增殖性视网膜病变玻璃体切割物的细胞学免疫组化研究

探讨不同视网膜增殖性疾病细胞增殖的特征及其异同。方法 采用３种特异性抗原，即抗人细胞角蛋白，抗胶质纤维酸性蛋白，抗肌动蛋白对２８例临床上不同的视网膜疾病的增殖膜及玻璃体切除液样本进行免疫细胞化学研究。增殖性玻璃体视网膜病变的增殖特征以胶质细胞和视网膜色素上皮细胞为主，二者在ＰＶＲ发病中作用不同。     

视网膜色素上皮（RPE）与增殖性玻璃体视网膜病变（PVR）

视网膜色素上皮（ＲＰＥ）与增殖性玻璃体视网膜病变（ＰＶＲ）上海医大眼耳鼻喉科医院眼科栾洁综述王文吉审阅ＰＶＲ是指增殖性玻璃体视网膜病变，表现为剥离后由视网膜在其内、外面及玻璃体内形成可以收缩的细胞性膜，常致视网膜剥离复位手术失败。许多学者认为，视网膜...     

维甲酸对视网膜色素上皮细胞增殖的影响

为探讨维甲酸（ＲＡ）对兔视网膜色素上皮（ＲＰＥ）细胞增殖的影响，我们在传代培养的兔ＲＰＥ细胞中分别加入不同浓度的ＲＡ，用溴脱氧嘧啶（ＢｒｄＵ）掺入标记法测定ＤＮＡ合成，计算各药物浓度下的ＢｒｄＵ标记率。结果表明：ＲＡ能明显抑制ＲＰＥ细胞的ＤＮＡ合成（Ｐ＜００１）。这种抑制作用呈剂量依赖趋势，为ＲＡ治疗增殖性玻璃体视网膜病变（ＰＶＲ）奠定理论基础。     

PVR药物治疗的研究进展

ＰＶＲ是视网膜复位手术失败的主要原因。为寻找防止ＰＶＲ发生的途径和方法，近年来眼科医师和研究人员做大量的工作。本复习有关献，就药物在抑制ＰＶＲ膜主要细胞成分：ＲＰＥ细胞、胶质细胞等细胞增殖的体外基础研究以及防止ＰＶＲ和牵引性视网膜脱离发生的动物实验研究进行综述。指出，免疫抑制剂（如环孢霉素Ａ）和抗代谢类药物（如５－ＦＵ）等能有效地抑制ＲＰＥ细胞的增殖及防止ＰＶＲ的发生；玻璃体内植入药物缓释胶囊     

PVR药物治疗的研究进展

ＰＶＲ是视网膜复位手术失败的主要原因。为寻找防止ＰＶＲ发生的途径和方法，近年来眼科医师和研究人员做了大量的工作。本文复习有关文献，就药物在抑制ＰＶＲ膜主要细胞成分：ＲＰＥ细胞、胶质细胞等细胞增殖的体外基础研究以及防止ＰＶＲ和牵引性视网膜脱离发生的动物实验研究进行综述。指出，免疫抑制剂（如环孢霉素Ａ）和抗代谢类药物（如５－ＦＵ）等能有效地抑制ＲＰＥ细胞的增殖及防止ＰＶＲ的发生；玻璃体内植入药物缓释胶囊可能是临床防治ＰＶＲ的有效途径。     

钙调素抑制剂对视网膜色素上皮细胞增殖的抑制作用的研究

目的：探讨三氟拉嗪（ｔｒｉｆｌｕｏｐｅｒａｚｉｎｅ,ＴＦＰ）对视网膜以素上皮（ｒｅｔｎａｌ ｐｉｇｍｅｎｔ ｅｐｉｔｈｅｌｉｕｍ,ＲＰＥ）细胞生长、增殖以及细胞周期的影响,为ＴＦＰ治疗增殖性玻璃体视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅ ｖｉｔｒｅｏｒｅｔｉｎｏｐａｔｈｙ,ＰＶＲ）提供理论依据。方法：通过细胞生长曲线的测定,观察不同浓度（５、１０、１５、２０μｍｏｌ／Ｌ）ＴＦＰ对ＲＰＥ细胞生长的影响     

