曲安奈德对缺氧条件下恒河猴视网膜血管内皮细胞增殖的抑制作用

目的:观察曲安奈德对正常和缺氧条件下体外培养恒河猴视网膜血管内皮细胞增殖的抑制作用。方法:将曲安奈德加入到培养的恒河猴视网膜血管内皮细胞(RF/6A)中,用MTT法测定曲安奈德对RF/6A细胞活性的影响,应用流式细胞仪观察曲安奈德对正常和缺氧条件下细胞增殖与凋亡的影响。结果:缺氧条件下可使内皮细胞周期中s期细胞相对减少,G2-M期细胞比例增加,曲安奈德对恒河猴视网膜血管内皮细胞周期有明显影响并可诱导内皮细胞凋亡。,可使正常及缺氧条件下内皮细胞周期中s期细胞相对增加,G2-M期细胞比例减少,说明曲安奈德可阻滞内皮细胞由s期进入G2-M期,从而降低其有丝分裂能力。结论:缺氧条件可促进视网膜内皮细胞的增殖;曲安奈德对正常及缺氧条件下内皮细胞的增殖有抑制作用并可诱导内皮细胞凋亡。     

PI3K抑制剂对体外培养的眼底血管内皮细胞的作用

目的 观察PI3K激酶抑制剂LY294002对体外培养的视网膜脉络膜血管内皮细胞增生和迁移抑制以及诱导凋亡的作用。方法 四唑盐（MTT）比色法检查对照组和LY294002干预组干预72h后RF／6A细胞增生的抑制，Transwell分析法检查不同浓度的LY294002对VEGF诱导的RF／6A细胞迁移的抑制效果，流式细胞仪检测细胞的凋亡。结果 LY294002以浓度依赖性方式抑制VEGF诱导的RF／6A细胞增生和迁移，各个LY294002干预组与对照组相比，增生和迁移能力降低，差异有统计学意义（P〈0．05），流式细胞仪分析结果显示LY294002可以诱导细胞的凋亡。结论 LY294002可抑制体外培养的RF／6A细胞的生长和迁移，并诱导细胞的凋亡，有望为临床上新生血管性疾病提供新的治疗方法。     

重组canstatin蛋白抑制体外视网膜微血管内皮细胞的迁移和增殖

目的 观察重组血管能抑素(canstatin)蛋白对体外培养的恒河猴视网膜微血管内皮细胞(RF/6A)迁移、增殖及磷酸化细胞外调节蛋白激酶(phospho-extracelluar regulated protein kinases,pERK)、磷酸化Akt表达的影响,初步探讨重组canstatin蛋白抑制视网膜新生血管的作用机制.方法 体外培养RF/6A细胞系,在培养基中分别添加重组canstatin蛋白和等量的PBS,采用Transwell小室法检测各组发生迁移的内皮细胞并计数;铺Matri-gel胶,行体外成管实验,观察canstatin蛋白对内皮细胞成管的抑制作用;用MTT法检测重组canstatin蛋白对内皮细胞增殖的影响;Western blotting检测重组canstatin蛋白对血清刺激30 min后RF/6A细胞pERK、磷酸化Akt蛋白表达的影响.结果 加入重组canstatin蛋白后,显著抑制了细胞的迁移,重组canstatin蛋白组迁移的细胞数每视野(7.75±1.50)个显著低于PBS组(25.25±2.36)个(t=12.505,P=0.000);两组内皮细胞成管数分别为重组canstatin组每视野(18.67±7.02)个,也显著低于PBS组每视野(44.67±2.52)个(t=6.036,P=0.004).与PBS组相比,重组canstatin蛋白组的内皮细胞的增殖也受到抑制,后者48 h后平均吸光度A490为0.286 9±0.014 0,低于前者0.3349±0.021 7(t=3.723,P=0.01).同时,重组canstatin蛋白降低了RF/6A细胞中pERK蛋白的表达水平.结论 重组canstatin蛋白能通过下调pERK蛋白的表达抑制RF/6A细胞的迁移和增殖,具有潜在抑制视网膜新生血管形成的作用.     

