糖尿病大鼠早期视网膜形态观察和Bcl-2、Bax及VEGF表达的意义

目的：探讨B-细胞淋巴瘤因子(B-cell lymphoma factor,Bcl-2),Bcl-2相关X蛋白(Bcl2-Associated X protein,Bax)和血管内皮生长因子(vascular endothelial growth factor,VEGF)在早期糖尿病大鼠视网膜上的表达及意义。

方法：用链脲佐菌素(streptozotocin,STZ)腹腔注射(60mg/kg)制作大鼠早期糖尿病模型。于造模后4、8、12wk颈椎脱臼法处死大鼠,取双眼全眼球组织做石蜡切片并做视网膜铺片,通过HE染色观察视网膜各层的形态学和血管分布变化; 取双眼视网膜组织石蜡切片,通过免疫组化法检测Bcl-2、Bax和VEGF在视网膜组织中的表达。行ADP酶视网膜血管染色,观察视网膜血管形态变化。应用激光共聚焦显微镜检测视网膜细胞的形态、细胞中Ca²⁺的荧光强度和分布变化。

结果：糖尿病组造模12wk突破内界膜的内皮细胞核个数呈递增趋势。糖尿病组视网膜中周部和周边部可见无血管区,无血管区面积也明显大于空白对照组,差异有统计学意义(P<0.05)。糖尿病组大鼠VEGF、Bcl-2和Bax光密度值与空白对照组比较,差异均有统计学意义(P<0.05)。糖尿病组大鼠造模4、8、12wk比较,RGCs内钙离子荧光浓度逐渐升高,荧光染色强度比值升高,差异均有统计学意义(P<0.05)。

结论：早期糖尿病大鼠视网膜的Bcl-2和Bax表达非常明显,从而上调 VEGF的表达。Bcl-2、Bax和VEGF是糖尿病视网膜病变中新生血管形成的重要影响因素。     

糖尿病大鼠眼内VEGF阶段性表达的Meta分析

目的：通过分析确定VEGF在糖尿病大鼠眼病各阶段表现,为临床治疗阶段提供最有效的参考。

方法：通过检索PubMed、EMBASE、Medline、中国知网、万方、维普等数据库,筛选以糖尿病大鼠为模型研究VEGF在糖尿病眼病病程中表达的随机对照实验。对入选文献用Jadad评分表(共5分)进行评分,经异质性检验后采用固定效应模型进行数据综合,按时间段进行亚组分析。

结果：纳入高质量文献8篇,异质性检验排除两篇。数据综合显示糖尿病大鼠眼内VEGF的表达上升了1.62%, 95%CI(1.20,2.03),P<0.01。

结论：VEGF在2～6月龄糖尿病大鼠眼内组织各个阶段中表达增强,但强度在逐渐下降。     

Ibrolipim对糖尿病小型猪视网膜血管内皮生长因子表达的影响

目的　观察血管内皮生长因子(VEGF)在早期糖尿病视网膜病变中的表达以及Ibrolipim (NO 1886)对 2型糖尿病小型猪血糖的影响;探讨药物Ibrolipim对视网膜VEGF的作用。 方法　采用高脂高蔗糖饲料喂养贵州小香猪,创建 2型糖尿病动物模型,并用Ibrolipim进行干预,用RT PCR、免疫组织化学和Westernblot技术检测VEGF转录和蛋白表达。 结果　正常组视网膜VEGFmRNA表达量少,免疫组织化学反应阳性较弱,VEGF蛋白表达较低;实验组视网膜VEGFmRNA表达上调,免疫组织化学反应呈强阳性 (P<0 01 ),VEGF蛋白表达增高;治疗组视网膜VEGFmRNA及VEGF蛋白表达介于其他两组之间。 结论　早期糖尿病视网膜中VEGF表达增高;Ibrolipim可通过降低血糖,改善视网膜组织缺氧,来抑制VEGFmRNA及蛋白的表达。     

血管内皮细胞生长因子在糖尿病性视网膜病变进展中的作用

糖尿病性视网膜病变是糖尿病微血管病变常见而严重的并发症,在很大程度上导致不可逆的视功能损害或完全失明.VEGF在DR发生、发展,尤其在视网膜新生血管形成过程中发挥重要作用,本文就血管内皮生长因子与糖尿病性视网膜病变的关系进行综述。     