前部增殖性玻璃体视网膜病变

前部增殖性玻璃体视网膜病变（ＡＰＶＲ）被认为是玻璃体切割术治疗增殖性玻璃体视网膜病变（ＰＶＲ）失败最主要的原因，临床上易与后极部增殖性玻璃体视网膜病变（ＰＰＶＲ）共存并且发病率高，根据牵引方向不同可分为前后、前部环形及前部垂直牵引三种类型。直接的手术切除大大增加了视网膜复位率并且可使部分低眼压眼球得到治疗。其病理结构与ＰＰＶＲ不同之处在于色素和无色素睫状上皮细胞的组成成分及纤维血管膜增殖的高发生率     

外伤眼视网膜色素上皮细胞的核型变化

目的：增殖性玻璃体视网膜病变（ＰＶＲ）是眼外伤及网脱术后最常见的并发症，也是手术失败的主要原因。视网膜色素上皮是ＰＶＲ形成的关键细胞。本实验培养ＰＶＲ眼的ＲＰＥ细胞，以探讨ＰＶＲ形成的机制，方法：本实验采用眼杯和组织片二种消化方法。对正常眼、新鲜外伤眼及ＰＶＲ眼的ＲＰＥ细胞进行培养。结果：ＰＶＲ眼的ＲＰＥ细胞分裂及增殖速度明显高于正常及新鲜外伤眼，同时发现ＰＶＲ眼的ＲＰＥ细胞发生基因突变，形成了三倍体核型。结论：ＰＶＲ眼的ＲＰＥ细胞的核型变化是细胞增殖的主要原因，但与外伤本身无关其基因突变机制有待于进一步探讨。     

细胞外基质在增殖性玻璃体视网膜病变形成中的作用

增殖性玻璃体视网膜病变（ＰＶＲ）增殖膜中有大量细胞外基质成分（ＥＣＭ）沉积，包括Ｉ～Ｖ型胶原及纤维粘连蛋白，层粘连蛋白，玻粘连蛋白等，这些ＥＣＭ成分由增殖膜中的视网膜色素上皮细胞，成纤维细胞和胶质细胞合成分泌，ＥＣＭ通过其受体（粘合素）与细胞发生粘附及信息交流，促使细胞变形，运动，增殖和分化，并在细胞的介导下发生收缩。对以上各环节进行调控能抑制基质收缩，从而是启示干预ＰＶＲ的新的药物疗法。     

前部增殖性玻璃体视网膜病变

前部增殖性玻璃体视网膜病变（ＡＰＶＲ）被认为是玻璃体切割术治疗增殖性玻璃体视网膜病变（ＰＶＲ）失败最主要的原因，临床上易与后极部增殖性玻璃体视网膜病变（ＰＰＶＲ）共存并且发病率高，根据牵引方向不同可分为前后、前部环形及前部垂直牵引三种类型。直接的手术切除大大增加了视网膜复位率并且可使部分低眼压眼球得到治疗。其病理结构与ＰＰＶＲ不同之处在于色素和无色素睫状上皮细胞的组成成分及纤维血管膜增殖的高发生率。病理发展可分收缩、混合及新生血管长入三期。充分认识ＡＰＶＲ临床表现及病理过程，加上良好的手术技术可使ＡＰＶＲ得到良好的控制和治疗     

uPA, tPA and PAI-1 mRNA expression in periretinal membranes

PURPOSE: Formation of periretinal membranes occurs in proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR) and includes cell migration, proliferation, extracellular matrix formation and tissue contraction, processes in which plasminogen activation (PA) system is involved. METHODS: Twenty PVR, PDR or pucker membranes were examined to identify the cells with cell specific markers and to detect the expression of urokinase (uPA), tissue-type plasminogen activator (tPA) or plasminogen activator inhibitor-1 (PAI-1) by in situ hybridization and by immunohistochemistry. RESULTS: In PVR, uPA, tPA and PAI-1 were expressed by retinal pigment epithelial (RPE) cells, macrophages or retinal glial cells. In PDR, PA components were also expressed by endothelial cells. Semiquantitative analysis in in situ hybridization and immunohistochemistry results demonstrated no notable differences in uPA, tPA or PAI-1 expression between PDR and PVR membranes. CONCLUSIONS: We conclude that local proteolytic activation is involved in extracellular matrix production both in diabetic and non-diabetic membranes.     

