增生细胞核抗原在视网膜色素上皮细胞表达及其反义寡核苷酸的抑制作用

目的 研究视网膜色素上皮(retinal pigment epithelium,RPE)细胞中增生细胞核抗原(proliferating cell nuclear antigen,PCNA)表达及其反义寡核苷酸(antisense oligodeoxynucleotides,AS ODN)对其表达和细胞增生的抑制作用，为增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)治疗探索基因治疗新途径。 方法 (1)体外培养兔眼RPE细胞，在不同时间采用链霉亲合素-生物素化过氧化物酶复合物(streptoavidin-biotin enzyme complex,SABC)免疫组织化学法检测PCNA的表达;(2)脂质体介导下不同浓度的PCNA AS-ODN和正义寡核苷酸(sense oligodeoxynucleotides,S-ODN)分别作用于体外培养的RPE细胞，采用免疫组织化学方法检测PCNA的表达;(3)四唑盐比色法(methyl thiazolyl tetrazolium,MTT)检测在不同浓度的PCNA AS-ODN和S-ODN作用下RPE细胞生长活性及其生长抑制率。 结果 (1)体外培养兔眼RPE细胞表达PCNA，表达高峰为培养后48 h ;(2)在0.28、1.12 μmol/L AS-ODN 作用下，PCNA的表达明显受抑制;(3)0.28、1.12 μmol/L的PCNA AS-ODN 能明显抑制RPE细胞增生活性，并呈剂量依赖性，其生长抑制率分别达53%、81%。 结论 一定浓度PCNA AS-ODN能序列特异性地抑制RPE细胞PCNA表达和增生活性，有望进一步用于PVR的治疗实验研究。 (中华眼底病杂志, 2002, 18: 231-233)     

Involvement of rho-kinase pathway in contractile activity of rabbit RPE cells in vivo and in vitro

PURPOSE: Increased alpha-smooth muscle actin (alpha-SMA) expression in epiretinal membranes causes tractional retinal detachment (TRD) in proliferative vitreoretinopathy (PVR). The Rho-A/Rho-associated kinase signaling pathway is a principal mediator of contractile force generation in nonmuscle cells. In the current study, the relation between the Rho-kinase pathway and alpha-SMA expression and type I collagen gel contractile activity in retinal pigment epithelial (RPE) cells was investigated, using Y27632, a specific inhibitor of p160ROCK, and the involvement of the Rho-kinase pathway was evaluated in a rabbit PVR model with cultured RPE cells and platelet-rich plasma (PRP). METHODS: RPE cells were obtained from rabbits and cultured. The number of passages and the effect of Y27632 on alpha-SMA expression were studied by immunohistochemistry and immunoblot analysis. An in vitro type I collagen gel contraction assay and MTT assay evaluated the effect of Y27632 on RPE cell contractile force and proliferation. Cultured sixth-passage rabbit RPE cells were coinjected with PRP intravitreally, followed by 50 micro M of Y27632, injected weekly. The presence of TRD was assessed until 28 days to evaluate the effect of Y27632 in vivo. RESULTS: Expression of alpha-SMA was increased according to the passages. Y27632 suppressed alpha-SMA expression in cultured RPE cells and impaired contractile force. Y27632 did not affect the proliferative potential. Y27632 significantly (P < 0.01) reduced TRD development. CONCLUSIONS: Y27632 decreased alpha-SMA expression and the contractile force generated by RPE cells and attenuated PVR, indicating the involvement of the Rho-kinase pathway in cell-dependent collagen contraction in vitro and in vivo. The drug may affect the biological event by inhibiting alpha-SMA expression, and Y27632 could be useful for preventing PVR.     

