The role of advanced glycation end products in retinal microvascular leukostasis

PURPOSE. A critical event in the pathogenesis of diabetic retinopathy is the inappropriate adherence of leukocytes to the retinal capillaries. Advanced glycation end-products (AGEs) are known to play a role in chronic inflammatory processes, and the authors postulated that these adducts may play a role in promoting pathogenic increases in proinflammatory pathways within the retinal microvasculature. METHODS. Retinal microvascular endothelial cells (RMECs) were treated with glycoaldehyde-modified albumin (AGE-Alb) or unmodified albumin (Alb). NFkappaB DNA binding was measured by electromobility shift assay (EMSA) and quantified with an ELISA. In addition, the effect of AGEs on leukocyte adhesion to endothelial cell monolayers was investigated. Further studies were performed in an attempt to confirm that this was AGE-induced adhesion by co-incubation of AGE-treated cells with soluble receptor for AGE (sRAGE). Parallel in vivo studies of nondiabetic mice assessed the effect of intraperitoneal delivery of AGE-Alb on ICAM-1 mRNA expression, NFkappaB DNA-binding activity, leukostasis, and blood-retinal barrier breakdown. RESULTS. Treatment with AGE-Alb significantly enhanced the DNA-binding activity of NFkappaB (P = 0.0045) in retinal endothelial cells (RMECs) and increased the adhesion of leukocytes to RMEC monolayers (P = 0.04). The latter was significantly reduced by co-incubation with sRAGE (P < 0.01). Mice infused with AGE-Alb demonstrated a 1.8-fold increase in ICAM-1 mRNA when compared with control animals (P < 0.001, n = 20) as early as 48 hours, and this response remained for 7 days of treatment. Quantification of retinal NFkappaB demonstrated a threefold increase with AGE-Alb infusion in comparison to control levels (AGE Alb versus Alb, 0.23 vs. 0.076, P < 0.001, n = 10 mice). AGE-Alb treatment of mice also caused a significant increase in leukostasis in the retina (AGE-Alb versus Alb, 6.89 vs. 2.53, n = 12, P < 0.05) and a statistically significant increase in breakdown of the blood-retinal barrier (AGE Alb versus Alb, 8.2 vs. 1.6 n = 10, P < 0.001). CONCLUSIONS. AGEs caused upregulation of NFkappaB in the retinal microvascular endothelium and an AGE-specific increase in leukocyte adhesion in vitro was also observed. In addition, increased leukocyte adherence in vivo was demonstrated that was accompanied by blood-retinal barrier dysfunction. These findings add further evidence to the thinking that AGEs may play an important role in the pathogenesis of diabetic retinopathy.     

Transition metal-catalyzed oxidation of ascorbate in human cataract extracts: possible role of advanced glycation end products

