玻璃体内注射贝伐单抗治疗早产儿视网膜病变的临床进展

血管内皮细胞生长因子(VEGF)在早产儿视网膜病变(ROP)的发生过程中发挥重要作用.在ROP的新生血管增生阶段,应用抗VEGF类药物抑制VEGF可以阻断视网膜血管的异常增生.本文就抗VEGF类药物贝伐单抗(Avastin)单纯玻璃体内注射、玻璃体内注射联合视网膜光凝或玻璃体切除手术等不同方式治疗ROP的临床研究进行回顾,并就贝伐单抗治疗ROP的安全性评价进行汇总.     

抗VEGF药物治疗早产儿视网膜病变的研究进展

早产儿视网膜病变(retinopathy of prematurity,ROP)是目前全世界儿童致盲的主要原因之一[1]。以往对于阈值期和高危阈值前期的ROP采用激光或冷凝治疗,因激光和冷凝术均是破坏性的,不可避免地导致周边视野永久丧失,且并发症较多,术后仍有部分患儿病情无法控制,最终视力完全丧失。因此需要寻找新的治疗方法,近年来有较多的临床数据表明,玻璃体内注射抗血管内皮生长因子(anti-vascular endothelial growth factor,anti-VEGF)治疗ROP是一种有效的治疗方法。我们将对抗VEGF药物在ROP治疗的研究进展进行近期的文献综述。     

Mechanism of angiostatin induced reduction of vascular leakage in retina and iris of rats with retinopathy of prematurity

AIM: To study the effect of an intravitreal injection of angiostatin on vascular leakage in the retina and iris of oxygen-induced retinopathy of prematurity (ROP). METHODS: Brown Norway rats at postnatal day 7 (P7) were exposed to hyperoxia (750mL/L O₂) for 5 days (P7-12) and then returned to normoxia to induce retinopathy. Angiostatin was reconstituted in sterile Phosphate Buffered Saline (PBS) and diluted to desired different concentrations. Angiostatin solution was injected into the vitreous of the right eye of the ROP rats at P14 and the age-matched normal rats through pars plana using a glass capillary, and the left eye received the same volume of sterile PBS as the control. Vascular permeability was quantified at 1, 2 and 3 days after the injection by measuring albumin leakage from blood vessels into the retina and iris using the Evans blue method and normalized by total protein concentrations. The expression of vascular endothelial growth factor (VEGF) in retina was evaluated using the Western Blot analysis and immunohis- tochemistry 24 hours following the injection. RESULTS: ROP rats showed significant increases of vascular permeability in the retina and iris (P <0.01). Angiostatin reduces vascular permeability in a dose-dependent manner in the retina of ROP rats. The reduction showed a time course trend. Angiostatin injection reduced retinal vascular permeability by approximately 1.5 and 2-fold at P15 (P <0.05) and P16 (P < 0.01), respectively. Angiostatin injection significantly reduced VEGF levels in the retina of ROP rats but did not affect retinal VEGF levels in normal rats. CONCLUSION: Angiostatin significantly decreases patholog- ical vascular permeability in the retina and iris of ROP rats but not in normal rats. Angiostatin down-regulates VEGF expression in retina of ROP rats. These results suggest that angiostatin may have a therapeutic potential in the treatment of ROP and other diseases with vascular leakage.     

氧诱导小鼠视网膜新生血管的实验研究

目的 制作氧诱导视网膜新生血管的小鼠动物模型并了解其视网膜血管内皮生长因子(VEGF)的变化.方法将出生后第7 d的C57BL/6J小鼠置于75%的高氧环境中5 d,再置于普通空气中5 d.在空气中饲养的小鼠为对照组.两组进行视网膜铺片,ADPase染色,组织切片染色,ELISA测定视网膜VEGF蛋白含量.结果实验组新生血管形成率为100%,对照组未见新生血管.实验组小鼠生后第17 d时突破视网膜内界膜的血管内皮细胞核数目达(46.7±11.1)个,对照组不足2个.第12 d实验组视网膜VEGF蛋白水平比对照组下降,第17 d比对照组升高. 结论该动物模型是研究早产儿视网膜病变(ROP)发病机制及治疗的合适模型.VEGF是造成视网膜新生血管发生的主要机制之一.     

