目的 研究低氧環(huán)境對人臍帶間充質(zhì)干細(xì)胞（hUCMSCs）增殖及生物學(xué)特性的影響。方法 分別在5% O₂和21% O₂環(huán)境下培養(yǎng)hUCMSCs，使用群體倍增時間（PDT）評價hUCMSCs的增殖情況；流式細(xì)胞術(shù)檢測細(xì)胞表面標(biāo)志CD73、CD90、CD105、CD34、CD45、HLA-DR的表達(dá)；培養(yǎng)基誘導(dǎo)分化后，茜素紅-S對含鈣骨細(xì)胞染色檢測成骨誘導(dǎo)分化，油紅O對脂滴染色檢測成脂誘導(dǎo)分化，阿爾新藍(lán)8GX對蛋白多糖檢測軟骨誘導(dǎo)分化；羧基熒光素二醋酸鹽琥珀酰亞胺酯（CFSE）染色法檢測hUCMSCs對植物血凝素P（PHA-P）刺激的外周血單個核細(xì)胞（peripheral blood mononuclear cell，PBMC）的增殖抑制作用，并應(yīng)用流式細(xì)胞術(shù)檢測對CD8+T細(xì)胞的抑制作用；實(shí)時熒光定量PCR（qRT-PCR）法檢測低氧誘導(dǎo)因子1α（HIF-1α）、血管內(nèi)皮生長因子（VEGF）、肝細(xì)胞生長因子（HGF）、胰島素樣生長因子2（IGF-2）、轉(zhuǎn)化生長因子β（TGF-β）、堿性成纖維細(xì)胞生長因子（bFGF）、基質(zhì)細(xì)胞衍生生長因子1（SDF-1）、神經(jīng)生長因子（NGF）mRNA表達(dá)；試劑盒法檢測細(xì)胞培養(yǎng)上清中IGF-2濃度。結(jié)果 21% O₂和5% O₂環(huán)境培養(yǎng)的hUCMSCs表面標(biāo)志CD73、CD90、CD105的表達(dá)均為陽性（＞95%），CD34、CD45、HLA-DR的表達(dá)均為陰性（＜2%），均具有成骨、成脂、成軟骨三系分化的能力；5% O₂組的PDT均顯著小于21% O₂組（P＜0.05）；PBMC經(jīng)PHA-P刺激后觀察到細(xì)胞增殖聚集，與hUCMSCs共培養(yǎng)后幾乎沒有聚集出現(xiàn)，與PBMC＋PHA-P組比較，PBMC＋PHA-P＋hUCMSCs（21%或5% O₂）組子代細(xì)胞明顯減少，PBMC＋PHA-P＋hUCMSCs（5% O₂）組PBMC抑制率為（61.44±0.92）%，與PBMC＋PHA-P＋hUCMSCs （21% O₂）抑制率（60.48±4.00）%相當(dāng)，無統(tǒng)計(jì)學(xué)差異，且兩組hUCMSCs對CD8+T細(xì)胞的抑制作用無統(tǒng)計(jì)學(xué)差異。與21% O₂組比較，5% O₂組hUCMSCs HIF-1α、IGF-2、SDF-1、HGF、VEGF、bFGF、NGF mRNA表達(dá)水平顯著升高（P＜0.05、0.001），TGF-β無明顯變化； 5% O₂組hUCMSCs培養(yǎng)上清IGF-2水平顯著高于21% O₂組（P＜0.001）。結(jié)論 5% O₂環(huán)境可使hUCMSCs的增殖能力和相關(guān)生長因子的表達(dá)增強(qiáng)，尤其是IGF-2，但依然保持著與21% O₂環(huán)境培養(yǎng)的hUCMSCs相似的表型、分化能力以及淋巴細(xì)胞增殖抑制能力。

Objective To study the effects of hypoxia on proliferation and growth factor secretion of human umbilical cord mesenchymal stem cells (hUCMSCs). Methods hUCMSCs were cultured at 5% O₂ and 21% O₂, respectively, and the proliferation of hUCMSCs was evaluated by population doubling time (PDT). The expression of CD73, CD90, CD105, CD34, CD45 and HLADR were detected by flow cytometry. After medium induced differentiation, alizarin red-S was used to detect osteoblast induced differentiation, oil red O was used to detect lipid-induced differentiation, alsinblue 8GX was used to detect cartilage induced differentiation. The inhibitory effect of hUCMSCs on peripheral blood mononuclear cells (PBMC) stimulated by PHA-P was determined by CFSE staining. Flow cytometry was used to detect the inhibition of CD8⁺ T cells. Real-time fluorescence quantitative PCR (QRT-PCR) was used to detect HIF-1 α, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulinlike growth factor 2 (IGF-2), transforming growth factor β (TGF- β), basic fibroblast growth factor (bFGF) and stromal cells Expression of SDF-1 and NGF mRNA; The concentration of IGF-2 in cell culture supernatant was detected by kit method. Results The expression of CD73, CD90, and CD105 was positive (>95%), and the expression of CD34, CD45, and HLA-DR was negative (<2%). HUCMSCs cultured in 21% O₂ and 5% O₂ environment had the ability of osteogenic, lipid-forming, and chondrogenic differentiation. The PDT in 5% O₂ group was significantly lower than that in 21% O₂ group (P<0.05). Cell proliferation and aggregation were observed after PHA-P stimulation of PBMC, and almost no aggregation was observed after co-culture with hUCMSCs. Compared with PBMC+PHA-P group, the progeny cells of PBMC+PHA-P+hUCMSCs (21% or 5% O₂) were significantly reduced. The PBMC inhibition rate of PBMC+PHA-P+hUCMSCs (5% O₂) group was (61.44±0.92) %, which was similar to that of PBMC+PHA-P+hUCMSCs (21% O₂) (60.48±4.00) % , with no statistical difference. There was no statistical difference in the inhibitory effect of hUCMSCs on CD8⁺ T cells between the two groups. Compared with 21% O₂ group, mRNA expression levels of hUCMSCs HIF-1α, IGF-2, SDF-1, HGF, VEGF, bFGF and NGF in 5% O₂ group were significantly increased (P<0.05, 0.001), while TGF-β had no significant change. The IGF-2 level in the supernatant of hUCMSCs cultured in 5% O₂ group was significantly higher than that in 21% O₂ group (P<0.001). Conclusion The expression of related growth factors, especially IGF- 2, and proliferation ability of hUCMSCs are enhanced when hUCMSCs are cultured in 5% O₂, but the phenotype, differentiation ability and lymphocytes proliferation inhibition ability of hUCMSCs are still similar to those cultured in 21% O₂.

R329.2

天津市科技計(jì)劃創(chuàng)新平臺專項(xiàng)(18PTSYJC00070);天津市博士后擇優(yōu)資助計(jì)劃項(xiàng)目(TJQYBSH2018030)