PURPOSE: With age, human lens crystallins become more pigmented, oxidized, modified by ascorbate oxidation and advanced glycation end products (AGEs), and bind copper. The hypothesis was tested that the major AGE and ascorbylation product in the human lens, N(epsilon)-carboxymethyl-L-lysine (CML), has an EDTA-like structure, which may predispose it to bind redox active copper. METHODS: Young, old, and cataractous human lens protein fractions were glycated with ascorbic acid and tested for their ability to bind Cu(II) by atomic absorption spectroscopy and oxidize (14C1)-ascorbate by radiometric thin-layer chromatography method. AGEs were assayed by high-performance liquid chromatography (HPLC). CML-rich proteins were immunoprecipitated from young, old, and cataractous crystallins using affinity-purified CML antibody and tested for their ability to oxidize ascorbate and generate hydroxyl radicals in the presence of H2O2 using 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) spin-trap and EPR spectroscopy. RESULTS: Ascorbate oxidizing activity at 24 hours of native crystallins was significantly increased in both the water soluble (WS; P < 0.001) and insoluble (WIS; P < 0.05) fractions from cataractous and normal lenses. The chelator DTPA completely prevented oxidation up to 24 hours of incubation but less effectively thereafter. Mean endogenous Cu content in pooled young, old, and cataract fractions increased from 0.016 to 0.026 nmol/mg protein, respectively, in WS (P < 0.05) and WIS (P < 0.001) fractions, and Cu(II) binding was 20% to 30% increased in cataractous versus old and young lenses in WS (P < 0.01) and WIS (P < 0.001) fractions. Mean levels of the AGEs, CML, and pentosidine were markedly elevated in WS and WIS fractions from cataractous versus old or young crystallins (20% to severalfold, P < 0.05 to P < 0.001). In a separate experiment, protein-bound Fe was not elevated. Crystallins ascorbylated in vitro showed an increase in CML as well as Cu(II) binding. CML-rich proteins (immunoprecipitated from cataractous lenses) oxidized ascorbate approximately 4 times faster than similar proteins from young and old normal lenses (P < 0.01) and generated hydroxyl radicals in the presence of H2O2 and DMPO. CONCLUSIONS: The association between CML formation, copper binding, and generation of free radicals by cataractous lens crystallins can be duplicated by ascorbylation in vitro. These effects are only in part attributable to CML itself, and other modifications (AGEs, conformational changes) may participate in the process. A vicious cycle between AGE formation, lipoxidation, and metal binding may exist in the aging lens, suggesting that chelation therapy could be beneficial in delaying cataractogenesis.     

Beta-N-acetyl-glucosaminidase: possible role in ocular neovascularization

The possibility that lysosomal enzymes might be involved as angiogenic factors in ocular neovascularization (NV) was investigated. Beta-N-acetyl-glucosaminidase (NAGase) activity, and that of two other glycosidases, were present in the retinal derived protein fraction (RDPF) reported by others (10) to be angiogenic. NAGase, but not the other glycosidases, was inhibited by vitreous. NAGase exhibited the same stability characteristics as RDPF. In diabetic rats there was a significant rise in vitreous but a fall in retinal NAGase activity. The sera of these animals, however, showed elevation in the activities of all five glycosidases. Preliminary experiments indicate that only the intermediate isoenzyme of NAGase, putatively insulin dependent, is elevated in the eyes of these diabetic rats. NAGase was also specifically elevated in the intraocular fluid from monkey eyes with retinal vein occlusion (RVO), and markedly so if NV was present. These results suggest the involvement of NAGase in the neovascular process in the eye.     

Expression of advanced glycation end products and related molecules in diabetic fibrovascular epiretinal membranes

Purpose: To investigate associations between expressions of advanced glycation end products (AGEs), transforming growth factor‐β (TGF‐β), tumour necrosis factor‐α (TNF‐α) and integrins and correlations between their expression and level of vascularization and proliferative activity in diabetic fibrovascular epiretinal membranes. Methods: Membranes from eight patients with active proliferative diabetic retinopathy and nine patients with inactive proliferative diabetic retinopathy were studied by immunohistochemistry. Results: Blood vessels expressed AGEs, TGF‐β, TNF‐α and α_vβ₃ integrin in 5, 13, 8 and 8 membranes, respectively. Stromal cells expressed AGEs, TNF‐α and α_vβ₃ integrin in 15, 13 and 3 membranes, respectively. There was no immunoreactivity for α_vβ₅, α₅β₁ and α₂β₁ integrins. There were significant correlations between number of blood vessels expressing CD34 and number of blood vessels expressing AGEs (r_s = 0.496; P = 0.043), TGF‐β (r_s = 0.777; P < 0.001) and TNF‐α (r_s = 0.699; P = 0.002). There were significant correlations between number of blood vessels expressing AGEs and number of blood vessels expressing TGF‐β (r_s = 0.532; P = 0.028) and TNF‐α (r_s = 0.626; P = 0.007). The correlation between number of blood vessels expressing TNF‐α and α_vβ₃ integrin was significant (r_s = 0.617; P = 0.008). Number of blood vessels expressing CD34 (P = 0.001), TGF‐β (P = 0.006) and TNF‐α (P = 0.002) and stromal cells expressing AGEs (P = 0.001) and TNF‐α (P = 0.004) were significantly higher in active membranes than in inactive membranes. Conclusion: Interactions of AGEs, TGF‐β, TNF‐α and α_vβ₃ integrin might be involved in pathogenesis of proliferative diabetic retinopathy fibrovascular proliferation.     

