周细胞对缺氧诱导下视网膜微血管内皮细胞增生的影响

目的探讨视网膜微血管内皮细胞（RMECs）与共培养的周细胞（PCs）及PCs条件培养液（PCM）对常氧和缺氧条件下RMECs增生的影响。方法传代培养大鼠RMECs和PCs,采用兔抗鼠Ⅷ因子多克隆抗体和小鼠抗大鼠CD31单克隆抗体免疫组织化学法鉴定RMECs,PCs采用小鼠抗大鼠desmin多克隆抗体和兔抗鼠抗血小板衍生生长因子受体-β（PDGFR-β）进行免疫荧光双标鉴定。Millicell小室建立PCs和RMECs共培养系统,PCs培养近融合期后制备PCM,向共培养系统或PCM中加入终浓度为200μmol/L的CoCl2诱导形成细胞化学缺氧模型,观察常氧和缺氧条件下PCs和PCM对RMECs增生的影响。使用MTT法、3H-TdR掺入法检测RMECs在不同培养条件下的增生数量,并用流式细胞仪检测RMECs在不同细胞周期的细胞率。结果近融合期的RMECs呈类纺锤状、单层生长,可见明显的接触抑制,Ⅷ因子和CD31均阳性表达。PCs形状不规则,细胞重叠生长无接触抑制,PDGFR-β和desmin抗体双标阳性。缺氧3～9d缺氧RMECs单独培养组较常氧RMECs单独培养组各时间点的RMECs均明显增生,缺氧6d时增生达高峰（P〈0.01）,细胞生长抑制率（GIR）为-24.9%。缺氧共培养组较缺氧RMECs单独培养组RMECs增生下降（P〈0.05）。缺氧3、6、9d时常氧共培养组较常氧RMECs单独培养组RMECs数目均明显下降（P〈0.05,P〈0.01,P〈0.01）。缺氧6d时S期RMECs数量明显增加,占各期细胞总数的（43.9±0.8）%;常氧或缺氧共培养组,PCs均可抑制RMECs的增生,S期细胞分别占各期细胞总数的（3.6±0.1）%、（15.1±0.9）%（P〈0.05,P〈0.01）。常氧PCM组RMECs的吸光度（A）值和3H-TdR掺入量4～24h均低于对照组（P〈0.05）。结论缺氧和常氧条件下RMECs和PCs共培养时PCs均可抑制RMECs的增生,常氧条件下PCM可抑制RMECs的增生。

Effects of pericytes on hypoxia-induced proliferation in retinal microvascular endothelial cells

Background Retinal neovascularization is common pathological procedure in widespread retinal diseases such as diabetic retinopathy and retinal vein occlusion.Recruitment and coverage of pericytes in microvessels are key processes in normal vascular development,maturation and maintenance.However,the effect of pericyte on proliferation of retinal endothelial cells is still unclear,especially in the hypoxia condition.ObjectivePresent study was to investigate the role of pericyte in growth of retinal microvascular endothelial cells with co-culture system or with pericyte conditioned medium under the hypoxia condition.MethodsRetinal microvascular endothelial cells（RMECs）were isolated according to modified protocol and identified by immunocytochemistry using anti-factor Ⅷ and CD31 antibodies.Rat retinal pericytes were isolated and identified by immunofluorescent staining with PDGFR-β and desmin antibodies.Co-culture system with RMECs and pericytes or pericyte conditioned medium were created respectively.The hypoxia cell models were established by adding 200μmol/L CoCl2 into co-culture medium and pericyte conditioned medium.Pericytes and RMECs were cultured in contact co-culture system by Millicell chambers under the normoxia and hypoxia conditions separately.Proliferation of RMECs in different culture conditions was evaluated by MTT,3H-thymidine incorporation assay.Cell cycle of RMECs was assessed with flow cytometry.ResultsRMECs formed a confluent monolayer and grow in a contact-inhibited way.The positive response for anti-factor Ⅷ and CD31 antibodies were seen in cultured RMECs.The pericytes with non-contact-inhibited growth presented with the irregular shape and fluorescence staining for both PDGFR-β and desmin.RMECs proliferated significantly under the hypoxia condition from day 3 to day 9 and reached maximum rate on day 6（P0.01）with the growth inhibition ratio of-24.9%.Under the hypoxia condition,the proliferation of RMECs was inhibited in co-culture system by pericytes in comparison with the normoxia culture condition（P0.05）.Under the normoxia condition,RMECs were less in co-culture group than in RMECs group from day 3 to day 9（P0.05,P0.01）.After 6days exposed to hypoxia,the numbers of S phase RMECs increased dramatically to 43.9%.In co-culture system,RMECs proliferation was inhibited by pericytes through decreasing the S phase cell number both under the normoxia（3.6%,P0.05）and hypoxia conditions（15.1%,P0.01）.Pericyte conditioned medium exhibited inhibitory effect on the proliferation of RMECs under the normoxia condition but not hypoxia condition from 4hours to 24hours after experiment（P0.05）.ConclusionPresent study demonstrated that co-cultured pericytes inhibit the growth of RMECs in both normoxia and hypoxia environment;while pericyte conditioned medium inhibit RMECs only in the normoxia environment.