选择性培养牛视网膜血管内皮细胞和周细胞

目的　建立选择性培养牛视网膜血管内皮细胞和周细胞的简要方法。方法　采用机械除杂法或等密度沉降分离法 ,培养皿用不同的基质成分包埋 ,培养液中加入有利于细胞生长的添加物。结果　原代培养的牛视网膜血管内皮细胞和周细胞纯净度 >95 % ,能连续传代。周细胞在传代后不规则形状及细胞内微丝更明显。视网膜血管内皮细胞融合后呈“铺路石”样改变。视网膜血管内皮细胞对Ⅷ因子相关抗体染色阳性。视网膜周细胞对单克隆抗体α 平滑肌肌动蛋白抗体染色阳性。结论　用明胶包被培养皿 ,在培养液中加入肝素、内皮细胞生长因子及高浓度的胎牛血清可获得较纯的内皮细胞。而周细胞最适合的培养液为DMEM加入 2 0 g·L-1胎牛血清。利用Percoll分离液也可分离培养出较纯的牛视网膜血管内皮细胞和周细胞     

Angiotensin II does not contract bovine retinal resistance arteries in vitro

The effect of angiotensin II was studied in vitro on ring segments of bovine retinal resistance arteries (i.d. 126-271 microns) and posterior ciliary arteries (i.d. 207-1153 microns). Although the retinal resistance arteries were responsive to 5-hydroxytryptamine, prostaglandin F2 alpha, and changes in extracellular K(+)-concentration, they did not, in contrast to the posterior ciliary arteries, contract to cumulative or single doses of angiotensin II. In the latter arteries, angiotensin II induced a small concentration dependent contraction, 5% of maximal 125 mM K(+)-induced response, with a pD2-value of 9.3. The single addition of 10(-6) M angiotensin II increased the maximal vessel response of the posterior ciliary arteries three times to angiotensin II. Tachyphylaxis was pronounced in the posterior ciliary arteries, in which the response to angiotensin II could not be repeated. Indomethacin (10(-5) M), methylene blue (3 x 10(-6) M), or removal of endothelium did not make the retinal resistance arteries responsive to angiotensin II. Retinal arteries precontracted with 30 mM potassium did not respond to angiotensin II. Angiotensin II did not potentiate the 5-hydroxytryptamine- and noradrenaline concentration-response characteristics of both retinal resistance and posterior ciliary arteries. Although angiotensin II-receptors have been detected in bovine retinal vascular smooth muscle using radioligand-binding technique, the present results suggest that these receptors are non-functional in respect to regulation of retinal resistance artery tone.     

牛视网膜微血管周细胞和内皮细胞的选择性培养

目的 建立视网膜微血管周细胞（ＰＣ）和内皮细胞（ＥＣ）选择性培养的简便方法。方法 采用含２０％胎牛血清（ＦＢＳ）的ＤＭＥＭ和在该培养基加入５％贫血小板人血浆（ＰＰＰ）及牛视网膜浸出液（２０ｕｌ／ｍｌ）,结合原代培养细胞克隆的分离、除杂。结果 获得的ＰＣ和ＥＣ能连续传代,纯净度〉９５％。ＰＣ形态不规则,无接触性抑制,对３Ｇ５和α－肌动蛋白单克隆抗体染色阳性,Ⅷ因子抗体染色阴性;ＥＣ呈鹅卵石状生长,有接触性抑制,对Ⅷ因子抗体染色阳性,３Ｇ５和α－肌动蛋白抗体染色阴性。结论 用２０％ＦＢＳ的ＤＭＥＭ或在该培养液中加入５％ＰＰＰ及视网膜浸出液,结合原代细胞克隆的分离、除杂,可分别获得较纯净的ＰＣ和ＥＣ培养物。     