糖基化终末产物与糖尿病瞳孔功能异常的关系

目的探讨虹膜中糖基化终末产物（AGEs）的表达及血浆AGEs质量浓度与糖尿病患者瞳孔功能障碍的关系,并探讨其发病机制。方法对糖尿病并发白内障患者的瞳孔散大难易度与单纯白内障患者进行对照研究。依据白内障手术前瞳孔散大是否能够达到6mm,将2型糖尿病并发白内障患者分为瞳孔功能异常组24例和瞳孔功能正常组21例,单纯白内障组患者21例作为对照。采用竞争性酶联免疫吸附分析法测定各组患者血浆AGEs的吸光度（A）值;采用免疫组织化学法分别检测上述各组患者虹膜组织中AGEs的表达强度。结果 3组在性别、年龄、眼压和糖尿病病程方面差异均无统计学意义（P〉0.05）。瞳孔功能异常组、瞳孔功能正常组和单纯白内障组血浆AGEs质量浓度分别为7.625±1.69、5.904±1.27和3.726±1.35,组间差异均有统计学意义（F=312.431,P〈0.05）;瞳孔功能正常组和单纯白内障组的血浆AGEs质量浓度明显低于瞳孔功能异常组,差异均有统计学意义（t1=2.017,P〈0.05;t2=2.018,P〈0.05）。瞳孔功能异常组、瞳孔功能正常组和单纯白内障组AGEs在虹膜组织中的表达强度值分别为109.13±19.47、79.71±13.06和40.92±11.91,差异有统计学意义（F=188.32,P〈0.05）,瞳孔功能正常组和单纯白内障组的AGEs表达值明显低于瞳孔功能异常组,差异均有统计学意义（t1=2.017,P〈0.05;t2=2.023,P〈0.05）。结论 AGEs在糖尿病瞳孔功能异常患者虹膜组织中呈高表达,其血浆AGEs质量浓度亦明显升高,提示AGEs在虹膜组织中的积聚与糖尿病患者瞳孔功能异常有关,可能是其发病机制之一。     

糖基化终产物对牛视网膜毛细血管周细胞内抗氧化能力的影响

目的 通过测量糖基化终产物对培养的牛视网膜毛细血管周细胞抗氧化物酶和脂质过氧化物含量的影响, 以探讨氧化应激在糖尿病视网膜病变进程中的作用。 方法 不同浓度的糖基化终产物（0，8，32，125，500，2 000 μg/ml）与周细胞作用4 d后，以分光光度法测量细胞内过氧化氢酶的活性及过氧化脂质丙二醛的含量。 结果 糖基化终产物能以剂量依赖的方式降低周细胞内过氧化氢酶的活性(r=-0.714, P＜0.01)，增加丙二醛的含量(r=0.748, P＜0.01)，与对照组相比，当糖基化终产物浓度达到32 μg/ml时，两者比较差异有显著性的意义（P＜0.01）。 结论 氧化应激的增加可能是早期糖尿病视网膜病变中周细胞丧失的原因之一。 (中华眼底病杂志, 2002, 18: 143-145)     