Enhancement of dedifferentiation and myoid differentiation of retinal pigment epithelial cells by platelet derived growth factor

AIMS: To clarify factor(s) involved in morphological dedifferentiation of retinal pigment epithelial (RPE) cells in vitro from mitotically quiescent hexagonal cells to flattened cells that lack epithelial characteristics and concurrent myoid differentiation. METHODS: RPE cells which retained their differentiated hexagonal morphology were isolated from bovine eyes by mechanical pipetting. Dedifferentiation and myoid differentiation of RPE cells were examined by microscopic observation and immunohistochemical analysis using antibodies against cytokeratin, an epithelial marker, and alpha smooth muscle actin, a marker of myoid differentiation. The contractile ability of RPE cells was evaluated by collagen gel contraction assay. RESULTS: Platelet derived growth factor (PDGF) enhanced morphological changes in the RPE from hexagonal-shaped cells to flattened cells. Coincident with this morphological alteration, the expression of cytokeratin in RPE cells decreased and expression of alpha smooth muscle actin began and was increased in a time dependent manner. These alterations were completely blocked by collagen synthesis inhibitors. Interleukin 1beta, transforming growth factor beta1, insulin-like growth factor I, and basic fibroblast growth factor had little or no effect on the dedifferentiation. PDGF also potentiated the RPE induced collagen gel contraction. CONCLUSIONS: These results demonstrate that PDGF enhanced the dedifferentiation of RPE cells, the initial step of proliferative vitreoretinopathy (PVR), as well as myoid differentiation and collagen gel contraction. PDGF may have a versatile role in the pathogenesis of PVR involving collagen synthesis.     

曲安奈德对培养人视网膜色素上皮细胞表达survivin的影响

目的 观察曲安奈德(TA)对体外培养的人视网膜色素上皮(RPE)细胞表达survivin基因的影响,探讨TA治疗增生性玻璃体视网膜病变(PVR)的机制.方法 四甲基偶氮唑盐比色(MTT)法和流式细胞术检测不同质量浓度(0.01、0.1、1.0 g/L)TA对人RPE细胞增生及凋亡的作用,免疫组织化学法、RT-PCR检测药物在蛋白和基因水平对survivin表达的影响. 结果 在不同质量浓度的TA作用下,人RPE细胞的增生受到抑制,凋亡率增加,在蛋白水平和基因水平,药物均对RPE细胞survivin的表达有明显的抑制作用,且均与药物的作用时间和剂量呈正相关.各组与对照组比较差异均有统计学意义(P＜0.05).结论 TA能抑制人RPE细胞的增生及survivin蛋白和mRNA在人RPE细胞的表达,诱导RPE细胞的凋亡.     

血小板源性生长因子对增生性玻璃体视网膜病变增生膜形成的影响

目的 探讨血小板源性生长因子（platelet-derived growth factor,PDGF)在增生性玻璃体视网膜病变（proliferative vitreoretinopathy,PVR)增生膜形成中的作用及PVR增生膜中是否存在PDGF的分泌细胞与靶细胞。方法 选择PVR患者的手术标本,其中视网膜前膜（epiretinal membrane,ERM)和视网膜下膜（subretinal membrane,SRM)标本各7例。采用免疫电镜方法检测PDGF及其受体在ERM、SRM中的表达及其与ERM、SRM中两种主要细胞成分即视网膜色素上皮细胞和神经胶质细胞的关系。结果 PDGF－A基7例ERM中均表达阳性,在7例SRM中5例表达阳性,标记物主要位于ERM、SRM部分细胞的胞质中。PDGF－B在7例ERM中均表达阳性,在6例SRM中5例表达阳性,标记物主要位于SRM、SRM的部分细胞胞质中,尤其集中于细胞质内一些电子密度高的椭圆形或不规则形的分泌颗粒中。提示PDGF可由ERM、SRM局部细胞产生,其在ERM、SRM的发病中起重要作用。本实验中PDGF－A和PDGF－B分别与细胞角蛋白和神经胶质纤维酸性蛋白双标记,结果显示细胞角蛋白标记阳性的细胞即视网膜色素上皮细胞和神经胶质纤维酸性蛋白标记阳性细胞－神经胶质细胞中的PDGF－A、PDGF－B蛋白表达阳性,表明视网膜色素上皮细胞和神经胶质细胞是PDGF的分泌细胞。结论 PDGF可由PVR增生膜中的细胞产生,在PVR的发病中起重要作用。视网膜色素上皮细胞和神经胶质细胞是PDGF的分泌细胞,从而为自分泌物、旁分泌机制提供依据。     