Early electroretinographic features of streptozotocin-induced diabetic retinopathy

Background: This study set out to document the early electrophysiological and immunohistochemical changes that occur in the retina of experimentally induced diabetic rats. Methods: Diabetes was induced in rats by intraperitoneal injection of 60 mg/kg of streptozotocin (STZ). Electroretinogram readings were taken monthly under either short‐duration or long‐duration stimuli for up to 3 months after STZ. Oscillatory potentials (OP) and the amplitudes and implicit times of a‐ and b‐waves were analysed, and b‐wave amplitudes were analysed using a Naka–Rushton fit. Scotopic a‐waves were analysed with photoreceptor models, and Rmp3 (the maximum a‐wave amplitude) and S (sensitivity) were calculated. Three months after STZ injection, immunohistochemistry for glial fibrillary acidic protein was performed on the retinas of the STZ‐treated rats and age‐matched controls. Results: The implicit OP times were significantly longer in the diabetic rats as compared with the controls, and this difference was noted as early as 1 month following STZ treatment. Other electrophysiological parameters, such as OP amplitudes, a‐ and b‐wave amplitude as well as the implicit times, did not differ from controls at this stage. The sacrificed STZ‐treated rats also demonstrated marked enhancement of glial fibrillary acidic protein immunoreactivity, suggesting that at least in experimentally induced diabetic retinopathy there is increased Müller cell reactivity. Conclusion: The results of this study indicated that functional alterations in the retina develop rapidly after the onset of diabetes. Analysis of each electroretinogram component may be useful in further investigating the development mechanisms of diabetic retinopathy.     

血管内皮生长因子及其受体与糖尿病视网膜病变

视网膜新生血管形成和黄斑水肿是糖尿病视网膜病变(diabeticretinopathy,DR)的主要临床表现,也是DR主要的致盲原因。目前研究表明血管内皮生长因子(vascularen-dothelialgrowthfactor,VEGF)在糖尿病视网膜微血管并发症的发生中发挥重要作用,因此VEGF成为当今DR治疗干预的一个研究热点。本文就VEGF及其受体在DR中的表达及其相关治疗措施进行综述。     

坎地沙坦对糖尿病大鼠视网膜VEGF和MCP-1表达的影响

目的:研究血管紧张素Ⅱ受体拮抗剂坎地沙坦对糖尿病(DM)大鼠视网膜组织VEGF和MCP-1表达的影响。方法:链脲佐菌素(STZ)制备DM大鼠动物模型36只,随机分为DM模型组和坎地沙坦治疗组,另取18只正常SD大鼠作为正常对照组,每组均随机分为4,8,12wk3个亚组。视网膜铺片联合PAS染色观察视网膜微血管形态学变化,应用SABC免疫组织化学法检测坎地沙坦对大鼠视网膜组织VEGF和MCP-1表达的影响。结果:正常对照组视网膜血管网结构清晰,走行规则;DM模型组血管迂曲阻塞,走行不规则;治疗组见血管网迂曲情况较模型组明显改善,走行较规则。在对照组和模型4wk组中大鼠视网膜组织无VEGF和MCP-1阳性表达或只呈弱阳性表达;模型8wk和12wk组两者阳性表达明显增强,且随着病程延长呈递增趋势。治疗组两者的表达则均较同时期模型组明显减弱,差异有统计学意义(P<0.05)。结论:研究血管紧张素Ⅱ受体拮抗剂坎地沙坦可降低DM大鼠视网膜VEGF和MCP-1的表达。     