增生性玻璃体视网膜病变膜剥离标本的免疫组化研究

目的 探讨不同增生性玻璃体视网膜病变(PVR)的增殖特征。方法采用5种特异性抗体对12例PVR膜样本进行免疫组织化学研究。结果成纤维细胞、神经胶质细胞为参与PVR膜的主要细胞成分,视网膜色素上皮细胞(RPE)、巨噬细胞、纤维连接蛋白和新生血管也参与了PVR的病理过程。结论新生血管主要参与了增生性视网膜血管病变的病理过程。增殖膜中增殖的细胞、细胞外基质和血管成分参与了PVR的病理过程并起着不同的作用。     

Upregulation of RAGE and its ligands in proliferative retinal disease

We sought to study the presence of the receptor for advanced glycation endproducts (RAGE) and its ligands, advanced glycation endproducts (AGEs), S100/calgranulins and amphoterin (high mobility group box 1 protein; HMGB1), in the vitreous cavity and epiretinal membranes (ERMs) of eyes of patients with proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). Undiluted vitreous specimens were collected from 30 eyes of 30 patients undergoing pars plana vitrectomy for repair of retinal detachment (RD) secondary to PDR (n = 15) or PVR (n = 15). The vitreous samples obtained from 10 eyes undergoing macular hole repair were used as controls. Epiretinal membranes were obtained from eight eyes with PDR and from 10 eyes with PVR. The levels of AGEs in the vitreous were measured using ELISA. The vitreous levels of soluble RAGE (sRAGE), S100/calgranulins and amphoterin were measured using Western blot analyses. The localization of RAGE and its ligands in ERMs was determined with immunohistochemistry. The vitreous levels of sRAGE were significantly increased in both PDR and PVR (p < or = 0.05) compared to control vitreous. In both PDR and PVR, the vitreous levels of AGEs (p < or = 0.01), S100/calgranulins (p < or = 0.05), and amphoterin (p < or = 0.01) were also elevated compared to control eyes. Expression of RAGE was detected in six of eight ERMs from eyes with PDR and eight of 10 ERMs from eyes with PVR. Many cells expressing RAGE also expressed vimentin, suggesting a glial cell origin. Ligands for RAGE were also detected in ERMs, with AGEs detected in five eyes with PDR and eight eyes with PVR. Similarly, S100 and amphoterin ERM expression was observed in six eyes with PDR; these ligands were also expressed in ERMs from eyes with PVR (8 and 7 cases, respectively). We conclude that RAGE and its ligands are increased in the vitreous cavity of eyes with PDR and PVR and are present in ERMs of eyes with these proliferative retinal disorders. These findings suggest a role for the proinflammatory RAGE axis in the pathogenesis of proliferative retinal diseases.     

EIU in the rat promotes the potential of syngeneic retinal cells injected into the vitreous cavity to induce PVR

PURPOSE: To determine whether syngeneic retinal cells injected in the vitreous cavity of the rat are able to initiate a proliferative process and whether the ocular inflammation induced in rats by lipopolysaccharide (LPS) promotes this proliferative vitreoretinopathy (PVR). METHODS: Primary cultured differentiated retinal Müller glial (RMG) and retinal pigmented epithelial (RPE) cells isolated from 8 to 12 postnatal Lewis rats were injected into the vitreous cavity of 8- to 10-week-old Lewis rats (10(5) cells/eye in 2 microlieter sterile saline), with or without the systemic injection of 150 microgram LPS to cause endotoxin-induced uveitis (EIU). Control groups received an intravitreal injection of 2 microliter saline. At 5, 15, and 28 days after cell injections, PVR was clinically quantified, and immunohistochemistry for OX42, ED1, vimentin (VIM), glial fibrillary acidic protein (GFAP), and cytokeratin was performed. RESULTS: The injection of RMG cells, alone or in combination with RPE cells, induced the preretinal proliferation of a GFAP-positive tissue, that was enhanced by the systemic injection of LPS. Indeed, when EIU was induced at the time of RMG cell injection into the vitreous cavity, the proliferation led to retinal folds and localized tractional detachments. In contrast, PVR enhanced the infiltration of inflammatory cells in the anterior segment of the eye. CONCLUSIONS: In the rat, syngeneic retinal cells of glial origin induce PVR that is enhanced by the coinduction of EIU. In return, vitreoretinal glial proliferation enhanced the intensity and duration of EIU.     