金雀异黄素衍生物对糖基化终产物损伤猴视网膜血管内皮细胞的保护作用

目的研究7-二氟亚甲基-5,4’-二甲烷氧基异黄酮（dFMGEN）对糖基化终产物损伤的猴视网膜血管内皮细胞的影响。方法制备RF／6A细胞糖基化终产物损伤模型,用不同浓度的dFMGEN（1、3、10、30、100μmol／L）进行干预,MTT法观察dFMGEN对细胞生长增生的影响,流式细胞术检测dFMGEN对细胞凋亡的影响。结果糖基化终产物孵育RF／6A细胞48h,可明显抑制细胞生长、促进凋亡;dFMGEN与糖基化终产物共同孵育RF／6A细胞48h,能有效降低增生抑制率,减少凋亡,并呈浓度依赖性。100μmol／L dFMGEN比相同浓度的先导化合物金雀异黄素（GEN）和阳性药物Vit B1作用更强。结论dFMGEN对糖基化终产物引起的视网膜血管内皮细胞的损伤具有保护作用。     

超声联合色素上皮源性因子免疫纳米脂质体对脉络膜新生血管内皮细胞靶向杀伤作用的体外研究

目的 体外实验研究超声联合色素上皮源性因子(pigment epithelium derived factor,PEDF)免疫纳米脂质体对脉络膜新生血管内皮细胞的靶向杀伤作用.方法 制备能与脉络膜新生血管内皮细胞特异性结合的PEDF免疫纳米脂质体.向培养恒河猴脉络膜视网膜内皮细胞系RF/6A细胞加入免疫纳米脂质体,观察其与RF/6A细胞结合的能力.激光共聚焦显微镜观察免疫纳米脂质体的细胞内化过程.分别将游离PEDF、载PEDF脂质体、载PEDF免疫脂质体和超声联合载PEDF免疫脂质体作用细胞后,MTT法检测对RF/6A细胞生长的抑制作用,流式细胞术检测凋亡,并与仅用PBS作用细胞的对照组比较.结果 制备的载PEDF免疫纳米脂质体可紧密结合RF/6A细胞,3 h时,载PEDF免疫脂质体布满细胞浆.包含PEDF终浓度为80 mg·L-1的游离PEDF、载PEDF脂质体、载PEDF免疫脂质体和超声联合载PEDF免疫脂质体对RF/6A细胞抑制率分别为(32.79±3.62)%、(48.71±3.84)%、(57.45±4.02)%和(90.83±5.61)%.凋亡率分别为(25.05±4.06)%、(30.21±3.13)、(36.85±4.12)%和(43.13±4.37)%.提示载PEDF免疫脂质体和载PEDF脂质体的抑制细胞生长和促进细胞凋亡作用均较游离PEDF强(均为P<0,05),且载PEDF免疫脂质体的抑制生长和促进凋亡作用更强(均为P<0.05).超声可显著提高载PEDF免疫脂质体的细胞内化能力,并增强其抑制细胞生长和促进细胞凋亡的作用.结论 体外实验证实超声联合PEDF免疫纳米脂质体可靶向杀伤脉络膜新生血管内皮细胞.     

白藜芦醇对视网膜血管内皮细胞增殖及VEGF表达的影响(英文)

目的:探讨白藜芦醇对缺氧诱导的视网膜血管内皮细胞增殖的影响。方法:用100μmol/LCoCl2模拟缺氧环境,以体外培养的人视网膜血管内皮细胞为模型,采用MTT法测定细胞增殖、SABC法检测血管内皮生长因子的表达,并以计算机图像分析仪进行分析,观察研究白藜芦醇对CoCl2诱导的视网膜血管内皮细胞增殖的影响。结果:白藜芦醇对CoCl2诱导的视网膜血管内皮细胞增殖具有显著抑制作用(P<0.01),且呈时间和剂量依赖关系;同时,各给药组均抑制VEGF的表达(P<0.01)。结论:白藜芦醇能抑制CoCl2诱导的视网膜血管内皮细胞增殖及VEGF的表达,将为视网膜新生血管疾病的药物治疗提供一条新思路。     

