内皮祖细胞条件培养基对间充质干细胞向内皮细胞分化的影响

Effect of conditioned medium of endothelial progenitor cells on differentiation of mesenchymal stem cells into endothelial cells

目的探讨体外小鼠内皮祖细胞(endothelial progenitor cells,EPCs)条件培养基(CM)对间充质干细胞(mesenchymal stem cells,MSCs)向内皮细胞(endothelial cells,ECs)分化的影响。方法全骨髓贴壁法和差速贴壁法提取培养MSCs和EPCs,流式细胞术检测MSCs和EPCs表面标记物,双荧光染色和体外成管实验对EPCs进行功能鉴定。将MSCs分为0%EPCs-CM组(对照组)、25%EPCs-CM组和50%EPCs-CM组。qRT-PCR检测各组CD31、vWF、eNOS的mRNA表达水平。体外成管实验检测各组细胞的体外成管能力;免疫荧光检测各组MSCs CD31、CD34表达情况;对各组上清中的NO进行检测。结果流式细胞术结果显示,第3代MSCs高表达Sca-1,低表达CD34、CD11b;EPCs高表达CD34、CD133和VEGFR2,可吞噬Dil-AC-LDL和结合UEA-1。在基质胶上可形成管腔样结构。qRT-PCR显示3周时50%EPCs-CM组CD31、vWF、eNOS表达(2.28±0.25, 1.76±0.71,8.27±1.65),较对照组显著升高( P <0.05)。50%EPCs-CM组MSCs可体外形成管腔样结构;免疫荧光显示50%EPCs-CM组MSCs CD31和CD34的表达明显高于对照组。对照组细胞与实验组细胞上清液中NO的含量(8.81+3.41,20.93±9.47)μmol/L,差异显著( t =3.96, P <0.05)。结论 EPCs-CM能提高MSCs体外成管能力,并促进MSCs向ECs分化。

Objective To investigate the effects of Endothelial progenitor cells (EPCs) conditioned medium (CM) on the differentiation of mesenchymal stem cells (MSCs) into endothelial cells (ECs) in vitro. Methods MSCs and EPCs were extracted by whole bone marrow adherence method and differential adherence method. The surface markers of MSCs and EPCs were detected by flow cytometry. The functions of EPCs were identified by double fluorescent staining and tube formation experiments in vitro. MSCs were divided into 0% EPCs-CM group (control group), 25% EPCs-CM group and 50% EPCs-CM group. qRT-PCR was used to detect the mRNA expression levels of CD31, vWF and eNOS in each group. In vitro tube formation experiment was used to to test the in vitro tube-forming ability of each group of cells;. Immunofluorescence was used to detect the expression of CD31 and CD34 in each group;Detection of NO in each group of supernatants. Results Flow cytometry showed that the third generation MSCs expressed high levels of Sca-1 and low expression of CD34 and CD11b. EPCs highly expressed CD34, CD133 and VEGFR2, which could engulf Dil-AC-LDL and bind UEA-1. Tumor-like structures were formed on Matrigel. qRT-PCR showed that CD31, vWF, and eNOS were expressed in 50% EPCs-CM group at 3 weeks (2.28±0.25, 1.76±0.71, 8.27±1.65), which was significantly higher than the control group.( P <0.05). 50% EPCs-CM group MSCs can form a lumen-like structure in vitro;immunofluorescence showed that the expression of CD31 and CD34 in MSCs in 50% EPCs-CM group was significantly higher than that in the control group. The content of NO in the cell culture supernatant of the control group and the experimental group (8.81 3.41, 20.93±9.47)μmol/L, the difference was significant ( P <0.05). Conclusion EPCs-CM can increase the ability of MSCs to form in vitro and promote the differentiation of MSCs into ECs.