牛视网膜微血管内皮细胞和周细胞的体外培养

目的 探讨牛视网膜微血管内皮细胞（ bovine retinal endothelial cells, BREC ）和周细胞（bovine retinal pericytes, BRP）的体外选择性培养方法。 方法 结合视网膜微血管的消化分离，采用含10％人血清、100 μg/ml 肝素（Heparin）的Dulbecco改良Eagle培养基(Dulbecco′s modified Eagle′s medium, DMEM)和含20％胎牛血清的DMEM培养基分别选择性培养BREC和BRP。通过荧光显微镜观察乙酰化低密度脂蛋白(acetylated low density lipoprotein, Dil-Ac-LDL)吞噬情况并应用Von Willebrand因子抗体通过免疫组织化学方法鉴定BREC；以及α-平滑肌肌动蛋白抗体免疫组织化学鉴定BRP。 结果 通过选择性培养获得的BREC和BRP的纯度达到98％以上，并能连续传代。BREC早期似小鲤鱼状聚集在微血管碎片周围，呈片状鹅卵石样单层生长，存在接触性抑制；周细胞多散布在距血管碎片稍远处，形状不规则，呈无接触性抑制的生长。BREC可吞噬Dil-Ac-LDL，细胞浆内有荧光表达 ；消化传代后BREC中Von Willebrand因子表达阳性，而α-平滑肌肌动蛋白表达阴性；BRP的α-平滑肌肌动蛋白表达阳性，而Von Willebrand因子表达阴性。 结论 选择性培养基的应用和培养皿的处理，可分别获得较高纯度的BREC和BRP，简单且具有良好的重复性，无需额外步骤来去除BREC中混杂的BRP。 （中华眼底病杂志，2004，20：23-26）     

Short-time application of latanoprost does not stimulate melanogenesis in bovine ocular melanin-containing cells in vitro

Topical use of latanoprost for glaucoma can lead to an increase in iris and eye lash pigmentation but the precise mechanism is unclear. To study the possible effect of this drug on ocular melanogenesis, we used cultures of bovine iris melanocytes, iris pigment epithelial cells, retinal pigment epithelial cells, and choroidal melanocytes. Latanoprost (at concentrations of 10(-8) and 10(-6) mol) was applied for 3 days, and cell numbers as well as melanin content were measured prior to and 10 days after exposure and compared to untreated controls. In none of the cell types examined a significant increase in melanin content or an increase in cell proliferation was observed. Additional treatment with the tyrosinase inhibitor alpha-methyl-p-tyrosine showed no significant effect either. Our results support the concept of a rather complex mechanism underlying the increased iris pigmentation after treatment with latanoprost.     

糖尿病视网膜病变中视网膜内皮细胞功能障碍的研究进展

视网膜内皮细胞(REC)是参与糖尿病视网膜病变和许多眼部疾病的主要细胞类型之一。视网膜微血管系统有助于血-视网膜屏障的维持,这对正常的视功能至关重要。REC的改变在视网膜疾病的发生发展中起着关键作用。高血糖是糖尿病微血管损伤的重要原因,通过不同的机制导致REC功能障碍,包括向衰老表型改变、迁移和增殖能力增强、炎性凋亡等,最终导致无细胞毛细血管及病理性新生血管形成。本文对糖尿病视网膜病变中REC的功能障碍作一综述。     

Effect of pan retinal photocoagulation on the serum levels of vascular endothelial growth factor in diabetic patients

This study tests the hypothesis that subjects with proliferative diabetic retinopathy (PDR) have a detectable rise in levels of serum vascular endothelial growth factor (VEGF), which is an important regulator of angiogenesis. Our investigation aims to evaluate plasma VEGF changes after pan-retinal photocoagulation (PRP) in diabetic patients. Twenty-nine type two diabetic patients (17 male, 12 female: mean age 53.13 ± 12.22 years) with PDR secondary to diabetes were studied. Blood samples were obtained before and at 2 months after the last PRP session. Serum VEGF levels were measured by ELISA. After PRP, the mean serum VEGF decreased, but this reduction was not remarkable (88.68 ± 71.09 vs. 77.01 ± 60.33 ng/ml) (P = 0.18). There was a statistically significant difference in serum VEGF changes between patients who had regressed PDR with patients who had progressed PDR (−25.98 ± 47.37 vs. 56.44 ± 31.7 ng/ml) (P = 0.003). Our results showed a significant reduction in levels of serum VEGF in the patients who had successful laser treatment. Our findings suggest that serum VEGF levels could be used for monitoring diabetic retinopathy outcome.     