视网膜微血管周细胞内明胶酶的表达及影响

目的　检测糖基化终产物 (AGE)对培养的牛视网膜微血管周细胞 (BRPs)分泌明胶酶的作用 ,以探讨糖尿病视网膜病变 (DRP)的发病机制。方法　不同质量浓度AGE( 0、8、3 2、12 5、5 0 0、2 0 0 0 μg/mL)与BRPs作用 4d后 ,明胶酶谱分析细胞所分泌的明胶酶。结果　正常BRPs有明胶酶 A的分泌 ,相对分子质量分别为 72 0 0 0的原酶及 62 0 0 0的活性酶 ;扫描单位分别为 13 3 73± 6 66及 160 18± 15 2 9,另有少量 92 0 0 0的明胶酶 B分泌 ,扫描单位 93 0 1± 5 89。AGE能以剂量依赖的方式抑制BRPs内明胶酶 A的分泌 (原酶和活性酶与AGE相关系数分别为r =-0 798及r =-0 73 4,P <0 0 1)。结论　周细胞分泌明胶酶 A的下降可能是DRP中基底膜增厚的重要原因之一     

糖基化终末产物在青光眼中的研究进展

糖基化终末产物(AGEs)体内多种组织中累积,通过调节相关因子表达及激活信号通路等诱发一系列生物学反应,引起年龄相关性疾病及神经退行性病变,如阿尔茨海默病、帕金森病、动脉粥样硬化。青光眼是一种视神经退行性病变,最终导致不可逆的视野缺失,是全球仅次于白内障导致视力丧失的主要原因。青光眼患者视网膜等眼组织中过多AGEs累积,通过激活信号通路、引发生物反应,对组织、细胞的结构及功能造成损伤,参与青光眼发生发展的病理过程。本文主要阐述AGEs在青光眼发病机制、治疗、筛查等相关研究中的最新进展,为青光眼的防治提供新的思路和研究方法。     

Immunohistochemical localization of advanced glycation end products in pinguecula

Background Advanced glycation end products (AGEs) are known to be deposited in the target organ of ageing. In addition, the deposition of AGEs accelerate the process of ageing. We investigated the immunohistochemical localization of AGEs in pinguecula, one of the ocular changes related with ageing process. Methods Surgical specimens of conjunctiva with or without pinguecula were prepared from nine patients, respectively. Immunohistochemical localization of AGEs was investigated using monoclonal antibodies to-(carboxymethyl)lysine, pentosidine, imidazolone, and pyrraline. Results Moderate to strong immunoreactivities to AGEs were detected in the subepithelial amorphous deposits of all the surgical specimens with pinguecula. In contrast, no or weak immunoreactivities to AGEs were detected in the surgical specimens without pinguecula. Conclusions Pinguecula is an aggregation of AGEs-modified proteins. The presence of pinguecula would be an index of local irradiation of ultraviolet rays and decreased antioxidant activities. Financial interest: none     

高级糖基化终末产物与糖尿病视网膜病变的关系

高级糖基化终末产物(advanced glycation end products,AGE)的堆积是糖尿病视网膜病变(diabetic retinopathy,DR)主要的致病因素,其可促进视网膜色素上皮(retinal pigment epithelium,RPE)细胞AGE受体(receptor of AGE,RAGE)的表达、血管内皮生长因子(vascular endothelial growth factor,VEGF)的分泌及细胞凋亡损伤.RPE细胞参与构成视网膜的外屏障,具有屏障、滤过、吞噬和分泌等作用,在维持视网膜正常生理功能方面具有重要作用,RPE细胞的功能损害会导致多种视网膜疾病.通过研究AGE对RPE细胞的损伤,探讨AGE在DR发生发展中的作用,有利于寻找有效防治DR的新途径.     

Receptor for advanced glycation end products and age-related macular degeneration