Apoptosis and cell proliferation in proliferative retinal disorders: PCNA, Ki-67, caspase-3, and PARP expression

PURPOSE: To assess the incidence of cell proliferation and apoptosis in epiretinal membranes from eyes with proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR), and macular pucker (MP) and to further investigate the potential involvement of key executors of apoptosis. METHODS: Epiretinal membranes were obtained from the eyes of 23 patients who underwent vitrectomy surgery for recurrent retinal detachment due to PVR (n = 16), traction retinal detachment due to PDR (n = 5), and macular pucker (n = 2). Cell proliferation was evaluated by Ki-67 and PCNA (proliferation cell nuclear antigen) immunostaining. Apoptosis was assessed by TUNEL (terminal deoxynucleotidyl transfrase-dUTP-nick end labeling). The expression of caspase-3 and PARP (poly-ADP-ribose-polymerase) was detected using antibodies against activated caspase-3 and p85 fragment of PARP. Cytokeratin and activated caspase-3/PARP, GFAP (glial fibrillary acidic protein) and activated caspase-3/PARP double staining were used to identify cell types in the membranes. RESULTS: There was no statistically significant difference in the cell proliferative index between PVR (70.1 +/- 4.2%), PDR (82.1 +/- 7.0%), and macular pucker (72.9 +/- 22.8%) by multivariate analysis (p = 0.39, ANOVA) and univariate analysis. Apoptotic nuclei were seen more frequently in chronic retinal detachments of greater than 2 months duration, but the difference, compared to shorter term retinal detachments was not statistically significant (p = 0.19). The apoptosis indices determined for PVR (2.3 +/- 0.7%), PDR (3.4 +/- 1.5%) and macular pucker (5.5 +/- 3.2%) were not significantly different (ANOVA, p = 0.41). Apoptotic nuclei were correlated, increased with expression of caspase-3 and PARP. Many apoptotic cells appeared to derive from retinal pigment epithelium cells. CONCLUSIONS: Cell proliferation and apoptosis appear to be key mechanisms regulating certain cell populations in epiretinal membranes of PVR, PDR, and macular pucker. Inhibition of proliferative regulators such as PCNA and/or activation of apoptotic executors such as caspase-3 may serve as therapeutic targets to halt progression of proliferative retinal disorders.     

Cellular components of proliferative vitreoretinal membranes.

To understand the pathogenesis of proliferative vitreoretinal membrane formation which occurs in proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), etc., accurate identification of the cellular components of the membrane is needed. This study was performed to identify cellular components of the membranes by means of immunohistochemical technique. 11 proliferative vitreoretinal membranes which were surgically obtained from 7 eyes with PVR and 4 eyes with PDR were stained with monoclonal antibodies against cytokeratin, glial fibrillary acidic protein (GFAP), or vimentin using immunoperoxidase technique (ABC method). In the PVR membranes, mean cell positivities for cytokeratin, GFAP and vimentin were 48%, 1% and 92%, respectively and in the PDR membranes, 0%, 5% and 93%, respectively. The above results suggest that retinal pigment epithelial cells and fibroblasts are major cellular components of PVR membranes, and that mesenchymal cells are major cellular components and glial cells are minor cellular components of PDR membranes.     

