年轻与老年小鼠血管内皮细胞microRNA的差异表达  

Differential expressions of microRNA between young and senescent endothelial cells

作　　者：张俊斌[1] 朱小南 崔进[1] 陈鹏[1] 王深明[1] 王劲松[1] 

机构地区：[1]中山大学附属第一医院血管外科,广州510080 [2]中山大学基础医学院

出　　处：《中华医学杂志》2012年第31期2205-2209,共5页National Medical Journal of China

基　　金：国家自然科学基金（81070257）;教育部留学回国启动基金（教外司留（2010））609号）

摘　　要：目的观察年轻与老年小鼠血管内皮细胞（EC）microRNA（miRNA）表达差异及意义。方法原代分离并培养雄性C57BL／6J小鼠年轻和老年EC，免疫组化Ⅷ因子染色对EC进行鉴定，体外比较年轻和老年EC增殖速度，采用miRNA基因芯片分析年轻与老年EC内miRNA表达差异谱，荧光定量PCR（qRT．PCR）进行验证。免疫印迹（Westernblot）检测EC内皮细胞型一氧化氮合成酶（eNOS）表达。结果免疫组化Ⅷ因子染色证实原代分离的细胞为EC；在0．5％、1％、5％、10％、20％血清浓度下，MrITr比色法测得年轻EC的吸光度值（A值）分别为0．260±0．006、0．350±0．011、0．404±0．027、0．519±O．034、0．590±0．040；老年EC的A值分别为0．360±O．011、0．480±0．019、0．532±0．025、0．641±0．036、0．710±0．038，发现老年EC增殖速度比年轻EC快（P〈0．05）；排除性别差异，经miRNA基因芯片，qRT—PCR证实miR一135a、miR-182、miR-96、miR-31、miR-126-3p和miR·362-5p在年轻EC表达上调（表达差异倍数分别为34．54±1．8、5．44±1、12．8±1、3．1±0．6和14．5±1．1），miR-335—3p和miR-335—5p在老年EC表达上调，表达差异倍数分别为3．3±0．6和2．1±0．3；其中，老年EC内miR-335-3p的上调，年轻EC内miR-135a，miR-96上调可能与老年EC内eNOS表达降低相关。结论随年龄增长，EC的miRNA发生变化，调节EC内的基因表达，与衰老相关。Objective To explore the differential expressions of microRNA （miRNA） between young and senescent endothelial cells. Methods Young and senescent aorta endothelial cells （EC） were isolated and cultured in young and old male C57BL/6J mice. Immunostaining of Wl~ factor was performed to identify the endothelial cells. The method of diphenyl tetrazolium bromide （MTT） was employed to compare the cell growth. Microarray was used to detect the differential expression of microRNA between young and senescent endothelial cells and the microarray results were confirmed by real-time polymerase chain reaction （PCR）. The expression of endothelial nitric oxide synthase （eNOS） was detected by Western blot. Results Primarily cultured endothelial cells were confirmed by the VI~ immunostaining factor. Senescent ECs grew more rapidly than young ECs in lower serum ex vivo. Excluding gender difference, miR-135a, miR-182, miR-96, miR-31, miR-126-3p and miR-362-5p were up-regulated over 2 folds in young ECs, and miR-335-3p and miR-335-Sp up-regulated over 2 folds in senescent ECs by miRNA microarray and RT- PCR. The up-regulation of miR335-3p in old ECs and the up-regulation of miR-135a, miR-96 in the young ECs might contribute to a lower expression of eNOS in senescent ECs. Conclusion The expression of miRNAs changes with advancing age and may result in differential expressions of downstream genes.

关 键 词：老年 血管内皮细胞 MICRORNA 内皮细胞型一氧化氮合成酶 

分 类 号：R363[医药卫生—病理学]