PURPOSE: Advanced glycation end products (AGE) exacerbate disease progression through two general mechanisms: modifying molecules and forming nondegradable aggregates, thus impairing normal cellular/tissue functions, and altering cellular function directly through receptor-mediated activation. In the present study receptor for AGE (RAGE)-mediated cellular activation was evaluated in the etiology of human retinal aging and disease. METHODS: The maculas of human donor retinas from normal eyes and eyes with early age-related macular degeneration (AMD) and advanced AMD with geographic atrophy (GA) were assayed for AGE and RAGE by immunocytochemistry. Cultured ARPE-19 cells were challenged with known ligands for RAGE, AGE, and S100B, to test for activation capacity. Immunocytochemistry, real-time RT-PCR, immunoblot analysis, and the TUNEL assay were used to determine the consequences of RPE cellular activation. RESULTS: Little to no immunolabeling for AGE or RAGE was found in photoreceptor and RPE cell layers in normal retinas. However, when small drusen were present, AGE and RAGE were identified in the RPE or both the RPE and photoreceptors. In early AMD and GA, the RPE and remnant photoreceptor cells showed intense AGE and RAGE immunolabeling. Both AGE and S100B activated cultured RPE cells, as revealed by upregulated expression of RAGE, NFkappaB nuclear translocation, and apoptotic cell death. CONCLUSIONS: Immunolocalization of RAGE in RPE and photoreceptors coincided with AGE deposits and macular disease in aged, early AMD, and GA retinas. Further, AGE stimulated RAGE-mediated activation of cultured ARPE-19 cells in a dose-dependent fashion. AGE accumulation, as occurs with normal aging and in disease, may induce receptor-mediated activation of RPE/photoreceptor cells, contributing to disease progression in the aging human retinas.     

Relationship between autofluorescence and advanced glycation end products in diabetic lenses

Autofluorescence and advanced glycation end product (AGE) levels were measured in the lenses of 9 diabetic Chinese hamsters and 6 age-matched controls. Lens autofluorescence also was measured in 37 diabetic patients and 14 age-matched controls. Lens autofluorescence values were measured noninvasively with a lens measurement system using color filters with peak transmission at 365- and 434-nm wavelengths (excitation and emission, respectively) that are characteristic of AGE fluorescence. The peak lens autofluorescence level was used as the lens autofluorescence value, and the mean lens autofluorescence values from both eyes of each subject were used for statistical analysis. The AGE levels in one lens from each hamster were measured by noncompetitive enzyme-linked immunosorbent assay with a polyclonal anti-AGE antibody. We found a 2.2 times increase of the mean lens autofluorescence value of diabetic hamsters in comparison with that of controls (P<0.01). We also found a 1.5 times increase of the mean AGE level from the lenses of diabetic hamsters in comparison with that of controls (P<0.01). Moreover, a statistically significant positive correlation between the AGE level and autofluorescence value in the same lenses was observed in all hamsters (rho=0.58, P<0.05). In human subjects, we found a 1.4 times increase of the mean lens autofluorescence value of diabetic patients in comparison with that of age-matched controls (P<0.01). Our results suggest that non invasive measurement of lens autofluorescence may be a guide to AGE levels in lenses.     

超氧化物歧化酶在视网膜毛细血管周细胞凋亡中的活性及意义

目的通过检测糖基化终产物（AGE）诱导培养的牛视网膜毛细血管周细胞凋亡及凋亡周细胞中超氧化物歧化酶（SOD）的活性及意义，进一步探讨糖尿病视网膜病变（DR）的发生机制。方法周细胞分别与不同浓度的AGE（0．47、1．88、7．5μmol／L）共同培养4d后，分别检测细胞凋亡、SOD活性，以及SOD对细胞凋亡及凋亡调节基因Bcl-2／Bax比率的影响。结果AGE能以浓度依赖的方式诱导周细胞凋亡（r=0．878，P〈0．01）、降低细胞内SOD的活性（r=-0．878，P〈0．01），而应用SOD能明显抑制AGE作用下的周细胞凋亡及提高凋亡调节基因Bcl-2／Bax的比率.结论凋亡及氧化应激的增加是DRP中周细胞选择性丧失的主要原因，SOD活性的下降是AGE诱导周细胞发生凋亡的关键因素。     

糖基化终末产物在眼部疾病中的作用

糖基化终末产物(AGEs)是由还原糖的醛或酮幕与蛋白质、脂质或核酸的氨基发生复杂的化学反应生成的终产物,是糖尿病并发症与年龄相关性疾病发生发展的重要因素.目前,AGEs在眼部的致病作用日益受到重视.研究表明,循环血中沉积在眼部的AGEs以及眼组织中的长寿蛋白糖化形成的AGEs与其受体相互作用,导致了糖尿病和年龄相关的角膜病变、晶状体混浊、玻璃体液化、视网膜病变,最终严重威胁视力.就AGEs在糖尿病和年龄相关性眼病中的作用进行综述.     

