mDCs的CYP27B1和VDR表达无显著影响(P>0.05)。④FITC和PI双染后,流式细胞术检测显示:rhTNF-α刺激48 h后,干预组DCs的早期凋亡率显著高于对照组(20.8%vs 9.23%,P<0.01);rhTNF-α刺激72 h后,干预组DCs的晚期凋亡率显著高于对照组(19.39%vs 7.27%,P<0.01)。结论 1,25(OH)2D3抑制了脐血单核细胞来源DCs的分化和成熟,促进脐血单核细胞来源DCs的凋亡。

Objective To investigate the effects of let, 25-dihydroxyvitamin D3 [ 1,25 （OH）2 D3] on the differentiation, maturation and apoptosis of cord blood monocyte-derived dendritic cells （ DCs）. Methods Cord blood monocytes were isolated from human umbilical cord, and were cultured in vitro in the presence of recombinant human granulocyte/ macrophage colony-stimulating factor （rhGM-CSF）, recombinant human interleukin-4 （rhlL-4） and recombinant human tumor necrosis factor-α （rhTNF-α） to obtain the immature DCs （iDCs） and mature DCs （ mDCs）, with addition of 1,25 （OH） 2D3 or not （treatment group or control group）. The differentiation and maturation surface markers on DCs were determined by immunofluorescence staining and flow cytometry. The expression of vitamin D receptor （VDR） mRNA and 1-α-hydroxylase （CYP27B1） mRNA was detected by Real-Time PCR. The apoptosis of DCs was measured using Annexin V-FITC and PI staining by flow cytometry. Results ①Compared with control group, the expression of CD14 on iDCs was significantly higher （40.05% vs 5. 14%, P 〈0. 001）, the expression of CDla and CDIIc on iDCs was significantly lower （12.73% vs30.07%, P〈0.01;91.27% vs 95.94%,P〈0.05）, and the expression of CD80, CD86 and HLA-DR on mDCs was significantly lower in treatment group （3.52% vs 17.75%, P 〈0.01;51. 10% vs 69.76%, P 〈0.01;69.38% vs92.35%, P 〈0.01）. ②With the differentiatiation and maturation of DCs, the expression of CYP27B1 in iDCs or mDC was significantly higher than that in monocytes （ P = 0. 005, P = 0. 002）, and the expression of CYP27B1 in mDCs was significantly higher than that in iDCs （P = 0.01）. The expression of VDR in iDCs or mDCs was significantly lower than that in monocytes （P = 0.01, P = 0. 003）, while there was no significant difference in the expression of VDR between iDCs and mDCs （P = 0.43）. ③The expression of CYP27B1 in iDCs in treatment group was significantly higher than that in control group （P 〈 0.01）, and the expression of VDR in iDCs in treatment group was significantly lower than that in control group （ P 〈 O. 01）, while 1,25 （OH） 2D3 did not have effect on the expression of CYP27B1 and VDR in mDCs （P 〉 0.05）. ④The early apoptosis rate of mDCs stimulated by rhTNF-α for 48 h in treatment group was signifieantly higher than that in control group （20. 8% vs 9.23%, P 〈0.01）, and the late apoptosis rate of mDCs stimulated by rhTNF-α for 72 h was significantly higher than that in control group （ 19.39% vs 7.27%, P 〈 O. 01）. Conclusion 1,25 （OH） 2 D3 inhibits the differentiation and maturation of DCs derived from eord blood monocytes, and promotes the apoptosis of DCs derived from cord blood monocytes.