VEGF和PEDF在糖尿病大鼠视网膜脉络膜中的表达

目的:通过免疫组织化学法比较正常大鼠及糖尿病大鼠视网膜、脉络膜组织中VEGF和PEDF的表达情况及相互关系。方法:选取健康雄性Wistar大鼠,随机分成糖尿病组和正常对照组(M0),用链脲佐菌素大剂量一次性腹腔注射诱导1型糖尿病模型。成模后大鼠随机平均分成1mo(M1),2mo(M2),3mo(M3)及5mo(M5)组,免疫组织化学法检测VEGF和PEDF在各组大鼠视网膜及脉络膜的表达。结果:M1大鼠VEGF在视网膜及脉络膜的表达均与M0无明显差别;M2大鼠VEGF在脉络膜有阳性表达(33.3%),在视网膜的表达与M0无明显差别;M3大鼠VEGF在脉络膜有阳性表达(55.6%),在视网膜的表达与M0比较有明显差别(33.3%);M5大鼠VEGF在视网膜及脉络膜阳性表达(88.9%);M1和M2大鼠视网膜PEDF表达与M0比较无明显差别,各组脉络膜均无PEDF表达;M3和M5大鼠视网膜PEDF表达均较M0减弱,且随病程延长表达逐渐减弱(P<0.05),各组脉络膜均无PEDF表达;随着糖尿病病程延长,VEGF/PEDF比值逐渐增大,与M0相比有显著统计学差异(P<0.01)。结论:随着糖尿病病程的延长,VEGF在视网膜及脉络膜的表达均逐渐增强,而PEDF与之相反,在视网膜的表达逐渐减弱,且VEGF/PEDF比值逐渐增大,提示VEGF和PEDF均与糖尿病病程密切相关。     

Placental growth factor and its potential role in diabetic retinopathy and other ocular neovascular diseases

The role of vascular endothelial growth factor (VEGF), including in retinal vascular diseases, has been well studied, and pharmacological blockade of VEGF is the gold standard of treatment for neovascular age‐related macular degeneration, retinal vein occlusion and diabetic macular oedema. Placental growth factor (PGF, previously known as PlGF), a homologue of VEGF, is a multifunctional peptide associated with angiogenesis‐dependent pathologies in the eye and non‐ocular conditions. Animal studies using genetic modification and pharmacological treatment have demonstrated a mechanistic role for PGF in pathological angiogenesis. Inhibition decreases neovascularization and microvascular abnormalities across different models, including oxygen‐induced retinopathy, laser‐induced choroidal neovascularization and in diabetic mice exhibiting retinopathies. High levels of PGF have been found in the vitreous of patients with diabetic retinopathy. Despite these strong animal data, the exact role of PGF in pathological angiogenesis in retinal vascular diseases remains to be defined, and the benefits of PGF‐specific inhibition in humans with retinal neovascular diseases and macular oedema remain controversial. Comparative effectiveness research studies in patients with diabetic retinal disease have shown that treatment that inhibits both VEGF and PGF may provide superior outcomes in certain patients compared with treatment that inhibits only VEGF. This review summarizes current knowledge of PGF, including its relationship to VEGF and its role in pathological angiogenesis in retinal diseases, and identifies some key unanswered questions about PGF that can serve as a pathway for future basic, translational and clinical research.     

Elevation of serum apelin-13 associated with proliferative diabetic retinopathy in type 2 diabetic patients

AIM:To compare apelin-13, a ligand of G-protein-coupled receptor which has been shown to be involved in retinal angiogenesis, and vascular endothelial growth factor (VEGF) serum levels in type 2 diabetes mellitus (T2DM) with or without retinopathy, and to investigate the relationship between the serum concentration of apelin-13 and diabetes retinopathy.METHODS: Sixty-nine patients with T2DM were enrolled. Of the 69 patients, 16 had proliferative diabetic retinopathy (PDR group), 23 had non-PDR (NPDR group) and 30 had no retinopathy (T2DM group). Subjects’ information, including demographics, medical history, and use of medications were recorded. Their serum samples were collected for measuring the levels of C-reactive protein (CRP), serum lipid and glycosylated hemoglobin. Apelin-13 and VEGF serum levels were measured by enzyme-linked immunosorbent assay. Kruskal-Wallis test and one-way ANOVA were used to compare the differences among these groups. Chi-square test was used to assess categorical variables. Correlations between variables were investigated by Spearman rho correlation test and stepwise regression analysis. All statistical analyses were performed through SPSS 17.0 software.RESULTS:Sex, age, body mass index (BMI), blood pressure, CRP, hemoglobin A1c (HbA1c) have no significantly difference in the three groups. Serum level of apelin-13 was significantly elevated in PDR group as compared with T2DM group (P=0.041). Differences of VEGF serum concentration in the three groups were statistically significant (P=0.007, P=0.007 and P<0.001, respectively). Spearman rho correlation test showed that serum apelin-13 was positively correlated with BMI, serum triglycerides, VEGF, but not with age, duration of diabetes, blood pressure, CRP, HbA1c and total-cholesterol. Stepwise regression analysis showed that BMI also significantly associated with serum apelin-13 (P=0.002), while VEGF and serum triglycerides were irrelevant.CONCLUSION: This study elucidated a positive association of apelin-13 serum level with PDR, but not with VEGF. Apelin-13 may influence the promotion of PDR but unrelated with VEGF.     