Effects of tenascin-C on normal and diabetic retinal endothelial cells in culture

PURPOSE: Tenascin-C (TN-C) is expressed in embryogenesis, tissue remodeling, and healing. It is up-regulated in retinas of patients affected by diabetic retinopathy (DR). Because TN-C may promote neovascularization, its potential angiogenic effects were examined in vitro in normal and diabetic retinal endothelial cells (RECs). METHODS: Bovine and human RECs were cultured on plastic or reconstituted basement membrane (BM) matrix. Production of TN-C, capillary-like tube formation, secondary sprouting, and cell migration, survival, and proliferation were measured with or without angiogenic growth factors (GFs). Antibodies and inhibitors were used to determine the involvement of specific TN-C receptors and signaling pathways. RESULTS: TN-C significantly delayed collapse of REC capillary-like tubes on BM matrix. It decreased tube involution associated with serum deprivation, high glucose, and exposure to TGF-beta. TN-C's enhancement of tube stability was mediated by alphavbeta3 integrin. TN-C increased REC viability in 0.5% serum and stimulated REC proliferation in 10% serum. It promoted REC secondary sprouting on BM matrix, which involved signaling through mitogen-activated kinase kinase (MEK) and p38 mitogen-activated protein kinase. TN-C also enhanced tube branching after treatment with VEGF and stimulated REC migration twofold. Angiogenic GF increased TN-C production by RECs in an additive manner, which may explain higher levels of TN-C deposition in DR cells. CONCLUSIONS: TN-C was overexpressed in diabetic and DR REC cultures. TN-C enhanced the sprouting, migratory, and survival effects of angiogenic GFs, and had distinct proliferative, migratory, and protective capacities. The data suggest that TN-C may act as a proangiogenic mediator in DR and other pathologic conditions involving neovascularization.     

Mediation of vascular endothelial proliferation by factors in fetal bovine retinal extracts and serum

The effect of fetal bovine retinal extract (FBR) and dialyzed fetal bovine serum (FBS) on in vitro proliferation of vascular endothelial cells was studied. Quantitatively, FBR, both dialyzable and nondialyzable, and dialyzed FBS exhibited a synergism in bringing about an enhanced proliferation of vascular endothelial cells. Assays on selected samples using both aortic and retinal vascular endothelial cells yielded similar results. In the absence of dialyzed FBS, FBR had only a negligible stimulatory effect. When compared at a protein concentration of 3 mg ml^?1, dialyzed FBS elicited vascular endothelial proliferation at a rate only 25% that of FBS (not dialyzed). However, under experimental conditions using 3 mg ml^?1 of dialyzed FBS and 0·5 mg ml^?1 of dialyzed retinal extract, a six-fold increase in the activity of vascular endothelial proliferation was demonstrated. The observed synergistic effect was dependent on the concentrations of both dialyzed FBS and FBR. The observed synergism rules out the possibility that the stimulatory effect of FBR was due to blood contamination in sample preparation. Qualitatively, major components of dialyzed FBS and dialyzed FBR that exerted a synergistic effect on the stimulation of vascular endothelial proliferation were isolated by permeation chromatography using Sephadex G-100, with M_r estimated at 150 000 and 80 000, respectively. One minor component in FBS and one in FBR were also present.     

Blocking ET-1 receptors does not correct subnormal retinal oxygenation response in experimental diabetic retinopathy

PURPOSE: To test the hypothesis that bosentan (a dual ET(A)/ET(B) receptor antagonist) corrects a subnormal retinal oxygenation response in the STZ-induced diabetic rat. METHODS: In benchtop experiments, ET-1 was acutely injected into the vitreous of control and 5- to 7-day bosentan-treated nondiabetic rats. Major retinal vessel diameters were analyzed from ADPase-stained flatmounts. Retinal oxygenation (DeltaPo(2)), an established early surrogate marker of drug treatment efficacy, was measured by MRI during a 2-minute carbogen inhalation challenge in four groups: control rats (n = 7), control rats treated with bosentan (n = 7), 3-month diabetic rats (n = 9), and 3-month diabetic rats treated with bosentan (n = 5). Effect of baseline differences was studied in control rats breathing either room air (n = 5) or 12% oxygen breathing (n = 5) before a 2-minute carbogen provocation. RESULTS: ET-1 produced a significant (P < 0.05) reduction in retinal arterial diameter that was suppressed (P > 0.05) in rats fed bosentan chow admix. For all groups, no MRI baseline signal intensity differences were found (P > 0.05). Also, comparisons between baseline room air and 12% conditions and control rats fed normal chow or a bosentan admix both produced similar (P > 0.05) panretinal DeltaPo(2). In treated and untreated diabetes groups, inferior hemiretinal DeltaPo(2) remained normal (P > 0.05), but superior hemiretinal DeltaPo(2) was subnormal (P < 0.05). CONCLUSIONS: Because subnormal retinal DeltaPo(2) after drug treatment is a biomarker of subsequent vascular histopathology, the present data raise the possibility that retinal ET-1 does not play a key role in the pathogenesis of diabetic retinopathy.     