肝细胞生长因子受体在视网膜前膜及培养的人视网膜色素上皮细胞中的表达

目的 观察增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)C、D两级视网膜前膜 (epiretinal membranes, ERM)和培养的人视网膜色素上皮(retinal pigment epithelium, RPE)细胞中肝细胞生长因子受体（hepatocyte growth factor receptor, HGFR）的表达情况。 方法 采用免疫组织化学染色方法对15例复杂孔源性视网膜脱离患者玻璃体切割术中剥离的ERM以及培养的人RPE细胞中HGFR的表达情况进行观察。 结果 在6例PVR C级和9例PVR D级ERM标本中分别有5、7例呈HGFR阳性表达；培养的RPE细胞胞浆中HGFR呈阳性表达。 结论 肝细胞生长因子有可能参与了 PVR的病理过程。 (中华眼底病杂志, 2002, 18: 221-223)     

Vitreous modulation of gene expression in low-passage human retinal pigment epithelial cells

PURPOSE: In proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cells enter the vitreous and proliferate. They become fibroblast-like and participate in the formation of contractile membranes, which can lead to retinal detachment. Vitreous treatment of RPE cells in vitro results in similar morphologic changes. This study was conducted to examine vitreous-induced modulation of gene expression in RPE cells. METHODS: Low-passage human RPE cell lines derived from three donors were each treated for 6, 12, 24, or 48 hours with complete medium or complete medium containing 25% vitreous. Changes in mRNA levels were examined by using microarrays. Real-time quantitative PCR (qPCR) was used to measure mRNA expression of a subset of genes in cells from three additional donors. Immunohistochemistry and immunoblot analysis were used to examine protein expression. RESULTS: Vitreous treatment caused a progressive reprogramming of gene expression. qPCR confirmed vitreous modulation of mRNA levels of 10 of 10 genes. Changes consistent with a transition from an epithelial to a mesenchymal phenotype were observed. Downregulated genes included genes associated with differentiated RPE cells. Upregulated genes included genes associated with stress and inflammation. Pathway analysis indicated that the transforming growth factor-beta/bone morphogenetic protein (BMP) pathway and the focal adhesion pathway may play a role in this process. BMP-2 protein and mRNA were increased. CONCLUSIONS: Despite the biological variation in vitreous and RPE donors, vitreous reproducibly modulated a limited number of mRNAs. Many of these changes were consistent with the more fibroblast-like appearance of vitreous-treated cells and with the pathobiology of PVR. TGF-beta and BMP-2 may be important modulators of vitreous-induced changes in gene expression.     

增生性玻璃体视网膜病变中组织型转谷氨酰胺酶的表达

目的:检测增生性玻璃体视网膜病变(PVR)视网膜前膜和视网膜色素上皮(RPE)细胞中组织型转谷氨酰胺酶(tTG)的表达情况,探讨tTG是否参与PVR的发病过程.方法:PVR患者21例,收集视网膜前膜,C级8例,D级13例,行tTG免疫组织化学染色.另对培养3-5代的人RPE细胞分别用含有1 00mL/L PVR玻璃体液、正常玻璃体液和PBS的DMEM培养液进行孵育24h后行tTG免疫细胞化学染色.光镜下观察,比较C级和D级膜的表达阳性率,显微图像分析测量3组RPE细胞染色的平均灰度值.结果:在PVR视网膜前膜中tTG呈阳性表达(81%),C级和D级膜表达阳性率无差异 (C级88%,D级77%,P＞0.05).培养的人RPE细胞表达tTG,在PVR患者的玻璃体液作用下,tTG表达的平均灰度值为137.0±2.6,与正常玻璃体液组(143.5±2.9)及PBS对照组(143.6±3.0)相比,表达水平升高(P＜0.05).结论:PVR视网膜前膜和培养的人RPE细胞tTG表达阳性,在PVR患者的玻璃体液作用下,RPE细胞的tTG表达水平升高,这表明tTG参与了PVR的发病过程,在PVR视网膜前膜的形成过程中可能有重要作用.     