单分子成像显示视网膜血管内皮细胞膜上血管内皮生长因子受体2聚集状态

目的:观察单个视网膜血管内皮细胞膜上血管内皮生长因子受体2 （VEGFR2）的聚集状态。方法:将猴视网膜血管内皮细胞（RF/6A）分为空白对照组（正常培养）、质粒转染组[转染VEGFR2-绿色荧光蛋白（GFP）重组质粒]。采用共聚焦显微镜观察质粒转染组GFP的表达;实时荧光定量聚合酶链反应（qPCR）、蛋白免疫印迹法（...     

生长因子诱导的血管内皮细胞增殖作用的研究

目的 研究生长因子对血管内皮细胞增殖活动的诱导和刺激作用。以进一步探讨生长因子在视网新生血管疾病中所起的作用。方法 采用血管内皮生长因子（VEGF）和碱性成纤维生长因子（bFGF）诱导离体培养的血管内皮细胞的增殖。研究VEGF及bFGF对血管内皮细胞DNA合成、细胞生长和细胞周期的调控，以及两种因子的协同效应。结果 VEGF及bFGF均可显著刺激血管内皮细胞的增生，其刺激效应与生长因子浓度呈剂量依赖关系。共同作用时，具有协同效应。两种因子均可显著增加^3H-TdR的掺入量，其刺激作用与浓度呈剂量依赖关系，共同作用时亦具有协同效应。VEGF和bFGF均可显著地产加S期细胞的比例。结论 VEGF及bFGF均可显著地刺激内皮细胞的增殖，促进细胞DNA的合成。两种因子联合应用时，具协同效应。提示：两种生长因子在视网膜新生血管的发生和发展中可能具有一定作用，且存在因子间的协同效应。     

水蛭提取液对恒河猴视网膜血管内皮细胞增殖和细胞周期的影响

目的观察渗滤法水蛭提取液对恒河猴视网膜血管内皮细胞RF/6A细胞增殖和细胞周期的影响。方法采用MTT法观察不同浓度水蛭提取液(0g.L-1、8g.L-1、16g.L-1、32g.L-1、64g.L-1、128g.L-1)对RF/6A细胞增殖的影响,筛选最佳抑制浓度,应用流式细胞仪观察水蛭提取液对凝血酶诱导的RF/6A细胞周期的影响。结果8g.L-1、16g.L-1浓度组水蛭提取液对血管内皮细胞RF/6A有促进生长作用,与空白对照组相比差异均有统计学意义(均为P<0.05);32g.L-1、64g.L-1、128g.L-1浓度组水蛭提取液对血管内皮细胞RF/6A均有明显抑制作用,与空白对照组相比差异也均有统计学意义(均为P<0.05);24hIC50为97g.L-1;64g.L-1为水蛭提取液的最佳抑制浓度,用于后期实验。流式细胞仪检测发现:水蛭提取液组G1期细胞比例(91.95±1.69)%高于其他各组,合药组(83.60±1.88)%高于凝血酶组(77.78±3.22)%,差异均有统计学意义(均为P<0.05)。凝血酶组S期细胞比例(21.75±2.94)%高于其他各组,水蛭提取液组(7.55±2.45)%...     

脂联素对RF/6A细胞增殖、迁移及管腔形成的影响

目的 研究脂联素(adiponectin,APN)对体外培养的猴脉络膜/视网膜内皮细胞(RF/6A)增殖、迁移和管腔形成的影响,探讨APN对脉络膜和视网膜新生血管的抑制作用.方法 取体外培养的生长良好的RF/6A细胞用于实验,将细胞随机分为对照组、不同浓度重组APN组(5 pg·mL-1、50 pg·mL-1、500 pg·mL-1预处理1h).培养24h后采用MTT法检测细胞增殖,细胞划痕法检测细胞迁移,基质胶(Matrigel)法检测管腔形成.结果 不同浓度重组APN组24 h的细胞增殖均弱于对照组(均为P＜0.05),不同浓度重组APN组细胞24 h的迁移面积均小于对照组(均为P＜0.05),不同浓度重组APN组管腔形成数均明显少于对照组(均为P＜0.05),且细胞增殖、迁移和管腔形成均随着重组APN浓度的升高而降低.结论 APN能够明显抑制RF/6A细胞的血管生成过程,提示APN对于脉络膜和视网膜新生血管具有一定的保护作用.     

