缺氧环境下核转录因子Sp1在体外培养的人视网膜色素上皮细胞中的表达  

Expression of nuclear factor Sp1 in cultured human retinal pigment epithelial cells in vitro under hypoxic conditions

作　　者：吕建萌[1] 王雨生[1] 侯慧媛[1] 李夏[1] 张朝霞[1] 蔡岩[1] 

机构地区：[1]第四军医大学西京医院眼科,全军眼科研究所,陕西省西安市710032

出　　处：《眼科新进展》2009年第9期649-652,658,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:30672291);德国洪堡基金会(Alexander von Humboldt Foundation)仪器设备捐赠基金(V-8151/02085)资助~~

摘　　要：目的研究缺氧对体外培养的人视网膜色素上皮(retinal pigment epithelium,RPE)细胞中核转录因子Sp1表达的影响。方法将体外培养的人RPE细胞置于含终浓度为200μmol.L-1CoCl2的培养液中培养以建立RPE细胞化学缺氧模型。在缺氧后即刻(即常氧状态下)、2h、4h、8h、12h和24h终止缺氧,用细胞免疫荧光和Western blotting分别检测RPE细胞中核转录因子Sp1蛋白表达部位和量的动态变化;RT-PCR检测RPE细胞中Sp1mRNA表达量的动态变化。结果缺氧后即刻(即常氧状态下)Sp1蛋白荧光主要分布于RPE细胞质内;随缺氧时间延长,细胞质内绿色荧光强度减弱,细胞核荧光强度明显增强,至缺氧后12h,细胞核荧光强度达到最高水平。随着缺氧时间延长,RPE细胞内Sp1mRNA和蛋白水平逐渐增高,至12h达峰值,24h表达已降低,但仍高于常氧状态。结论缺氧早期可使RPE细胞内Sp1的表达水平增高,并促使其蛋白从细胞质向细胞核内转移,提示Sp1可能在视网膜脉络膜缺血缺氧性疾病的发生中起着一定作用。Objective To explore the influence of hypoxia on the expression of nuclear factor Sp1 in the cultured human retinal pigment epithelial（RPE） cells in vitro. Methods To set up the hypoxic model, human RPE ceils in vitro were cultured in the medium containing 200 μmol ·L^-1 CoCl2. At 0 hour （under normoxic condition）, 2 hours,4 hours,8 hours,12 hours and 24 hours after hypoxia,the location and dynamic changes of nuclear factor Sp1 in RPE cells were observed with cell immunofluorescence method and Western blotting,respectively, and the dynamic expression of Sp1 mRNA was detected by RT-PCR. Results The fluorescence of Sp1 protein mainly distributed in the cytoplasm of cultured RPE ceils under normoxic conditions. With the time prolonged under hypoxic condition, green fluorescence intensity decreased within the cytoplasm, while the fluorescence intensity in the nucleus increased and to the highest level at 12 hours after hypoxia. The expression of Sp1 mRNA and protein were enhanced gradually with the time prolonged under hypoxic conditions ,peaked at 12 hours and decreased at 24 hours in the cultured RPE ceils but still higher than that under normoxic condition. Conclusion Hypoxia at early stage can increase Sp1 expression in cultured human RPE cells,and promote Sp1 protein transferring from the cytoplasm to the nucleus,which implies that Sp1 may play a role in the developing of hypoxic-ischemic chorioretinal diseases.

关 键 词：视网膜色素上皮 缺氧 核转录因子SP1 

分 类 号：R774.1[医药卫生—眼科]