[关键词]
[摘要]
目的 研究低氧环境对人脐带间充质干细胞(hUCMSCs)增殖及生物学特性的影响。方法 分别在5% O2和21% O2环境下培养hUCMSCs,使用群体倍增时间(PDT)评价hUCMSCs的增殖情况;流式细胞术检测细胞表面标志CD73、CD90、CD105、CD34、CD45、HLA-DR的表达;培养基诱导分化后,茜素红-S对含钙骨细胞染色检测成骨诱导分化,油红O对脂滴染色检测成脂诱导分化,阿尔新蓝8GX对蛋白多糖检测软骨诱导分化;羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)染色法检测hUCMSCs对植物血凝素P(PHA-P)刺激的外周血单个核细胞(peripheral blood mononuclear cell,PBMC)的增殖抑制作用,并应用流式细胞术检测对CD8+T细胞的抑制作用;实时荧光定量PCR(qRT-PCR)法检测低氧诱导因子1α(HIF-1α)、血管内皮生长因子(VEGF)、肝细胞生长因子(HGF)、胰岛素样生长因子2(IGF-2)、转化生长因子β(TGF-β)、碱性成纤维细胞生长因子(bFGF)、基质细胞衍生生长因子1(SDF-1)、神经生长因子(NGF)mRNA表达;试剂盒法检测细胞培养上清中IGF-2浓度。结果 21% O2和5% O2环境培养的hUCMSCs表面标志CD73、CD90、CD105的表达均为阳性(>95%),CD34、CD45、HLA-DR的表达均为阴性(<2%),均具有成骨、成脂、成软骨三系分化的能力;5% O2组的PDT均显著小于21% O2组(P<0.05);PBMC经PHA-P刺激后观察到细胞增殖聚集,与hUCMSCs共培养后几乎没有聚集出现,与PBMC+PHA-P组比较,PBMC+PHA-P+hUCMSCs(21%或5% O2)组子代细胞明显减少,PBMC+PHA-P+hUCMSCs(5% O2)组PBMC抑制率为(61.44±0.92)%,与PBMC+PHA-P+hUCMSCs (21% O2)抑制率(60.48±4.00)%相当,无统计学差异,且两组hUCMSCs对CD8+T细胞的抑制作用无统计学差异。与21% O2组比较,5% O2组hUCMSCs HIF-1α、IGF-2、SDF-1、HGF、VEGF、bFGF、NGF mRNA表达水平显著升高(P<0.05、0.001),TGF-β无明显变化; 5% O2组hUCMSCs培养上清IGF-2水平显著高于21% O2组(P<0.001)。结论 5% O2环境可使hUCMSCs的增殖能力和相关生长因子的表达增强,尤其是IGF-2,但依然保持着与21% O2环境培养的hUCMSCs相似的表型、分化能力以及淋巴细胞增殖抑制能力。
[Key word]
[Abstract]
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.
[中图分类号]
R329.2
[基金项目]
天津市科技计划创新平台专项(18PTSYJC00070);天津市博士后择优资助计划项目(TJQYBSH2018030)