血管抑素降低ROP幼鼠视网膜及虹膜血管渗漏性的作用机制研究

目的：探讨血管抑素（angiostatin）玻璃体注射对氧诱导的早产儿视网膜病变（retinopathy of prematurity,ROP）的视网膜及虹膜血管渗漏的作用及其机制。方法：将出生7d（P7）的Brown Norway鼠置高氧环境（750mL／L O2）5d后再置正常氧环境诱导ROP,建立ROP动物模型,并以年龄相匹配的正常鼠作为正常对照。所有ROP鼠（P14）及正常鼠均右眼玻璃体腔注射血管抑素,左眼注射相同剂量的PBS（磷酸盐缓冲生理盐水）作为对照。用Evans蓝微血管渗透性检测法及总蛋白标准化分别于注射后1,2和3d检测视网膜和虹膜的血管渗透性;用Western blot蛋白印迹分析和免疫组化方法检测注射24h后血管内皮生长因子（vascular endothelial growth factor,VEGF）在视网膜的表达。结果：ROP鼠视网膜及虹膜的血管渗透性明显增加（P〈0．01）;中剂量（3．75ug/眼）和高剂量（7．5ug/眼）血管抑素降低ROP鼠视网膜血管渗透性（P〈0．05,P〈0．01）,而低剂量组（1．88ug／眼）没有引起明显改变,呈现剂量依赖型;三种不同剂量的血管抑素玻璃体注射后ROP鼠的虹膜均未发生明显的血管渗透性的改变。血管抑素注射后第1d和第2d视网膜血管渗透性明显降低（P〈0．05。P〈0．01）,而第3d无明显降低,其作用呈现出时间进程。Western blot蛋白印迹和免疫组化分析表明血管抑素显著降低了ROP鼠视网膜的VEGF水平,但对正常鼠无影响。结论：血管抑素可以降低ROP鼠视网膜的病理性血管渗漏,其血管渗透性下降可能与血管抑素下调VEGF的表达有关。血管抑素可能对ROP等其他视网膜血管渗漏性疾病具有潜在的治疗作用。     

GM6001抑制视网膜新生血管形成VEGF和MMP2的表达

目的:探讨基质金属蛋白酶抑制剂GM6001对鼠视网膜新生血管形成的抑制作用及机制。方法:采用高氧使鼠视网膜血管阻塞建立早产儿视网膜病变模型后,将鼠分为两组,玻璃体腔内注射75μmol/L的GM60010.5μL,另一组不治疗。5d后处死,行视网膜HE染色计数矢状面5μm视网膜切片中突破内界膜的血管内皮细胞核数,和血管内皮生长因子(VEGF)和基质金属蛋白酶-2(MMP-2)免疫组织化学染色,以及视网膜铺片。同时设立正常对照组。结果:GM6001治疗组视网膜发生新生血管的眼数较高氧对照组少,但较正常对照组多。GM6001治疗组、高氧对照组、正常对照组平均每个切面突破内界膜的血管内皮细胞核数为2.38±0.90,21.25±1.12和1.23±0.46,高氧对照组与正常对照组具有显著统计学差异(P<0.01),GM6001治疗组与正常对照组无统计学差异(P>0.05),与高氧对照组具有显著统计学差异(P<0.01)。视网膜铺片正常对照组血管发育成熟,深浅两层血管网结构清晰,可见螺旋动脉,高氧对照组血管迂曲阻塞,大量结构异常新生血管,丧失了血管网结构,GM6001治疗组见两层血管网结构清晰,仍有少部分深层血管阻塞。视网膜免疫组织化学检测GM6001治疗组VEGF及MMP-2与正常对照组有统计学差异(P<0.05),与高氧对照组也有显著统计学差异(P<0.01)。结论:GM6001可抑制视网膜病的血管新生,机制可能是抑制了MMP-2对VEGF的作用。     

Phosphorylation of alphaB-crystallin in epiretinal membrane of human proliferative diabetic retinopathy

AIM: To examine phosphorylation of alphaB-crystallin (p-?BC), a vascular endothelial growth factor (VEGF) chaperone, and immunohistochemically investigate relationship between p-?BC, VEGF and phosphorylated p38-mitogen-activated protein kinase (p-p38 MAPK) in the epiretinal membrane of human proliferative diabetic retinopathy (PDR). METHODS: Eleven epiretinal membranes of PDR surgically excised were included in this study. Two normal retinas were also collected from enucleation tissues due to choroidal melanoma. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with anti-p-?BC, VEGF, CD31, and p-p38 MAPK antibodies. RESULTS: Immunoreactivity for p-?BC was observed in all of the epiretinal membranes examined, where phosphorylation on serine (Ser) 59 showed strongest immunoreactivity in over 70% of the membranes. The immunolocalization of p-?BC was detected in the CD31-positive endothelial cells, and co-localized with VEGF and p-p38 MAPK in PDR membranes. Immunoreactivity for p-?BC, however, was undetectable in endothelial cells of the normal retinas, where p-p38 MAPK immunoreactivity was less marked than PDR membranes. CONCLUSION: Phosphorylation of ?BC, in particular, phosphorylation on Ser59 by p-p38 MAPK may play a potential role as a molecular chaperon for VEGF in the pathogenesis of epiretinal membranes in PDR.     