Beneficial effects of protocatechuic acid on diabetic retinopathy in streptozocin-induced diabetic rats

AIM: To determine the effects of protocatechuic acid (PCA) on streptozocin-induced diabetic retinopathy (DR) in rats.METHODS: Wistar rats were given a 50 mg/kg intraperitoneal injection of streptozocin to induce diabetes. Animals were assigned randomly one of four groups (8 rats per group): control, diabetic, diabetic plus PCA (25 mg/kg•d), and diabetic plus PCA (50 mg/kg•d). After inducing diabetes, treatments were started one week later and continued for eight weeks. After the experiment, the rats were sacrificed, and their retinas were taken for biochemical and molecular analysis.RESULTS: PCA administration diminished the blood glucose and glycated haemoglobin levels relative to the diabetic group. In diabetic rats, PCA lowered elevated levels of advanced glycosylated end products (AGEs) and receptor for AGEs (RAGE). In the retina of diabetic rats, PCA effectively decreased inflammatory cytokine, nuclear factor-κB, tumour necrosis factor-α, interleukin-1β, and vascular endothelial growth factor, and increased antioxidant markers glutathione, superoxide dismutase, and catalase.CONCLUSION: The protective benefits of PCA against DR may be attributable to its suppression of the AGEs and RAGE and its antioxidant and anti-inflammatory properties.     

贝那普利对糖尿病大鼠视网膜微血管病变的保护作用

目的:探讨贝那普利(benazepril,BZ)对糖尿病大鼠视网膜微血管病变的保护作用及机制。方法:链脲佐菌素ip制备糖尿病大鼠模型,分为糖尿病组(DM)、贝那普利治疗组(BZ),同时设立正常对照组(Con)。治疗组每天灌胃给予BZ10mg/kg。24wk后放射免疫法测定血浆AngⅡ水平,免疫组织化学法和Westernblot技术检测视网膜VEGF蛋白表达,透射电镜观察视网膜血管基底膜厚度变化。结果:与Con组相比,DM组大鼠血浆AngⅡ水平明显增高(P<0.01),视网膜VEGF蛋白表达显著增强(P<0.01),视网膜微血管基底膜明显增厚;与DM组相比,BZ治疗后大鼠血浆AngⅡ水平明显降低(P<0.05),视网膜VEGF蛋白表达则显著减弱(P<0.01),视网膜微血管基底膜增厚明显减轻。结论:BZ可通过抑制血浆AngⅡ水平,减少糖尿病大鼠视网膜组织VEGF表达,抑制视网膜微血管基底膜增厚。     

糖尿病视网膜病变药物治疗的研究进展

随着糖尿病视网膜病变(diabetic retinopathy,DR)发病机制的深入研究,目前对DR的药物治疗取得了一定的成果,包括糖皮质激素、抗血管内皮细胞生长因子、抗血小板源性生长因子、非甾体类消炎药以及改善视神经功能的药物.目前治疗糖尿病黄斑水肿,玻璃体注药治疗特别是抗血管内皮细胞生长因子药物已逐渐推广.     