Simplified methods for consistent and selective culture of bovine retinal endothelial cells and pericytes

Different matrix components, in combination with various media and serum supplements, were evaluated for their ability to promote selectively the growth of bovine retinal endothelial cells in primary culture. The optimal setting for the selective growth of retinal endothelial cells was a fibronectin/hyaluronic acid matrix, Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% pooled human serum and 100 micrograms/ml heparin. These conditions consistently yielded virtually homogeneous cultures of endothelial cells, assessed using specific endothelial markers. Thus obtained, the retinal endothelial cells could be subcultured and maintained in phenotypically stable long-term serial cultivation. Homogeneous cultures of retinal pericytes were obtained when microvessel isolates were seeded to uncoated or gelatin-coated culture dishes and grown in DMEM supplemented with 20% fetal bovine serum. The retinal pericytes could also be subcultured and cultivated for numerous populations doublings. Additionally, observations from this study suggest that two populations of pericytes may be obtained in culture and distinguished on the basis of their relative size and antigenic properties.     

Autocrine regulation of bovine retinal capillary endothelial cell (BREC) proliferation by BREC-derived basic fibroblast growth factor

Fibroblast growth factors (FGFs) are mitogenic for bovine retinal capillary endothelial cells (BREC) seeded at a low density. Seeding BREC cells at a high density greatly reduces their requirement for basic FGF (bFGF) in order to proliferate actively. We show here that monolayers of BREC cells synthesize and release into the culture medium a growth factor, which on the basis of biological activity, heparin affinity, immuno-cross reactivity with anti-bFGF antibodies and mRNA analysis, has been identified as basic fibroblast growth factor. These data indicate that BREC cells are able to synthesize and release bFGF, which can act as a promoting-growth factor for these cells by a para- and/or autocrine mechanism. We suggest thus, that this para- and/or autocrine mechanism involving bFGF may play a key role in preretinal neovascularization, particularly in diabetic patients presenting a proliferative retinopathy.     

Transendothelial electrical resistance of bovine retinal capillary endothelial cells is influenced by cell growth patterns: an ultrastructural study

The purpose of this study was to describe the ultrastructural features of an in vitro capillary endothelial cell model of blood-retinal barrier permeability and to relate morphological features with transendothelial electrical resistance. The electrical resistance of endothelial cell monocultures on small and large pore size polycarbonate Transwell filters was measured and compared with cocultures of endothelial cells and Müller cells. There was a wide variation in electrical resistance measurements with many preparations not achieving a functional barrier. The ultrastructural features associated with barrier function in vitro were studied by comparing cultures that exhibited a 'tight' or 'leaky' barrier when measured immediately prior to processing for electron microscopy. Preparations with low transendothelial electrical resistance were associated with irregular cell growth when studied morphologically. It was concluded that parallel light and electron microscopic studies are important for validation of in vitro models of vascular endothelial permeability.     

牛视网膜毛细血管周细胞的选择性培养

目的 :选择性分离、培养牛视网膜毛细血管周细胞。方法 :采用有限胶原酶消化和筛网过滤法培养周细胞。结果 :原代培养时 ,周细胞形态不规则 ,呈现出典型的非接触性抑制重叠融合生长 ;传代培养后增长速度加快 ,第 3天进入对数生长期 ,第 10天进入平台期。结论 :获得了较纯净的视网膜毛细血管周细胞 (纯净度 >98% ) ,并能连续传代。     

牛视网膜毛细血管周细胞的选择性培养

目的：选择性分离，培养牛视网膜毛细血管周细胞。方法：采用有限的胶原酶消化和筛网过滤，辅以细胞的克隆分离和除杂，培养近乎纯净的视网膜毛细血管周细胞。结果：原代培养时，视网膜毛细血管周细胞形态不规则，呈现出典型的非接触性抑制重叠融合生长，传代培养后增长速度明显加快，第3天进入对数生长期，第10天进入平台期。培养的视网膜毛细血管周细胞对单克隆抗体α-平滑肌肌动蛋白抗体染色显阳性，而对Ⅷ因子相关抗原抗体，抗GFAP抗原抗体染色显阴性。结果：此方法能简单、经济、有效地获得了较纯净的视网膜毛细血管周细胞（纯净度＞98％），并能连续传代，为研究与视网膜毛细血管周细胞有关的各种正常生理或病理过程提供了极大的方便。     