一氧化氮在早产儿视网膜病变中的作用

目的 研究一氧化氮(NO)在早产儿视网膜病变(ROP)中的作用及其与血管内皮生长因子(VEGF)的相互关系.方法 建立高浓度氧诱导的SD新生鼠ROP模型,腹腔分别注射结构型一氧化氮合酶(cNOS)抑制剂L-NNA 150mg/(kg·d)和诱导型NOS(iNOS)抑制剂L-NIL 25 mg/(kg·d).一段时间后,取新生鼠眼球制备切片计数视网膜新生血管内皮细胞数,Western blot法检测VEGF.结果 氧气组视网膜cNOS活力较空气组显著增高(P＜0.05),iNOS活力明显降低(P＜0.05).L-NNA治疗组与对照组比较,视网膜新生血管内皮细胞数显著减少(P＜0.05),VEGF在高氧期表达增加60.38%,相对低氧期表达减少17.97%,以上指标L-NIL治疗组与对照组差异均无统计学意义(P＞0.05).结论 抑制cNOS产生的NO能减少后者引起的血管活性以及细胞毒性效应,从而减轻对血管内皮细胞的损伤,并且能下调视网膜VEGF的表达.     

血管内皮生长因子受体与眼新生血管及增生性病变

血管内皮生长因子（VEGF）是一种特异性刺激血管内皮细胞增殖和新生血管形成的生长因子,是最直接的血管内皮细胞促分裂素,与血管内皮生长因子受体（VEGFR）结合而发挥各项功能。在视网膜细胞、色素上皮细胞、表皮细胞、M(?)ller细胞内均有VEGFR表达。缺氧可上调其基因表达,直接造成视网膜新生血管形成及增生性玻璃体视网膜病变。对VEGFR结构、功能、与新生血管的关系,VEGFR表达增高所导致的病理变化,VEGFR高表达的调节因素,如何抑制VEGFR阻断VEGF/VEGFR信号传导途径抑制VEGF作用等方面进行了较为全面的综述。     

玻璃体腔注射抗VEGF药物治疗早产儿视网膜病变的研究进展

早产儿视网膜病变(ROP)是发生于早产儿的一种未成熟视网膜血管增殖性眼病,是一种严重的儿童致盲性眼病。视网膜激光光凝术是治疗ROP的经典方法,然而激光治疗是破坏性的,尤其是在ROP Ⅰ区病变的情况下,视网膜激光光凝治疗会引起严重的并发症。研究表明,血管内皮细胞生长因子(VEGF)在ROP发生及发展过程中具有关键作用,而玻璃体腔注射抗VEGF药物不仅能有效控制ROP,并且为视网膜继续发育争取了机会。本文就玻璃体腔内注射抗VEGF药物治疗ROP的治疗指征、药物筛选、注射部位、给药剂量、疗效观察以及并发症对其进行综述。     

胎盘生长因子在早产儿视网膜病变纤维血管膜中的表达

目的　研究胎盘生长因子（ｐｌａｃｅｎｔａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＰＬＧＦ）在早产儿视网膜病变（ｒｅｔｉｎｏｐａｔｈｙｏｆｐｒｅｍａｔｕｒｉｔｙ,ＲＯＰ）纤维血管膜的血管内皮细胞、周细胞、巨噬细胞和胶质细胞中的表达和分布。方法　玻璃体切割术中取ＲＯＰ患者视网膜表面纤维血管膜保存备用,冰冻切片,ＨＥ染色、免疫组织化学染色和免疫荧光染色观察ＰＬＧＦ在纤维血管膜上的表达和分布。结果　ＨＥ染色及免疫组织化学染色结果显示ＰＬＧＦ在纤维血管膜中可见阳性表达。免疫荧光双标记法实验结果显示纤维血管膜的ＰＬＧＦ可能由血管内皮细胞、周细胞、巨噬细胞和胶质细胞表达。结论　ＰＬＧＦ可能参与了ＲＯＰ的发病过程,ＰＬＧＦ在ＲＯＰ发病中的作用及其分子机制需要进一步研究。     

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.     