Increased levels of advanced glycation end products in human cataractous lenses

PURPOSE: To investigate the occurrence of advanced glycation end products (AGEs) formed oxidatively (pentosidine and N(epsilon)-carboxymethyl lysine [CML]) or nonoxidatively (imidazolone) in human lenses and the relation of AGEs to lens coloration, cataract type, and patients' diabetic state. SETTING: Departments of Ophthalmology and Internal Medicine III, University of Jena, Jena, Germany. METHODS: Pentosidine, CML, and imidazolone concentrations were measured in the water-soluble protein fraction of 44 cataractous lenses (from 24 nondiabetic and 20 diabetic donors) and 6 noncataractous control lenses. RESULTS: Pentosidine, CML, and imidazolone were higher in cataractous lenses than in control lenses (pentosidine, 3.7 pmol/mg +/- 5.3 (SD) and 1.9 +/- 1.7 pmol/mg, respectively; CML, 3.0 +/- 2.2 nmol/mg and 1.3 +/- 0.7 nmol/mg, respectively; imidazoline, 80.4 +/- 93.3 AU/mg and 19.6 +/- 18.5 AU/mg, respectively). Among the cataractous lenses, the highest AGE concentrations were found in mature cataracts, with a statistically significant increase in CML. The AGE content increased relative to the intensity of brown coloration of the lens; the brown coloration also indicated the highest rise of imidazolone compared to pentosidine and CML. Lenses from diabetic donors had generally similar pentosidine values and elevated CML and imidazolone levels compared to lenses from nondiabetic donors. The pentosidine, CML, and imidazolone levels in the lenses correlated significantly with one another but not with patient age. CONCLUSION: Advanced glycation end products formed oxidatively and nonoxidatively occurred to a higher degree in cataractous lenses than in noncataractous lenses. The strong relationship between the lenses' AGE content, color/opacity, and the state of the cataract may indicate that advanced glycation plays a pivotal role in cataract formation.     

半胱氨酸天冬氨酸蛋白酶在视网膜毛细血管周细胞凋亡中的活性及意义

目的探讨糖基化终产物（AGE）在糖尿病视网膜病变（DR）发生中的作用。方法培养的牛视网膜毛细血管周细胞分别与不同浓度（0．47、1．88、7．50μmol／L）的AGE共同培养4d后,分别检测细胞凋亡、半胱氨酸天冬氨酸蛋白酶（caspase-3）活性及caspase-3抑制剂Z—DEVD-fmk对周细胞凋亡及凋亡调节基因Bcl-2／Bax比率的影响。结果AGE能以剂量依赖的方式诱导培养的牛视网膜毛细血管周细胞凋亡（r=0．867,P〈0．01）、增加细胞内caspase-3的活性,而选择性caspase-3抑制剂Z—DEVD—fmk能明显抑制AGE作用下的周细胞凋亡及提高凋亡调节基因Bcl-2／Bax的比率。结论凋亡是DR中视网膜毛细血管周细胞选择性丧失的机制之一,caspase-3活性的增加是AGE诱导周细胞发生凋亡的关键因素。     

Induction of an aging mRNA retinal pigment epithelial cell phenotype by matrix-containing advanced glycation end products in vitro

