摘要
目的探讨过表达Jagged1对老龄大鼠来源内皮祖细胞增殖和迁移能力的影响。方法 PBS冲洗1～2月龄和19～26月龄SD大鼠股骨和胫骨骨髓,Ficoll密度梯度离心法分离单个核细胞,应用含10%FBS的DMEM/F12培养基差速贴壁法进行体外培养,以Dil-ac-LDL与FITC-UEA-1荧光双染和vWF免疫组织化学染色进行鉴定。实验分为四组:对照组(未进行基因转染的老龄大鼠内皮祖细胞组)、PIRES2-EGFP转染组、PIRES2-EGFP-Jagged1转染组和未转染的年轻大鼠来源内皮祖细胞组。荧光显微镜下计数GFP表达细胞数并计算转染效率;免疫荧光、RT-PCR和Western blot检测Jagged1 mRNA和蛋白表达;Transwell培养和MTT法分别检测细胞迁移和增殖能力。结果免疫组织化学、RT-PCR和Western blot检测均显示转染后Jagged1显著增高,PIRES2-EGFP-Jagged1转染组mRNA和蛋白表达均较对照组显著增强(P<0.01);与对照组相比,过表达Jagged1显著增强老龄大鼠来源内皮祖细胞迁移和增殖能力(P<0.05或P<0.01)。结论过表达Jagged1增强老龄大鼠来源内皮祖细胞增殖和迁移能力。
Aim To investigate the effects of Jaggedl over-expression on aged rat-derived endothelial progenitor cells migration and proliferation. Methods Mononuclear cells were obtained from bone marrow of young （ 1 to 2 month-old） and aged （19 to 26 month-old） Sprague-Dawley rats by Ficoll density gradient centrifugation and cultured with medium DMEM/F12, 48 h later, the suspending cells were translocated to be cultured in new flasks coated with fibronectin, the secondary attached cells were used to perform the further experiments. EPCs were characterized as double posi- tive for Dil-ac-LDL uptake and lectin binding and cells were further identified by vWF expression. The experiments were divided into four groups： control group （ aged rat-derived EPCs without gene transfection）, PIRES2-EGFP transfection group, PIRES2-EGFP-Jaggedl transfection group and young rat-derived EPCs group in which transfection was also not performed. The GFP expression cell number was acquired through fluorescence microscope and transfection efficiency was calculated. Immunofluorescence, RT-PCR and Western blot were used to detect the mRNA and protein expression respectively. Cell migration and proliferation were tested by Transwell culture and MTI＂ assay respectively. Results Immunofluorescence showed that gene transfection markedly promoted Jaggedl expression; RT-PCR and Western blot demon- strated that Jaggedl mRNA and protein expression significantly increased in aged rat-derived EPCs （P〈0.01） compared with control; Over-expression of Jaggedl markedly enhanced aged rat-derived EPCs migration and proliferation compared with control group （P〈0.05 or P〈0.01 respectively）. Conclusion Over-expression of Jaggedl accelerates aged rat-derived EPCs migration and proliferation.
出处
《中国动脉硬化杂志》
CAS
CSCD
北大核心
2013年第5期391-396,共6页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金(81170316)资助
973基础研究规划项目(2012CB518100)资助
