Abstract

目的探讨miR-155在低氧条件下对间充质干细胞(MSC)免疫因子表达的调控以及作用机制。方法通过Lipofectamine™ 2000转染试剂将miR-155模拟物转染至MSC中,构建miR-155高表达的MSC细胞。在水合氯化钴(CoCl2·6H2O)模拟的化学低氧环境中,用脂多糖(LPS)刺激MSC的免疫作用,采用实时荧光定量PCR方法鉴定其转染效果,并检测IL-6、IL-8、诱导型一氧化氮合酶(iNOS)、TGF-β、低氧诱导因子1α(HIF-1α)mRNA的表达。用流式细胞术检测细胞表面抗原,ELISA法检测上清中IL-6、IL-8、TGF-β及SDF-1α的表达水平,Western blot法检测iNOS、HIF-1α蛋白的表达。结果较阴性对照组,miR-155转染组的IL-6、IL-8表达上调(24.201±1.536对1.802±0.058,P<0.01;24.406±4.611对7.407±1.553,P<0.01),iNOS mRNA表达下调(0.151±0.035对32.925±1.632,P<0.01)。同时,HIF-1α在低氧环境下高表达(45.093±3.371对2.210±0.498,P<0.01),且低氧miR-155转染组较低氧对照组上调更为显著(102.965±4.449对45.093±3.371,P<0.01)。低氧miR-155转染组IL-6、IL-8上调较miR-155转染组更加显著(65.670±10.613对24.201±1.536,P<0.01; 35.537±2.285对24.406±4.611,P< 0.01);miR-155下调iNOS表达(0.235±0.003对0.612±0.043,P<0.01),低氧条件下更为显著(0.087± 0.002对0.235±0.003,P<0.01)。低miR-155组SDF-1α、TGF-β mRNA表达均上调(5.690±1.655对0.841±0.194,P<0.01; 6.982±1.353对0.632±0.184,P<0.01),上清中细胞因子SDF-1α表达水平为24.609±2.584对25.359±2.455(P=0.760);、TGF-β表达水平为0.568±0.019对0.345±0.037(P=0.002)。结论低氧条件下,通过上调HIF-1α的表达,从而下调iNOS基因及蛋白,由此促进miR-155正向调控MSC免疫因子的表达。

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