Ruboxistaurin, a PKCβ inhibitor, inhibits retinal neovascularization via suppression of phosphorylation of ERK1/2 and Akt

Ruboxistaurin, a protein kinase C (PKC) β inhibitor, exhibits significant anti-angiogenic activity that reduces the response of vascular endothelial cells to stimulation by vascular endothelial growth factor (VEGF). In addition, the activation of PKC, specifically PKCβ, plays a key role in the retinal neovascularization. However, the effect of ruboxistaurin on oxygen-induced retinopathy (OIR), an experimental murine model of proliferative retinopathy, has not yet been investigated. In this study, we assessed the efficacy of ruboxistaurin both in vitro and in vivo and also evaluated its anti-angiogenic mechanisms. Ruboxistaurin inhibited formation, proliferation, and migration of VEGF-induced human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. It also inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and serine/threonine protein kinase family protein kinase B (Akt). In in vivo retinal neovasuclarization experiments, induced in neonatal mice by returning the retina to normoxia (21% O₂) after exposure to hyperoxia (75% O₂), ruboxistaurin given subcutaneously significantly reduced pathologic vascular changes. No effect was seen on revascularization of the capillary-free area. These findings indicate that ruboxistaurin has anti-angiogenic effects both in vitro and in vivo that are exerted partly via suppressing the phosphorylation of ERK1/2 and Akt. Ruboxistaurin may be a candidate for treatment of angiogenesis in retinal neovascularization diseases.     

骨髓间充质干细胞治疗视网膜新生血管性疾病的研究进展

视网膜新生血管性疾病并非独立的一种眼病,常见于许多眼病中,如早产儿视网膜病变、糖尿病视网膜病变、年龄相关性黄斑变性、视网膜中央静脉阻塞和视网膜静脉周围炎等都会形成新生血管,是严重损害视力的病变。此类疾病丧失正常血管的结构和功能,引起病理性出血、渗出、水肿和视网膜脱离等病理性改变,是视力丧失的主要原因,已经成为世界范围的致盲性疾病。目前主要的治疗方法为针对病因进行激光封闭,或行玻璃体切除术,或是反复、多次玻璃体腔注射抗血管内皮生长因子,虽然短期效果好,但不能防止复发,目前仍没有长期有效的治疗方法。干细胞治疗的出现为此提供了潜在的替代疗法。本文将对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)在视网膜新生血管疾病中的最新应用进展作一综述,展示其移植优势和良好的临床应用前景。     

Inhibition of LY294002 in retinal neovascularization via down-regulation the PI3K/AKT-VEGF pathway in vivo and in vitro

AIM: To investigate the effects of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 on retinal neovascularization (RNV) in the oxygen-induced retinopathy (OIR) mouse model and human umbilical vein endothelial cells (HUVECs). METHODS: C57BL/6J mice were randomly divided into normoxia-control, OIR-control and LY294002 treatment groups. LY294002 or phosphate-buffered solution was intraperitoneally injected daily into mouse pups from P6 to P9 in LY294002 treatment group or OIR-control group. Morphological and pathological changes in RNV, as well as expression levels of PI3K, serine-threonine kinase (AKT) and vascular endothelial growth factor (VEGF) were observed. HUVECs treating with LY294002 were exposed to hypoxia; the expression of PI3K, AKT and VEGF were examined by Western blot and RT-PCR analyses. RESULTS: Compared with the OIR-control group, LY294002 significantly inhibit RNV. Adenosine diphosphatase (ADPase) staining and hematoxylin and eosin staining indicated that the clock hour scores of neovascularization and the nuclei of pre-retinal neovascular cells in the LY294002 treatment group were clearly less than those in the OIR-control group (1.41±0.52 vs 6.20±1.21; 10.50±1.58 vs 22.25±1.82, both P<0.05). Intravitreal injection of LY294002 (in the LY294002 treatment group) markedly decreased PI3K/AKT-VEGF expression compared with the OIR-control group by immunohistochemistry, Western blotting and RT-PCR (all P<0.05). In HUVECs treated with hypoxia, expression of PI3K, AKT and VEGF were downregulated in the hypoxia-LY294002 group (all P<0.05). CONCLUSION: The PI3K inhibitor LY294002 can inhibit RNV by downregulating PI3K, AKT, and VEGF expression in vivo and in vitro. LY294002 may provide an effective method for preventing retinopathy of prematurity (ROP).     

血管内皮生长因子与病理性脉络膜新生血管

脉络膜新生血管(choroidal neovascularization,CNV)是造成许多眼底疾病和视力严重下降的主要原因。而血管内皮生长因子(vascular endothelial growth factor,VEGF)与CNV的形成密切相关,VEGF的过度表达将导致CNV形成。抗VEGF治疗能有效抑制CNV形成,从而达到治疗效果。本文就VEGF在眼内的分布及其在病理性CNV生成中的表达,和病理性CNV抗VEGF治疗的研究现状做一综述。     

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。