抗血管内皮生长因子药物治疗糖尿病视网膜病变的研究现状

糖尿病可引起眼内血管内皮生长因子（vascularendothelialgrowthfactor,VEGF）水平病理性升高,导致眼内新生血管形成、黄斑水肿的发生。抗VEGF药物最早被用于湿性年龄相关性黄斑变性,现也被试验性地用于糖尿病视网膜病变（diabeticretinopathy,DR）。VEGFl65选择性拮抗剂Pe—gaptanib与VEGF—A单抗Ranibizumab被批准玻璃体内注射,且对DR有较好的疗效;VEGF．A全长抗体Bevacizumab被标示外用于玻璃体内注射治疗DR,也能达到较好的效果;重组融合蛋白Aflibercept针对DR的疗效也得到一些试验的支持。玻璃体内注射抗VEGF药物治疗DR已被证实短期有效且安全,但其长期的疗效与安全性有待更多的大规模临床试验来验证。     

VEGF localisation in diabetic retinopathy

AIM—To determine the staining pattern of vascular endothelial growth factor (VEGF) at different stages of diabetic retinopathy (including post-laser photocoagulation) and to compare staining in excised fibrovascular and fibrocellular (non-diabetic) preretinal membranes.
METHODS—Immunohistochemical localisation of VEGF, using antibodies raised against VEGF₁₆₅ and VEGF_121,165,189, was carried out on specimens of normal human retina (n=15), diabetic retinas ((a) with no overt retinopathy (n=19), (b) with intraretinal vascular abnormalities but no proliferative retinopathy (n=6), (c) with active proliferative retinopathy (n=6), (d) with no residual proliferative retinopathy after photocoagulation therapy (n=15)), excised diabetic fibrovascular membranes (n=19), and non-diabetic fibrocellular membranes (n=7). The degree and pattern of immunostaining was recorded.
RESULTS—In general, VEGF was absent from the majority of normal retinas. VEGF staining was apparent in most diabetic tissues but the staining pattern was dependent on both the specificity of the antibody used and the category of tissue. Staining with the VEGF₁₆₅ antibody was generally confined to endothelial cells and perivascular regions while the VEGF_121,165,189 antibody was also associated with extravascular components of the inner retina. Intensity of immunostaining of diabetic eyes was dependent on the severity of retinopathy being least in diabetics with no overt retinopathy and greatest in retinas with proliferative retinopathy. Interestingly, the intensity of immunostaining in diabetic retinas which had undergone laser surgery for proliferative retinopathy was reduced to basal levels. Moderate to intense immunostaining was observed in all fibrovascular and fibrocellular membranes examined.
CONCLUSIONS—This study supports a circumstantial role for VEGF in the pathogenesis of both the preclinical and proliferative stages of diabetic retinopathy.

Keywords: vascular endothelial growth factor; VEGF; diabetes; diabetic retinopathy     

糖尿病大鼠视网膜中缺氧诱导因子-1α和血管内皮生长因子表达的研究

目的:检测缺氧诱导因子-1α(HIF-1α)与血管内皮生长因子(VEGF)在糖尿病大鼠视网膜中的表达,探讨其在糖尿病视网膜病变(DR)发生发展中的作用。方法:SD大鼠120只随机分为对照组与DR组,DR组采用链脲佐菌素(STZ)一次性建立糖尿病模型。应用免疫组化SP法和RT-PCR技术分别于1,3,6mo检测视网膜中HIF-1α与VEGF蛋白及其mRNA的表达变化。结果:HIF-1α与VEGF蛋白及其mRNA在对照组视网膜中不表达,在DR组中呈进行性表达增强(P<0.05);DR组中HIF-1α与VEGF的表达呈正相关(r=0.605,P<0.05)。结论:DR视网膜中HIF-1α与VEGF表达增强,与DR的发生发展密切相关。     

糖尿病性视网膜病变患者VEGF与微血管损伤的相关性

目的：探讨糖尿病视网膜病变(diabetic retinopathy, DR)患者血管内皮生长因子(vascular endothelial growth factor, VEGF)水平与微血管损伤程度的相关性。

方法：回顾性分析本院收治的糖尿病患者71例,根据有无DR及病变程度分为三组：单纯糖尿病组(n=31)、单纯型DR组(n=22)、增殖型DR组(n=18),比较各组微血管病变发生率。同时,取患者空腹肘静脉血,采用ELISA试剂盒测定血清VEGF水平,采用流式细胞仪检测内皮细胞(ECs)、内皮祖细胞(EPCs)、循环祖细胞(CPCs)计数。