Minocycline protects photoreceptors from light and oxidative stress in primary bovine retinal cell culture

PURPOSE: To determine whether minocycline, a compound known to protect the retina against light-induced damage in rodent models, and its structurally related analogues would protect photoreceptor cells in primary bovine retinal cell culture against light and oxidative stress. METHODS: Minocycline and its analogues were tested in primary retinal cell culture to see whether they would inhibit light or oxidative stress-induced cell death. Primary cell cultures composed of photoreceptors, bipolar cells, and glial cells were prepared from bovine retinas. The extent of cell death induced by light or oxidative stress was assessed by using Sytox Green (Invitrogen-Molecular Probes, Eugene, OR) a nucleic acid dye uptake assay. Differential protection of photoreceptor cells from stress were examined using immunocytochemistry. RESULTS: Minocycline and methacycline were cytoprotective against light- or oxidative stress-induced damage of bovine primary photoreceptors in culture with an EC(50) < 10 microM. In contrast, structurally related analogues such as demeclocycline, meclocycline, and doxycycline were phototoxic at >3 to >10 microM. Though demeclocycline was found to be phototoxic, it was cytoprotective (EC(50) = 5 microM) against oxidative stress in the absence of exposure to light. CONCLUSIONS: The protective action of minocycline against light-induced damage in the cell-based assays agrees with earlier reports in animal models and suggests that the in vitro assay using bovine primary retinal cell culture is a suitable model for evaluating compounds for retinal protection. Cellular protection or toxicity produced by structurally related compounds show that minor structural modifications can alter the function of minocycline and lead to potent retinal protective compounds.     

血清骨膜蛋白、血管内皮生长因子与糖尿病视网膜病变的关系

目的　探讨骨膜蛋白（periostin,PN）与血管内皮生长因子（vascular endothelial growth factor,VEGF）在糖尿病视网膜病变（diabetic retinopathy,DR）患者血清中的变化及其临床意义。方法　收集糖尿病（diabetes mellitus,DM）（病例组53例）患者血清样本,以同期健康体检对象的血清为对照（对照组50名）,其中病例组包括无眼底病变的DM患者15例（DM组）,NPDR患者18例（NPDR组）,PDR患者20例（PDR组）,用酶联免疫吸附试验测定各组样本血清中PN、VEGF含量,并分析血清中PN、VEGF与DR的关系。结果　病例组和对照组中PN和VEGF差异均有统计学意义（均为P<0.05）。PN、VEGF在四组间差异均有统计学意义（均为P<0.05）。组间两两比较显示,PN在DM组与NPDR组间比较差异无统计学意义（P＞0.05）,在其余组间比较差异均有统计学意义（均为P＜0.05）。VEGF在各组间两两比较差异均有统计学意义（均为P<0.05）。NPDR组和PDR组血清中PN与VEGF均呈正相关（r=0.483、0.509,均为P＜0.05）。结论　血清PN、VEGF参与了DR的发生发展,并与DR后期新生血管及纤维血管膜形成关系密切。     

Presence of endothelial cell growth factor activity in normal and diabetic eyes

Two classes of growth factors affecting endothelial cell proliferation have been found previously in ocular tissues: a heat labile mitogen from retina (RDGF) and a heat stable inhibitor of proliferation from vitreous. The relative amounts of these growth factors in normal and diabetic cadaver eyes were investigated using fetal bovine aortic endothelial cell proliferation as an assay. Equivalent levels of RDGF activity were extracted from diabetic and normal sensory retinas. An extract from pigment epithelium and choroid was found to have similar levels of mitogenic activity, but this activity was not as heat labile as RDGF. Like RDGF, equivalent amounts of mitogen were extracted from diabetic and normal tissue. Normal human vitreous inhibited endothelial cell proliferation, and this activity was enhanced by heating the material (10 min., 95 degrees C). Four of the five individual diabetic vitreous samples of identical postmortem times were mitogenic when not heated, and exhibited little or no inhibitory activity when heated. Vitreous of identical postmortem times was pooled and fractionated by heparin-Sepharose chromatography to determine if the heat labile mitogen in vitreous was RDGF. From the insulin-dependent diabetic (IDDM) pooled vitreous sample, a prominent protein of 18 Kd was eluted from the column with 1.2 M NaC1, a characteristic of RDGF. This work suggests that both RDGF and the vitreous inhibitor are found in human vitreous, but their relative concentrations may change in the diabetic state so that retinal neovascularization from retina can occur.