目的 研究局部乳酸堆积对人脐带来源的间充质干细胞（hUC-MSCs）功能的影响。方法 采集足月婴儿脐带，分离hUC-MSCs，流式细胞术进行hUC-MSCs表面标志物检测，分别应用骨茜素红S、油红O、阿尔新蓝进行成骨、成脂、成软骨诱导分化染色。采用CCK-8法检测乳酸（0、1、3、5、10、15 mmol·L^-1）处理24、48、72 h后对hUC-MSCs增殖能力的影响；乳酸各浓度处理hUC-MSCs 72 h，流式细胞术检测增殖指数（PI），流式细胞术检测hUC-MSCs表面标志物，ELISA试剂盒法检测hUC-MSCs的血管内皮生长因子（VEGF）分泌功能；取健康志愿者外周血，用Ficoll淋巴细胞分离液分离外周血单个核细胞（PBMC），hUC-MSCs经0、10、20 mmol·L^-1的乳酸处理72 h后，与PBMC共培养72 h，ELISA法检测上清液中炎症因子肿瘤坏死因子-α（TNF-α）、前列腺素E₂（PGE₂）水平，流式细胞术检测PBMC的CD³⁺/ki6⁷⁺水平。结果 hUC-MSCs高表达CD73、CD90、CD105，表达率≥95%； CD34、CD45、CD19、HLA-DR表达率≤2%；成脂分化14 d后有明显脂肪滴形成；成骨分化21 d后可见明显的矿化结节；软骨方向分化14 d后有软骨球形成。当乳酸作用hUC-MSCs 24、48 h时，随着乳酸浓度的升高hUC-MSCs的增殖能力增强（P<0.05、0.01、0.001），但当作用时间达到72 h，乳酸由促增殖作用转为抑增殖作用（P<0.01、0.001），PI减小；不同浓度的乳酸处理hUC-MSCs，其表面分子表达没有明显改变；与0 mol· L-1组比较，随着乳酸浓度的提高，hUC-MSCs上清中VEGF的分泌量逐渐减少（P<0.001）。PBMC的CD^3＋百分率为66.5%，符合行业标准。与PBMC组比较，PBMC＋hUC-MSCs组上清TNF-α水平显著降低、PEG₂水平显著增加（P<0.001）；与PBMC＋hUC-MSCs组比较，10、20 mmol·L^-1的乳酸可以显著抑制hUC-MSCs抗炎因子PEG₂的分泌、而对促炎因子TNF-α的分泌显著促进（P<0.001）。与PBMC组比较，hUC-MSCs可以显著抑制CD3阳性细胞的增殖（P<0.001）；与PBMC＋hUC-MSCs组比较，10、20 mmol·L^-1的乳酸预处理可以显著减弱hUC-MSCs对CD3阳性细胞的抑制作用（P<0.001）。结论 慢性皮肤伤口微环境中的高浓度乳酸长时间作用，可能通过影响hUC-MSCs的增殖和炎症调节能力，阻碍伤口的愈合进程。

Objective Study the effect of local lactate accumulation on the function of human umbilical cord derived mesenchymal stem cells (hUC-MSCs). Methods The umbilical cord of term infants was collected, hUC-MSCs were separated, and the surface markers of hUC-MSCs were detected by flow cytometry. Bone alizarin red S, Oil Red O, and alcian blue were respectively used for osteogenesis, adipogenesis, and chondrogenesis induced differentiation staining. The CCK-8 method was used to detect the effect of lactic acid (0, 1, 3, 5, 10, 15 mmol·L^-1) treatment on the proliferation ability of hUC-MSCs after 24, 48, and 72 hours. The proliferation index (PI), surface markers of hUC-MSCs and the secretion of vascular endothelial growth factor (VEGF) of hUCMSCs were detected by flow cytometry and ELISA respectively after treatment with different concentrations of lactic acid for 72 h. Peripheral blood mononuclear cell (PBMCs) were isolated from peripheral blood of healthy volunteers with Ficoll lymphocyte isolate. After being treated with 0, 10, and 20 mmol·L^-1 lactic acid for 72 h, hUC-MSCs were co-cultured with PBMCs for 72 h. The inflammatory factor, tumor necrosis factor-α (TNF-α) in the supernatant was detected by ELISA. The levels of prostaglandin E₂ (PGE₂) and CD³⁺/ki6⁷⁺ of PBMCs were detected by flow cytometry. Results hUC-MSCs were highly expressed in CD73, CD90, and CD105, with an expression rate ≥ 95%, expression rate of CD34, CD45, CD19, and HLA-DR ≤ 2%. After 14 days of adipogenic differentiation, obvious fat droplets formed. After 21 days of osteogenic differentiation, obvious mineralized nodules could be observed. After 14 days of differentiation in the cartilage direction, chondrocytes formed. When lactic acid acts on hUC-MSCs for 24 and 48 hours, the proliferation ability of hUC-MSCs became stronger with the increase of lactic acid concentration (P < 0.05, 0.01, 0.001). However, when the action time reaches 72 hours, lactic acid shifted from a pro-proliferation effect to an inhibitory effect (P < 0.01, 0.001), and PI decreased. The surface molecule expression of hUC-MSCs treated with different concentrations of lactic acid did not significantly change. Compared with the 0 mol·L^-1 group, with the increase of lactate concentration, the secretion of VEGF in the supernatant of hUC-MSCs gradually decreased (P < 0.001). The CD³⁺ percentage of PBMC was 66.5%, which meets industry standards. Compared with the PBMC group, the supernatant level of TNF-α in PBMC + hUC-MSCs group significantly decreased and the PEG₂ level significantly increased (P < 0.001). Compared with the PBMC + hUC-MSCs group, 10 and 20 mmol·L^-1 lactate significantly inhibited the secretion of anti-inflammatory factor PEG₂ in hUC-MSCs, while inhibiting the proinflammatory factor TNF-α secretion was significantly promoted (P < 0.001). Compared with the PBMC group, hUC-MSCs significantly inhibited the proliferation of CD3 positive cells (P < 0.001). Compared with the PBMC + hUC-MSCs group, lactate pretreatment with 10 and 20 mmol·L^-1 significantly inhibited the inhibitory effect of hUC-MSCs on CD3 positive cells (P < 0.001). Conclusion The long-term effects of high concentrations of lactic acid in the microenvironment of chronic skin wounds may hinder the healing process of wounds by affecting the proliferation and inflammatory regulation ability of hUC-MSCs.

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