结果：各组糖尿病肾病和糖尿病神经病变发生率有明显差异,增殖型DR组高于单纯型DR组和单纯糖尿病组,差异有统计学意义(P<0.05)。各组VEGF水平有明显差异,增殖型DR组高于单纯型DR组和单纯糖尿病组,单纯型DR组高于单纯糖尿病组; 合并糖尿病肾病及糖尿病神经病变者VEGF水平高于非合并者,差异均有统计学意义(P<0.05)。各组之间ECs、EPCs、CPCs水平有明显差异,增殖型DR组ECs高于单纯型DR组和单纯糖尿病组,单纯型DR组高于单纯糖尿病组; 增殖型DR组EPCs、CPCs水平低于单纯型DR组和单纯糖尿病组,单纯型DR组低于单纯糖尿病组,差异有统计学意义(P<0.01)。增殖型DR患者的VEGF水平与ECs水平呈正相关性(P<0.01),与EPCs、CPCs水平呈负相关性(P<0.01)。

结论：VEGF在糖尿病视网膜病变,尤其是增殖型糖尿病视网膜病变的临床诊断和医疗中,有重要意义。     

VEGF基因多态性与糖尿病视网膜病变的关系

糖尿病视网膜病变（ｄｉａｂｅｔｉｃｒｅｔｉｎｏｐａｔｈｙ,ＤＲ）是多因素共同作用的复杂疾病,是糖尿病最严重的微血管并发症之一,其发生发展不仅与疾病本身及血糖的控制有关,还表现出明显的家族聚集现象和种族差异。目前,生长因子基因与ＤＲ相关性的研究是国内外的研究热点,但该类具有代表性的大样本量研究仍很缺乏,很多涉及与ＤＲ病理相关的候选基因中,血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）基因在ＤＲ的早期预防、治疗以及延缓ＤＲ发生、发展中起着不可或缺的作用。准确把握ＶＥＧＦ基因多态性在ＤＲ发生发展过程中的作用,将对ＤＲ的早期防治具有重要的意义。本文就ＶＥＧＦ基因多态性与ＤＲ的相关性进行综述。     

Effect of estrogen on electroretinographic responses in streptozotocin-induced diabetic female rats

The aim of this study is to investigate the effects of estrogen on functional changes in the retinas of streptozotocin (STZ)-induced diabetic rats by using an electroretinography. Female rats were randomly divided into four treatment groups: (1) Control (sham operation and vehicle administration); (2) STZ (sham operation and STZ administration); (3) OVX (ovariectomy and vehicle administration); and (4) OVX + STZ (ovariectomy and STZ administration). Full-field electroretinograms (ERGs) were recorded before OVX and STZ administration and 4 and 12 weeks after STZ administration. At 4 weeks after STZ administration, although there were no differences in the STZ and OVX groups compared with the Control group, the amplitude of the cone-response was significantly lower in the OVX + STZ group than in the Control group (P = 0.013). At 12 weeks after STZ administration, this response showed a similar tendency in the STZ and the OVX + STZ groups. At 12 weeks after STZ administration, the implicit times of OP3 and OP4 and of the cone-response were significantly delayed in the STZ and OVX + STZ groups (OP3: P = 0.030 and 0.050, OP4: P = 0.0060 and 0.0053, cone-response: P = 0.014 and 0.039), compared with in the Control group. Thus, the retinal functions in STZ-induced diabetic female rats were aggravated by OVX. OVX-induced estrogen deficiency resulted in earlier changes in the amplitudes of cone-response, especially in the diabetes, although this is a transient effect and it is difficult to explain. Recognizing the early neurosensory change would enable a better understanding of the effect of estrogen in the retina.     

PDR中血管内皮生长因子与纤维化相关细胞因子的相关性

血管内皮生长因子在视网膜新生血管生成中是必不可少的重要诱导因子。增殖性糖尿病视网膜病变（ proliferative diabetic retinopathy, PDR）患者视网膜新生血管形成后,随着病情进一步加重,可造成纤维血管膜形成、视网膜前膜的纤维化加重,造成牵拉性视网膜脱离。目前研究认为,细胞因子具有促进成纤维细胞增殖、移动、黏附和分泌细胞外基质的功能,在糖尿病环境状态下,转变至促纤维化状态,导致了细胞外基质的积聚和纤维化。本文对血管内皮生长因子与纤维化相关细胞因子相关性的研究现状予以综述。