白藜芦醇对高糖环境下视网膜血管内皮细胞增殖及HMGB1乙酰化的影响

目的：探讨白藜芦醇对高糖环境下视网膜血管内皮细胞增殖的影响,并对其机制进行探讨。

方法：人视网膜血管内皮细胞培养于低糖或高糖环境,通过MTT法测定各组细胞增殖,研究白藜芦醇对高糖培养的视网膜血管内皮细胞增殖的影响。通过Western-blot及免疫共沉淀检测SIRT1表达及HMGB1的乙酰化。

结果：白藜芦醇对高糖环境下的视网膜血管内皮细胞增殖具有显著抑制作用(P<0.05),且随白藜芦醇剂量增加,抑制作用增强。高糖抑制SIRT1表达,提高HMGB1的乙酰化,白藜芦醇能逆转上述改变。

结论：白藜芦醇可能通过SIRT1-HMGB1通路抑制高糖环境下的视网膜血管内皮细胞增殖。     

A simple method for the in vitro culture of human retinal capillary endothelial cells.

A simple method for the culture of human retinal capillary endothelial cells in vitro is described in this report. Through a process of gentle mechanical disruption and sieving, a sufficient yield of retinal microvessels was obtained from one or two human eyes to allow the culture of endothelial cells in abundance. The cells were factor VIII-positive and demonstrated typical vascular endothelial morphology. Pericyte contamination was prevented by using human platelet-poor serum in primary culture and passaging cells with a low concentration of trypsin. Proliferation assays revealed that the cells grew best in Iscove's modified Dulbecco's modified Eagle's medium with 15% fetal calf serum (FCS). There was no difference in induced proliferation when FCS was compared to human platelet-poor serum. The method seemed to be as good as and much simpler than other recently described techniques. The study of human retinal capillary endothelial cells cultured in this way may shed light on the earliest stages of diabetic retinopathy and other diseases of the retinal microvasculature, particularly AIDS-related retinopathy and radiation retinopathy.     

Generation and characterization of a recombinant adenovirus expressing vascular endothelial growth factor for studies of neovascularization in the eye.

There is accumulating evidence that an increased expression of vascular endothelial growth factor from retinal pigment epithelial cells may be important in choroidal neovascularization. In vivo studies have demonstrated that subretinal injection of recombinant adenovirus vectors produces long-term transgene expression specifically within retinal pigment epithelial cells. A recombinant adenovirus encoding of vascular endothelial growth factor (Ad.RSV.VEGF) was therefore produced and characterized in order to determine whether an upregulation of vascular endothelial growth factor expression is sufficient to induce choroidal neovascularization. Ad.RSV.VEGF was produced by homologous recombination and its identity confirmed by restriction enzyme analysis. Ad.RSV.VEGF was characterized in vitro by the transduction of cultured retinal pigment epithelial cells. The in vitro characterization confirmed vascular endothelial growth factor mRNA and protein expression from Ad.RSV.VEGF and demonstrated the biological activity of the vascular endothelial growth factor protein. A preliminary in vivo study suggested that the subretinal injection of Ad.RSV.VEGF induced vascular leakage.     

