TNF-α增强人脐带间充质干细胞条件培养基的体外造血支持功能

TNF-α enhances hematopoiesis-supportive effect of conditioned culture medium from human umbilical cord-derived MSCs in vitro

目的:研究肿瘤坏死因子α(tumor necrosis factorα,TNF-α)刺激后所得的脐带间充质干细胞条件培养基对脐血CD34+细胞在半固体培养基中集落形成个数及种类的影响。方法:将3～6代人脐带来源间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)以2×106接种到75cm2培养瓶中,其中刺激组加入TNF-α(10 g/L),48 h后收集上清作为条件培养基。Real-time PCR检测hUC-MSCs中各类造血因子mRNA的表达量。密度梯度离心法分离脐血单个核细胞,磁珠分选CD34+细胞,流式细胞术检测细胞纯度后分5组接种到6孔板内:TNF-α刺激hUC-MSC上清+不完全甲基纤维素培养基;hUC-MSC上清+不完全甲基纤维素培养基;TNF-α+DMEM/F12完全培养基+不完全甲基纤维素培养基;完全甲基纤维素培养基;DMEM/F12完全培养基+不完全甲基纤维素培养基。10 d后显微镜下计数各类集落形成单位(colony-forming unit,CFU)的数目,收集集落形成细胞,流式细胞术检测其表型特征。结果:(1)TNF-α刺激后hUC-MSCs中粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)和白细胞介素6(interleukin-6,IL-6)mRNA表达上调。(2)两种条件培养组均可见粒系CFU(granulocyte CFU,CFU-G)、巨噬系CFU(macrophage CFU,CFU-M)和粒巨噬系CFU(granulocyte-macrophage CFU,CFU-GM),但TNF-α刺激组CFU-G和CFU-M的数目约为未刺激组的1.5倍,CFU-GM约为未刺激组的2倍;阳性对照组中除粒系、巨噬系集落外还可见红系集落;而DMEM/F12完全培养基加或不加TNF-α组10 d后均未见集落形成。(3)流式细胞术检测TNF-α刺激组与未刺激组集落细胞表型CD14、CD45和CD11b,未见明显差异。结论:hUC-MSC上清作为条件培养基可在体外促进CD34+细胞分化增殖为髓系细胞,具有造血支持作用,TNF-α刺激后此作用增强。

AIM：To study the influence of tumor necrosis factor-alpha （TNF-α）-stimulated conditioned culture medium from human umbilical cord-derived mesenchymal stem cells （hUC-MSCs） on the colony-forming ability of umbilical cord blood CD34＋ cells in semisolid medium. METHODS：The hUC-MSCs were cultured in 75-cm2 culture flasks at a concentration of 2×106 cells per flask, with or without TNF-α （10 μg/L）, and their culture supernatants were harvested as the conditioned culture medium 48 h later. The hUC-MSCs were collected and their RNA was extracted. Real-time PCR was performed to detect the mRNA expression of hematopoietic factors. Umbilical cord blood mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation, and then CD34＋ cells were isolated using Human Cord Blood CD34 Positive Selection Kit. The CD34＋ cells were divided into the following five groups： TNF-α group （TNF-α-stimulated hUC-MSC culture supernatant added into incomplete methylcellulose medium）, control group （unstimulated hUC-MSC culture supernatant added into incomplete methylcellulose medium）, positive group （complete methylcellulose medium with recombinant human cytokines）, TNF-α＋DMEM/F12 group （TNF-α and DMEM/F12 medium with 10% FBS added into incomplete methycellulose medium） and DMEM/F12 group （DMEM/F12 medium with 10% FBS added into incomplete methycellulose medium）. Ten days later, the number of the colony-forming units （CFU） was counted, and the cells were collected to detect the surface markers by flow cytometry. RESULTS：（1） TNF-α stimulation significantly up-regulated the mRNA expression of granulocyte colony-stimulating factor （G-CSF） and interleukin-6 （IL-6） in hUC-MSCs. （2） Granulocyte CFU （CFU-G）, macrophage CFU （CFU-M） and granulocyte-macrophage CFU （CFU-GM） were observed in both TNF-α and control groups. The numbers of CFU-G and CFU-M in TNF-α group were 1.5 times as large as those in control group, and the number of CFU-GM in TNF-α group was even twice as large as that in control group. Granulocyte-erythroid-macrophage-megakaryocyte CFU （CFU-GEMM） and erythroid burst-forming units （BFU-E） were only observed in positive group, and no CFU was observed in TNF-α＋DMEM/F12 and DMEM/F12 groups. （3） Flow cytometry showed no differences of CD14, CD45 and CD11b expression on the colony-forming cells between TNF-α and control groups. CONCLUSION：TNF-α can enhance the hematopoiesis-supportive effect of conditioned culture medium from hUC-MSCs in vitro.