PURPOSE: To determine an extensive mRNA phenotype of the established RPE cell line ARPE-19 when grown on a matrix modified by advanced glycation end products (AGEs). METHODS: Growth Factor Reduced Matrigel (Collaborative Biomedical Products, Bedford, MA) was nonenzymatically glycated with glycolaldehyde. ARPE-19 cells were seeded on both AGE-Matrigel and Matrigel and grown to confluence, and serum was withdrawn for 3 days. RNA was extracted, and microarray analysis was performed to characterize the genes, which are altered by a matrix modified by AGEs. Gene expression changes were confirmed by RT-PCR/Southern and Northern blot analysis. Apoptosis was measured by annexin V/propidium iodide labeling. RESULTS: Clusters of genes with altered expression were found related to cell differentiation, growth factors that regulate the RPE cell and basement membrane, and apoptosis. RT-PCR/Southern and Northern blot analysis confirmed the expression patterns of selected genes, and flow cytometry showed increased annexin V/propidium iodide-labeled cells when grown on AGE-Matrigel. CONCLUSIONS: Microarray analysis identified clusters of genes that could promote an aging RPE phenotype in vitro induced by a matrix modified with AGEs.     

糖尿病性白内障糖基化终末产物及其抑制剂的研究进展

糖尿病性白内障是一类重要的代谢型白内障,其形成机制目前尚不十分明确。近年的研究表明,糖基化终末产物（AGEs）在糖尿病性白内障发生发展中发挥着重要作用,而糖基化与血糖水平密切相关,血糖值的明显增加可加速糖基化反应速率,糖尿病性白内障形成也明显加快。鉴此,近来关于AGEs及糖基化抑制剂的研究也日益引起学者的关注。就AGEs的概念、在糖尿病性白内障发病中的作用及AGEs抑制剂对糖尿病性白内障发生发展的影响及其药物治疗方面的研究进展进行综述。     

糖基化终末产物对牛视网膜微血管内皮细胞和周细胞存活和形态的影响

目的 观察体外孵育的牛血清白蛋白(BSA)非酶促糖基化终末产物（AGEs）对牛视网膜微血管内皮细胞（BREC）和周细胞（BRP）存活和形态的影响。 方法 取终浓度为50mg/ml的牛血清白蛋白（BSA）、500 mmol/L D-葡萄糖，于37℃孵箱内避光孵育12周，制备外源性AGEs-BSA，经Sephacryl S-300层析纯化，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS PAGE）蛋白电泳鉴定AGEs-BSA和coomassie 蛋白质定量法测定蛋白浓度。分设不同浓度梯度的AGEs-BSA实验组和BSA对照组，以及空白对照组，分别观察体外孵育的AGEs-BSA对体外培养的BREC和BRP的毒性作用。相差倒置显微镜观察500μg/ml AGEs-BSA和BSA作用48 h对BREC和BRP形态的影响。 结果 随着AGEs-BSA剂量的增加，细胞被抑制呈上升趋势。500μg/ml AGEs-BSA抑制BREC为空白对照组的（72.8±15.9）%，抑制周细胞为空白对照组的（64.8±9.0）%。低浓度AGEs-BSA对BREC有一定促增生作用，但与空白对照组比较无统计学意义（P=0.231）。相差倒置显微镜观察结果显示AGEs-BSA处理组细胞增殖受抑制，失去正常细胞形态，而BSA对照组细胞同空白对照组，细胞形态正常。 结论 AGEs-BSA在高浓度时，无论是对BREC还是BRP，都产生生长抑制作用，从而导致BRP的丢失，损伤血管功能。进一步证实了非酶糖化是糖尿病微血管并发症的一个重要原因。 (中华眼底病杂志, 2006, 22: 11-15)     

晚期糖基化终末产物与糖尿病性白内障的基础与临床研究

晚期糖基化终末产物(advanced glycation end-products,AGE)是一类结构复杂的化学分子,它的形成与糖尿病性白内障的发生有着密切的联系。研究表明,AGE可以直接或者通过相应受体的作用导致糖尿病性白内障的形成。通过药物抑制AGE的生成等则可以明显延缓糖尿病性白内障的发生。本文就AGE的生化特征、导致糖尿病性白内障发生的机制、检测手段、药物研究及药物治疗方法作一综述。