不同年龄段大鼠血清影响内皮祖细胞向成熟内皮诱导分化  

Serum from Age-Related Rats Affects the Ability of Endothelial Progenitor Cells Differentiating into Mature Endothelial Cells

作　　者：朱光旭[1] 黄岚[1] 武晓静[1] 宋明宝[1] 晋军[1] 赵刚[1] 于世勇[1] 赵晓辉[1] 方玉强[1] 

机构地区：[1]第三军医大学新桥医院全军心血管内科研究所,重庆市400037

出　　处：《中国动脉硬化杂志》2007年第9期647-652,共6页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金(30400517和30470729);军队医学杰出人才基金(CSTC2005bb5304)

摘　　要：目的观察不同年龄段大鼠血清对骨髓内皮祖细胞向成熟内皮细胞诱导分化的影响。方法无菌取1-2月龄、19-26月龄Sprague-Dawley大鼠股骨和胫骨,获取骨髓单个核细胞,用含20%胎牛血清的DMEM/F12培养基培养,经差速贴壁法对二次贴壁细胞在纤维连接蛋白包被的培养板或培养瓶进行体外培养,以DiI-ac-LDL、FITC-UEA-1双荧光染色和FITC-CD31染色进行鉴定;收集制备相应年龄段（1-2月龄、19-26月龄）大鼠血清,将内皮祖细胞分为老年大鼠内皮祖细胞＋老年大鼠血清组、老年大鼠内皮祖细胞＋年轻大鼠血清组、年轻大鼠内皮祖细胞＋老年大鼠血清组、年轻大鼠内皮祖细胞＋年轻大鼠血清组。体外培养的内皮祖细胞经含20%不同年龄段大鼠血清的内皮条件培养基诱导培养后,激光共聚焦显微镜检测DiI-acLDLF、ITC-UEA-1双染色阳性率,免疫组织化学检测假血友病因子表达、逆转录聚合酶链反应分别检测内皮一氧化氮合酶、血管内皮生长因子受体2表达,体外血管生成实验观察内皮祖细胞参与管形生成能力。结果年轻大鼠血清显著提高老年大鼠内皮祖细胞在内皮条件培养基诱导培养后的DiI-acLDLF、ITC-UEA-1双染色阳性率（P〈0.05）,而老年大鼠血清显著降低年轻大鼠内皮祖细胞双染阳性率（P〈0.05）;年轻大鼠血清明显促进老年大鼠内皮祖细胞在内皮条件培养基诱导培养后的血管性血友病因子、内皮型一氧化氮合酶（P〈0.01）及血管内皮生长因子受体2表达（P〈0.05）;年轻大鼠血清显著减少内皮条件培养基诱导培养后未参与血管生成的老年大鼠内皮祖细胞数（P〈0.05）。结论年轻大鼠血清可显著增强老年大鼠内皮祖细胞向内皮细胞诱导分化的能力,而老年大鼠血清可部分抑制年轻大鼠内皮祖细胞向成熟内皮细胞诱导分化;年轻大鼠血清中可能存在激发衰老个体内皮祖细胞活力的系统性血To investigate the effects of serum from rats of different age.son endothelial progenitor cells （EPC） ability to differentiate into mature endothelial cells （EC）. Methods Mononuclear cells were obtained from young （1 to 2 month-old） and old （ 19 to 26 month-old） Sprague-Dawley rats bone marrow by FicoU density gradient centrifugation and cultured with medium DMEM/F12 in cultttre dishes, 48 h later, the suspending cells were translocated to be cultured in other new flasks coated with fd3ronectin, the secondary attached cells were characterized as adherent cells which were double positive for DiI-acLDL uptake and lectin binding by direct fluorescent staining demonstrated under a laser scanning confocal microscope; serums from young and aged rats were collected and used to culture EPC. The experiments were divided into four groups： aged rat EPC ＋ aged rat serum, aged rat EPC ＋ young rat serum, young rat EPC ＋ aged rat serum and young rat EPC ＋ young rat serum. After incubation with DMEM/F12 containing 20% serum from young or aged rats （no fetal blood senml was added）, in vitro cultttred EPC double positive for DiI-ac-LDL uptake and lectin binding were tested by laser scanning confocal microscopy, also were documented by demonstrating the expression of von WiUebrand factor （vWF） with inmlunofluorescence, vascular endothelial growth factor receptor-2 （KDR） and endothelial nitric oxide synthase （eNOS） nRNA expression with reverse transcription-polymerase chainreaction （RT-PCR） methods; in vitro vasculogenesis kit was used to test the participation of EPC to tube formation. Results Young rat sermn significantly increases percentage of double-positive cells for Dil-ac-LDL and lectin binding in aged rat EPC cultured in EC conditioned-medium whereas aged rat serum obviously decreases such percentage in young rat EPC; young rat serun also markedly promotes EC conditioned-medium to induce vWF and eNOS （P 〈 0.01） and KDR （P 〈 0.05） expression in aged rats EPC; young r

关 键 词：内科学 内皮祖细胞 诱导分化 一氧化氮合酶 血管内皮生长因子受体2 假血友病因子 10(r7—3949(20071 15—09—0647—06 

分 类 号：R5[医药卫生—内科学]