脐带间充质干细胞经TLR4-NF-κB通路诱导产生regDC治疗急性肝衰竭

A study on the role of umbilical cord mesenchymal stem cells in the regulation of inflammatory environment through regulating the maturation and differentiation of dendritic cells in patients with acute hepatic failure

目的探讨脐带间充质干细胞(hU-MSC)对急性肝衰竭患者(FHF)单核细胞衍生的树突状细胞(DC)的免疫调节作用及炎性环境调节机制。方法选取5例急性肝衰竭患者外周血DC与hU-MSC进行共培养。检测DC成熟和活化水平、调节性DC(regDC)细胞比例,细胞上清细胞因子水平。蛋白质印迹检测TLR4、IKKα与NF-κB-p65蛋白表达变化,全转录组测序分析筛选新的治疗靶点。结果hU-MSC与DC共培养后,regDC增加,未成熟DC(imDC)表面共刺激分子MHC-II、CD80及CD86表达水平升高,共培养后IL-1α、IL-1β和IL-6因子表达均降低,IL-1α为(754.68±22.65)pg/mL比(76.67±11.25)pg/mL;IL-1β为(336.49±55.75)pg/mL比(85.33±12.58)pg/mL;IL-6为(38027.85±5231.14)pg/mL比12000.75±6793.69)pg/mL;抑炎因子IL-10表达上调,为(0.41±0.01)pg/mL比(0.61±0.01)pg/mL;差异均有统计学意义(P<0.05)。WB结果显示,hUC-MSC共培养后显著下调NF-κB-P65(0.777±0.01比0.55±0.02)与TLR4(1.12±0.01比0.54±0.01)蛋白表达,同时IKKα蛋白表达上调(0.12±0.01比0.88±0.01),NS398处理后在hUC-MSC作用基础上,下调NF-κB-p65与TLR4蛋白表达,蛋白表达量分别为0.14±0.01、0.12±0.01,均显著低于其他两组,而IKKα蛋白表达再次显著上调,蛋白表达量为1.18±0.01,高于其他两组,差异均有统计学意义(P<0.05)。全转录组测序分析结果显示,与非共培养组相比,共培养组有2127个基因上调表达,663个基因下调表达,差异表达明显的基因大多与炎症发生密切相关。结论在急性肝衰竭中,hU-MSC能够在体外诱导imDC分化生成CD11b high 1a low CD11c low regDCs,表现出免疫抑制作用,从而促进hU-MSC发挥抗炎作用,其可通过TLR4/NF-κB/COX-2途径抑制炎性级联反应的产生。

Objective To investigate the role of umbilical cord mesenchymal stem cells(hU-MSCs)in the regulation of inflammatory environment by regulating the maturation and differentiation of dendritic cells(DCs)in patients with acute hepatic failure(AHF).Methods DCs derive from five patients with AHF were co-cultured with hU-MSCs.The maturation and activation levels of DCs and the proportion of regulatory DC cells(regDCs)and the cytokine levels in the cell culture supernatants were measured with or without the administration of NS398,a selective cyclooxygenase-2(COX-2)inhibitor,to the co-culture system.The expression of Toll like receptor 4(TLR4),inhibitor of nuclear factor kappa-B kinase subunit alpha(IKKα)and nuclear factors-κB p65(NF-κB-p65)were then detected by Western blot.New therapeutic targets were screened by whole transcriptomic sequencing analysis on the DCs before and after co-culture with hU-MSCs.Results After co-cultured with hU-MSCs,regDCs increased.The expression levels of surface markers MHC-II,CD80 and CD86 on immature DCs(imDCs)increased.The levels of IL-1 and IL-6 decreased in cocultured DCs compared to DCs that cultured alone[IL-1α:(754.68±22.65)pg/ml vs(76.67±11.25)pg/mL;IL-1β:(336.49±55.75)pg/mL vs(85.33±12.58)pg/mL;IL-6:(38027.85±5231.14)pg/mL vs(12000.75±6793.69)pg/mL;P<0.0500)],whereas the level of IL-10 increased significantly[(0.41±0.01)pg/mL vs(0.61±0.01)pg/mL;P<0.05].After co-cultured with hU-MSCs,the down-regulation of NF-κB-p65[(0.777±0.01 vs 0.55±0.02;P<0.05)]and TLR4[(1.12±0.01 vs 0.54±0.01;P<0.05)]expression and up-regulation of IKKαexpression in DCs were significant[(0.12±0.01 vs 0.88±0.01;P<0.05)].After the administration of NS398 to DCs that cocultured with hU-MSCs,the expression of NF-κB-p65 and TLR4(0.14±0.01,0.12±0.01)in DCs were down-regulated,and IKKαwas significantly up-regulated(1.18±0.01).By transcriptomic sequencing analysis it was found that the expressions of 909 genes were up-regulated and 363 genes were down-regulated in DCs co-cultured with hU-MSCs when compared with those without.Most of the genes played important roles in inflammation.Conclusion hU-MSCs showed an immuno-suppressive effect by inducing the differentiation of imDCs derived from AHF to CD11bhigh1alowCD11clowregDCs in vitro,and played an anti-inflammatory role by suppressing the inflammatory cascade reaction through TLR4/NF-κB/COX-2 pathway.