鼠视网膜血管内皮细胞体外培养的改良及鉴定

背景视网膜血管内皮细胞（RVECs）的体外培养是进行视网膜血管性疾病研究的基础,人眼球供体的缺乏是限制人血管内皮细胞培养的主要原因,与人高度同源的大鼠血管内皮细胞的培养方法已经建立,但其分离方法对细胞损伤较大,影响细胞的培养效率。目的建立和改良大鼠RVECs的培养方法。方法选取5只SD大鼠眼球,获得视网膜,用组织块培养法,将大鼠视网膜用质量分数0．1％胶原酶消化,并加入质量分数20％胎牛血清、血管内皮生长因子（VEGF）、血管内皮细胞生长添加物（ECGS）,用血管内皮细胞培养基中和后充分吹打,制备细胞及组织悬液,接种于人纤维粘连蛋白（FN）包被的培养瓶中。用Ⅷ因子相关抗体鉴定培养的内皮细胞。结果组织块法消化培养2d可见细胞从组织块游出,培养4d可伸出触角呈梭形,5～6d可融合生长,培养14d后的细胞呈单层生长,培养的细胞Ⅷ因子相关抗体染色阳性。传代培养的细胞接种2h可重新贴壁,恢复融合生长状态。结论视网膜组织块培养并用胶原酶消化可获得活性较强的细胞和碎片,再用高选择性的血管内皮细胞培养基和FN促贴壁进行选择性培养,可获得纯度较高的RVECs。     

Vascular endothelial growth factor upregulates expression of ADAMTS1 in endothelial cells through protein kinase C signaling

PURPOSE: ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs) has been demonstrated to inhibit angiogenesis in vivo and to suppress endothelial cell proliferation in vitro. The purpose of this study was to investigate the expression of ADAMTS1 in endothelial cells and in a mouse model of ischemia-induced retinal neovascularization and to study the regulation of ADAMTS1 expression in endothelial cells by vascular endothelial growth factor (VEGF). In addition, the potential function of endothelial cell-derived ADAMTS1 on cell proliferation was investigated. METHODS: Expression of ADAMTS1 in human retinal endothelial cells (HRECs), human umbilical vein endothelial cells (HUVECs), and the mouse model of ischemia-induced retinal neovascularization was assayed by real-time PCR and Western blot analysis. The effect of ADAMTS1 on endothelial cell proliferation was evaluated using siRNA knockdown and [3H] thymidine incorporation. RESULTS: ADAMTS1 mRNA and protein levels were increased in a mouse model of ischemia-induced retinal neovascularization, and VEGF induced time- and dose-dependent increases in ADAMTS1 mRNA and protein expression in endothelial cells. This upregulation was inhibited by the VEGF receptor (VEGFR)2 inhibitor SU1498, anti-VEGFR2 neutralizing antibody, and the phospholipase C (PLC)-gamma inhibitor U73122. VEGF upregulation of ADAMTS1 expression was completely abolished by the inhibition of protein kinase C by calphostin C and largely blocked by the specific inhibition of PKCbeta. Knockdown of endogenous ADAMTS1 resulted in increased proliferation of endothelial cells. CONCLUSIONS: These results indicate that VEGF significantly induces ADAMTS1 expression in endothelial cells in a PKC-dependent fashion. ADAMTS1 expression is also increased, along with VEGF expression, in vivo in ischemia-induced retinal neovascularization. In addition, ADAMTS1 appears to be an endogenous regulator of endothelial cell proliferation. Therefore, VEGF upregulation of ADAMTS1, a potent angiogenesis inhibitor, may represent a mechanism for feedback inhibition of angiogenesis and retinal neovascularization.     

