Jagged1过表达促进老龄大鼠来源的内皮祖细胞向成熟内皮细胞分化

Overexpression of Jagged1 promotes aged rat-derived endothelial progenitor cells differentiating into mature endothelial cells

目的:探讨上调Jagged1表达对内皮培养条件下老龄大鼠来源的内皮祖细胞(EPC)向内皮细胞分化的影响。方法:脱臼处死1～2月龄和19～26月龄SD大鼠,PBS冲洗股骨和胫骨骨髓,Ficoll密度梯度离心分离单个核细胞,应用含10%FBS的DMEM/F12培养基以差速贴壁法进行体外培养,DiI-ac-LDL与FITC-UEA-1荧光双染进行EPC特性鉴定。实验分为4组:对照组、PIRES2-EGFP转染组、PIRES2-EGFP-Jagged1转染组和未转染的年轻大鼠来源EPC组。荧光显微镜下计数GFP阳性细胞数并计算转染效率;免疫荧光、RT-PCR和Western blotting检测Jagged1 mRNA和蛋白、von Willebrand因子(vWF)及血管内皮生长因子激酶插入区受体(KDR)mRNA表达,体外血管生成实验检测EPC的血管形成能力。结果:转染后Jagged1在EGFP-Jagged1组表达较对照组显著增强(P<0.01);Jagged1过表达显著促进老龄大鼠EPC vWF与KDR mRNA表达(P<0.01)和体外血管生成能力(P<0.01);vWF与KDR mRNA表达以及体外血管生成能力在Jagged1转染组与年轻大鼠EPC组间未见有显著差别。结论:Jagged1过表达促进内皮培养条件下老龄大鼠来源EPC向成熟内皮细胞分化。

AIM： To investigate the effect of Jaggedl overexpression on endothelial cell-directional differentia- tion of aged rat-derived endothelial progenitor cells （EPC）. METHODS： Mononuclear cells were obtained from bone mar- row of young （ 1 to 2 months old） or aged （ 19 to 26 months old） Sprague-Dawley rats and cultured in DMEM/F12 medium supplemented with 10% FBS. EPC were characterized as double positive for DiI-ac-LDL uptake and lectin binding. The experiments were divided into control group, PIRES2-EGFP transfection group, PIRES2-EGFP-Jaggedl transfection group and young rat-derived EPC group in which transfection was not performed. The GFP expression positive cell number was ac- quired by fluorescence microscopy and the transfection efficiency was calculated , RT-PCR and West- em blotting were used to detect the mRNA and protein expression. In vitro vasculogenesis kit was used to test the tube for- mation ability of EPC. RESULTS ： EGFP-Jaggedl transfection induced a significant increase in the expression of Jaggedl in aged rat-derived EPC （P 〈 0.01 ）. Compared with the control, Jaggedl overexpression markedly enhanced the mRNA expres- sion of yon Willebrand factor （vWF） and kinase insert domain receptor （KDR） of vascular endothelial grouth factor vWF in aged rat-derived EPC （P 〈0. 01 ） and improved the EPC-related tube formation （P 〈 0. 01 ）. No significant difference be- tween Jaggedl transfection and young rat-derived EPC groups in vWF and KDR mRNA expression and the ability of tube for- mation was found. CONCLUSION： In endothelial cell-conditioning medium, Jagged1 overexpression significantly pro-motes aged rat-derived EPC differentiation into mature endothelial cells.