共培养系统中视网膜微血管周细胞经KDR/Flk-1途径对内皮细胞增生的影响(英文)  

Inhibition of proliferation of retinal microvas -cular endothelial cells by pericytes through down-regulating KDR/Flk-1 in a co-culture system

作　　者：王应利[1] 惠延年[1] 郭斌[1] 张晓光[1] 侯旭[1] 马吉献[1] 

机构地区：[1]第四军医大学西京医院眼科,中国陕西省西安市710032

出　　处：《国际眼科杂志》2006年第2期255-263,共9页International Eye Science

摘　　要：目的:采用细胞共培养模型研究缺氧诱导视网膜新生血管生成过程中周细胞对微血管内皮细胞增生的作用及其相关血管内皮细胞生长因子受体2(KDR/Flk-1)调节机制。方法:免疫磁珠法原代分离培养大鼠视网膜微血管内皮细胞和周细胞,分别采用抗VIII因子、CD31抗体以及抗血小板源性生长因子受体β、结蛋白抗体免疫细胞化学法鉴定内皮细胞和周细胞。通过Millicell小室建立周细胞和内皮细胞共培养模型,使用MTT法和流式细胞仪检测内皮细胞增殖数量及周期,观察缺氧和常氧下周细胞对血管内皮细胞增生的影响,并采用RT-PCR法检测内皮细胞KDR/Flk-1的mRNA水平变化,探讨相关调节机制。结果:经免疫细胞化学法鉴定,免疫磁珠法可获得高纯度的视网膜微血管内皮细胞和周细胞。MTT法显示,缺氧3～9d内皮细胞增生明显,6d增生达高峰(24.9%,P<0.01);共培养时内皮细胞的增生可被周细胞抑制。流式细胞仪检测发现,缺氧6dS期内皮细胞数量明显增加(43.9%,P<0.01);常氧(3.6%,P<0.05)或缺氧(15.1%,P<0.01)共培养时,周细胞均可抑制内皮细胞增生。缺氧下内皮细胞KDR/Flk-1的mRNA水平是常氧下的1.3倍;常氧(45.1%,P<0.05)和缺氧(27.7%,P<0.05)共培养下,周细胞均可下调内皮细胞KDR/Flk-1的mRNA表达。结论:缺氧和常氧共培养时周细胞均可抑制微血管内皮细胞增生,此抑制作用部分是通过下调内皮细胞KDR/Flk-1mRNA表达实现。AIM：To investigate the role of pericytes in growth of retinal microvascular endothelial cells with a co-culture system in order to understand some mechanism of angiogenesis in hypoxia induced retinal neovascular disorders. METHODS： Retinal microvascular endothelial cells （RMECs） were isolated by a modified protocol using CD31 coated Dynabeads, and identified by immunocytochemical staining with anti-Factor Ⅷ and CD31 antibodies. Rat retinal pericytes were isolated and characterized by immunofluorescent staining with PDGFR-β ; and desmin antibodies. Pericytes and RMECs were cultured in a contact co-culture system both under normoxia and hypoxia by Millicell chamber. RMECs proliferation was evaluated by MIT and cell cycle assay with flow cytometry. RT-PCR was used to detect the alteration of KDR/FIk-1 mRNA level in RMECs under normoxia or hypoxia in the co-culture system. RESULTS： Highly pure rat RMECs and pericytes were harvested with the modified isolating method. The two cell types were identified by positive Factor Ⅷ, CD31 and PDGFR-β, desmin cytochemical staining respectively. RMECs proliferated significantly under hypoxia from 3 to 9d with a maximal rate on day 6 （24.9%, P 〈 0.01） by MTT. In the co-culture system, the proliferation of RMECs was inhibited by pericytes. After 6d exposure to hypoxia,the fraction of S-phase RMECs number was greatly increased by 43.9% （P 〈 0.01）. In the co-culture system, RMECs proliferation was inhibited by pericytes through decreasing the fraction of S-phase cell number both under normoxia （3.6%, P〈0.05） and under hypoxia （15.1%,P〈0.01）. KDR/FIk-1 mRNA level in single cultured RMECs was shown to increase approximately 1.3-fold when exposed to hypoxia. Compared with single cultured RMECs, co-culture with pericytes could decrease KDR/FIk-1 mRNA by 45.1% （P〈0.05） and 27.7% （P 〈 0.05） under normoxia and hypoxia condition respectively. CONCLUSION： The present study demonstrated that pericytes could inhibit proliferation of RMEC

关 键 词：周细胞 视网膜微血管内皮细胞 共培养 增生 KDR/FLK-1 血管生成 缺氧 

分 类 号：R774.1[医药卫生—眼科] R331.32[医药卫生—临床医学]