Tenomodulin蛋白对血管内皮细胞增生的抑制作用

目的研究tenomodulin（TeM）蛋白对血管内皮生长因子（VEGF）诱导的人视网膜血管内皮细胞（RECs）和人脐静脉内皮细胞（UVECs）增生及其对体外血管样结构形成的作用。方法将传代至3～6代的人RECs和人UVECs生长至融合时用含质量分数0.5%胎牛血清（FBS）的培养基孵箱中培养6h,加入不同质量浓度的VEGF或VEGF＋TeM混合物,继续培养12h,ELISA法检测TeM对VEGF诱导的血管内皮细胞增生的影响。将含10%FBS、1%FBSDMEM培养基的人RECs和人UVECs悬液分别接种于预先放置基质胶的24孔培养板表面,含1%FBS培养基的细胞培养板内分别加入VEGF（100μg/L）或VEGF＋TeM混合物37℃继续培养6h,共焦显微镜下观察,图像处理分析软件量化分析TeM蛋白对毛细血管样结构形成的作用。结果随着VEGF质量浓度的增加,血管内皮细胞DNA合成增加,其吸光度（A）值呈明显上升曲线,而添加TeM组的A值比VEGF组降低,差异有统计学意义（P〈0.01）。含10%FBS和1%FBS培养基的血管内皮细胞在基质胶表面形成毛细血管样结构,并连接成网状;在基质胶表面同时添加VEGF的血管内皮细胞,其毛细血管样结构明显增多,长度明显变长。添加TeM组的血管内皮细胞和其毛细血管样结构破坏,每个视野内血管内皮细胞毛细血管样结构显著破坏,管形细胞总长度明显变短。结论 TeM蛋白对血管内皮细胞的增生及体外血管样结构的形成有明显抑制作用。     

视网膜微血管内皮细胞培养及血管二维模型的建立

目的 培养人视网膜微血管内皮细胞，建立人视网膜血管体外二维模型。 方法 人视网膜血管内皮细胞在纤维连接蛋白包被的细胞培养池内，以无血清人内皮细胞培养基培养，形成二维血管模型。用辣根过氧化酶检测其通透性。部分血管模型加入5 ng/ml的血管内皮生长因子培养，与无血管内皮生长因子培养形成的血管模型比较通透性的变化，观察血管内皮生长因子对血管通透性的影响。 结果2～4 d左右内皮细胞亚融合形成网状血管样结构，6 d左右形成较完整的二维血管模型。血管内皮生长因子可增大血管的通透性，促进血管生成。 结论 用人内皮细胞培养基可成功培养人视网膜血管内皮细胞；利用细胞培养池和无血清人内皮细胞培养基培养，可建立标准的体外视网膜二维血管模型。 (中华眼底病杂志, 2006, 22: 110-112)     

Pharmacologic manipulation of sphingosine kinase in retinal endothelial cells: implications for angiogenic ocular diseases

PURPOSE: The increased vascular permeability and pathogenic angiogenesis observed in diabetic retinopathy are induced, at least in part, by local inflammation and vascular endothelial growth factor (VEGF). Therefore, inhibition of signaling from VEGF and tumor necrosis factor-alpha (TNFalpha) is a promising approach to the treatment of this disease, as well as ocular diseases with similar etiologies, including age-related macular degeneration. A growing body of evidence demonstrates that sphingosine kinase (SK) plays an important role in cellular proliferation and angiogenesis. This study was undertaken to examine the effects of SK inhibitors on the responses of retinal endothelial cells (RECs) to VEGF and TNFalpha and their therapeutic efficacy in a diabetic retinopathy model. METHODS: The expression and function of SK in bovine and human RECs were examined by immunoblot analysis. The involvement of SK in mediating responses to VEGF and TNFalpha was examined by using pharmacologic inhibitors of SK in cellular and in vivo assays, including a 3-month streptozotocin-induced diabetic retinopathy model in rats. RESULTS: SK was present and active in human and bovine RECs, and SK activity in these cells was stimulated by VEGF. Inhibitors of SK blocked VEGF-induced production of sphingosine 1-phosphate and markedly attenuated VEGF-induced proliferation and migration of RECs. In addition, SK inhibitors were shown to block TNFalpha-induced expression of adhesion proteins, suppress VEGF-induced vascular leakage in an in vivo mouse model, and reduce retinal vascular leakage in the rat diabetic retinopathy model. CONCLUSIONS: Overall, these studies demonstrate that inhibitors of SK attenuate the effects of proliferative and inflammatory stimuli on RECs both in vitro and in vivo, and so could be significant therapeutics in the treatment of diabetic retinopathy.