目的 研究局部乳酸堆積對(duì)人臍帶來(lái)源的間充質(zhì)干細(xì)胞（hUC-MSCs）功能的影響。方法 采集足月嬰兒臍帶，分離hUC-MSCs，流式細(xì)胞術(shù)進(jìn)行hUC-MSCs表面標(biāo)志物檢測(cè)，分別應(yīng)用骨茜素紅S、油紅O、阿爾新藍(lán)進(jìn)行成骨、成脂、成軟骨誘導(dǎo)分化染色。采用CCK-8法檢測(cè)乳酸（0、1、3、5、10、15 mmol·L^-1）處理24、48、72 h后對(duì)hUC-MSCs增殖能力的影響；乳酸各濃度處理hUC-MSCs 72 h，流式細(xì)胞術(shù)檢測(cè)增殖指數(shù)（PI），流式細(xì)胞術(shù)檢測(cè)hUC-MSCs表面標(biāo)志物，ELISA試劑盒法檢測(cè)hUC-MSCs的血管內(nèi)皮生長(zhǎng)因子（VEGF）分泌功能；取健康志愿者外周血，用Ficoll淋巴細(xì)胞分離液分離外周血單個(gè)核細(xì)胞（PBMC），hUC-MSCs經(jīng)0、10、20 mmol·L^-1的乳酸處理72 h后，與PBMC共培養(yǎng)72 h，ELISA法檢測(cè)上清液中炎癥因子腫瘤壞死因子-α（TNF-α）、前列腺素E₂（PGE₂）水平，流式細(xì)胞術(shù)檢測(cè)PBMC的CD³⁺/ki6⁷⁺水平。結(jié)果 hUC-MSCs高表達(dá)CD73、CD90、CD105，表達(dá)率≥95%； CD34、CD45、CD19、HLA-DR表達(dá)率≤2%；成脂分化14 d后有明顯脂肪滴形成；成骨分化21 d后可見(jiàn)明顯的礦化結(jié)節(jié)；軟骨方向分化14 d后有軟骨球形成。當(dāng)乳酸作用hUC-MSCs 24、48 h時(shí)，隨著乳酸濃度的升高h(yuǎn)UC-MSCs的增殖能力增強(qiáng)（P<0.05、0.01、0.001），但當(dāng)作用時(shí)間達(dá)到72 h，乳酸由促增殖作用轉(zhuǎn)為抑增殖作用（P<0.01、0.001），PI減?。徊煌瑵舛鹊娜樗崽幚韍UC-MSCs，其表面分子表達(dá)沒(méi)有明顯改變；與0 mol· L-1組比較，隨著乳酸濃度的提高，hUC-MSCs上清中VEGF的分泌量逐漸減少（P<0.001）。PBMC的CD^3＋百分率為66.5%，符合行業(yè)標(biāo)準(zhǔn)。與PBMC組比較，PBMC＋hUC-MSCs組上清TNF-α水平顯著降低、PEG₂水平顯著增加（P<0.001）；與PBMC＋hUC-MSCs組比較，10、20 mmol·L^-1的乳酸可以顯著抑制hUC-MSCs抗炎因子PEG₂的分泌、而對(duì)促炎因子TNF-α的分泌顯著促進(jìn)（P<0.001）。與PBMC組比較，hUC-MSCs可以顯著抑制CD3陽(yáng)性細(xì)胞的增殖（P<0.001）；與PBMC＋hUC-MSCs組比較，10、20 mmol·L^-1的乳酸預(yù)處理可以顯著減弱hUC-MSCs對(duì)CD3陽(yáng)性細(xì)胞的抑制作用（P<0.001）。結(jié)論 慢性皮膚傷口微環(huán)境中的高濃度乳酸長(zhǎng)時(shí)間作用，可能通過(guò)影響hUC-MSCs的增殖和炎癥調(diào)節(jié)能力，阻礙傷口的愈合進(jìn)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.

R965