增殖性玻璃体视网膜病变细胞凋亡的信号传导途径研究

目的 :探讨增殖性玻璃体视网膜疾病细胞凋亡的信号传导途径 ,以寻求新的药物治疗途径。方法 :2 3例增殖性玻璃体视网膜病变 (PVR) ,增殖性糖尿病性视网膜病变 (PDR) ,黄斑裂孔 (MH)及黄斑前膜 (MP)的视网膜前膜 (epiretinalmem brane,ERM )由玻璃体切割术中取得。细胞凋亡的情况由terminaldeoxynucleotidyltransfrase dUTP nickendlabeling(TUNEL法 )进行评估。Caspase 3及PARP的表达由特异性抗体抗活性Caspase 3和抗P 85片段的PARP检测。Cytokeratin与抗活性的Caspase 3双染色法进行凋亡细胞类型的鉴别。结果 :大多数发生凋亡的细胞为RPE细胞 ,而凋亡细胞与抗活性Caspase 3和抗P 85片段的PARP表达增加相关。细胞凋亡的数目与发生慢性视网膜脱离 (>2个月 )的病例有关 ,但凋亡系数 (apopto sisindex ,AI)在两组间无显著性差异 (1 4 4 2 9vs 3 2 2 86 ,P =0 1877)。PVR ,PDR ,MP各组的凋亡系数分别为 2 32 5 % ,3 4 2 % ,5 5 % ,P值分别为PPVR&PDR>0 1(0 16 85 ) ,PPDR&MP>0 1(0 5 380 ) ,PPVR&MP>0 1(0 8333)。结论 :此项研究发现细胞凋亡在PVR、PDR、MH及MP发病中的重要调节作用。诱导Caspase 3活性表达可作为一种治疗增殖性视网膜疾病的新的尝试     

Development of traction retinal detachments following intravitreal injections of retinal Muller and pigment epithelial cells

We injected varying numbers of retinal Muller glia into the rabbit vitreous in an established model of traction retinal detachment. We used indirect ophthalmoscopy to observe the changes elicited during the following 1 month. Although the severity of the tractional changes increased with increasing numbers of the glial cells, the pathology produced stabilized within the 1st week of injury. Muller glia were less effective at eliciting retinal detachments than retinal pigment epithelial cells (RPE) or mixtures of glia and RPE. Intravitreal tissue membranes derived from the glia differed morphologically from those derived from RPE. The glial membranes had fewer fibroblast-like cells, synthesized less extracellular matrix, and showed lower intravitreal cell proliferation, as determined by³H-thymidine radioautography. Our findings indicate that membranes composed only of Muller glial cells promote less severe retinal pathology than those membranes composed of RPE or mixed cell types.This study was supported in part by National Eye Institute grant EY04799 (J.M.B.), Core Center Grant EY01931 (J.M.B.), an unrestricted grant from Research to Prevent Blindness, Inc., and the Good Samaritan Foundation for Ophthalmic Research (Portland, Oregon)     

Interleukin (IL)-6, interleukin (IL)-8 levels and cellular composition of the vitreous humor in proliferative diabetic retinopathy, proliferative vitreoretinopathy, and traumatic proliferative vitreoretinopathy

PURPOSE: To investigate the interleukin (IL)-6 levels, IL-8 levels, and cellular composition of the vitreous humor in patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and traumatic PVR. METHODS: Vitreous samples from 14 patients with PDR, 10 patients with PVR, and 10 patients with traumatic PVR were analyzed. Fifteen cadaver eyes were used as controls. Cytokine levels were measured by ELISA. RESULTS: Elevated IL-6 levels were detected in the vitreous of 12 (85.7%) of the PDR patients, eight (80%) of the PVR patients, and all (100%) of the traumatic PVR patients. None of the control IL-6 results were elevated. Vitreous IL-8 levels were elevated in 12 (85.7%) of the PDR patients, six (60%) of the PVR patients, all (100%) of the traumatic PVR patients, and one (6.7%) of the control eyes. Cytological examination of the vitreous specimens revealed a predominance of macrophages (50%) in the PDR samples and a predominance of retinal pigment epithelial (RPE) cells (60%) in the PVR samples. In contrast, neutrophils predominated (88%) in the traumatic PVR samples. CONCLUSION: The findings suggest that IL-6 and IL-8 may be involved in the pathogenesis of PDR, PVR, and traumatic PVR. High proportions of RPE cells and macrophages are associated with elevated IL-6 and IL-8 levels in the vitreous of PDR and PVR patients; however, the fact that these cells are not predominant in traumatic PVR suggests that different immune response mechanisms may be active in the pathogenesis of these disorders.