Macrophage modulation of retinal pigment epithelial cell migration and proliferation

Macrophages are fully differentiated cells that do not synthesize an extracellular matrix and do not contract; they do, however, produce substances that modify the behavior and functions of other cells, particularly those involved in the inflammatory and immune responses. Since macrophages are a ubiquitous component of periretinal membranes, we sought to determine whether they modulate proliferation and/or migration of retinal pigment epithelial (RPE) cells, functions that may be essential for the development of proliferative vitreoretinopathy (PVR). Using an in vitro assay, we found that macrophage supernatant contains factors that stimulate proliferation and migration of cultured human RPE cells. Since interleukin-1 (IL-1) is a product of activated macrophages that modulates a number of cellular functions, we also examined its effect on RPE proliferation and migration. We found that IL-1 increased migration but did not affect proliferation, and thus could not duplicate the effect of macrophage supernatant. Injection of activated macrophages into the vitreous of rabbits which had a retinal hole stimulated RPE cell proliferation in the area of the retinal hole, where the RPE cells were exposed. These findings suggest the ability of macrophages to modulate functions of RPE cells that are thought to be critical for the development of PVR. Macrophages may thus be an important part of the vitreous environment that favors the development of PVR.     

Targeting of integrin-linked kinase with a small interfering RNA suppresses progression of experimental proliferative vitreoretinopathy

Integrin-linked kinase (ILK) is a serine/threonine kinase that interacts through its COOH terminus with β1 and β3 integrins, which mediates a diversity of cell functions by coupling integrins and growth factors to cascades of downstream signaling events. The purpose of this work was to investigate the effects of ILK on development of experimental proliferative vitreoretinopathy (PVR). Cultured human RPE cell line D407 was knocked down for ILK using a small interfering RNA (siRNA). For this, cellular ILK expression was quantified by real-time quantitative PCR, Western blot analysis and immunocytochemical assay, and cytotoxicity of transfection was determined by MTT assay. Moreover, cell attachment, spreading, migration, microfilament dynamics, and cell cycling assays were performed. Furthermore, the impact of the ILK-specific siRNA on PVR was tested using a rabbit model in which PVR was induced by the injection of human RPE cells. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection. The results showed that blocking the expression of ILK by siRNA significantly inhibited human RPE cell attachment, spreading, migration and proliferation. The knockdown of ILK also disturbed F-actin assembly and induced a cellular arrest in the G1 phase of the cell cycle. Though the eyes injected with ILK-specific siRNA also developed features of PVR, the severities of day 28 post-injection were significantly lower than those in the control eyes (P < 0.01). We conclude that targeting of ILK with a small interfering RNA not only inhibits human RPE cell attachment, spreading, migration and proliferation in vitro, but also effectively suppresses development of proliferative vitreoretinopathy in a rabbit model. This may be a potential therapeutic usefulness in treating PVR.     

Homologous serum-stimulated fibronectin synthesis in human retinal pigmented epithelial cells.

PURPOSE: Fibronectin expression has been recorded in subretinal membranes from patients with proliferative vitreoretinopathy (PVR). Retinal pigmented epithelial (RPE) cells are a major cellular component of PVR membranes and synthesize fibronectin. We investigated the effects of human serum (as a model of vascular leakage in vivo) on the expression of fibronectin by cultured human RPE cells and compared the responses to those stimulated by fetal bovine serum (FBS). METHODS: Human RPE cells were incubated in M199 with 1, 10 and 40% human adult serum or FBS for 72 h. RESULTS: Retinal pigmented epithelial cells expressed maximum extracellular matrix fibronectin when exposed to 40% human serum using immunohistochemistry. Using ELISA to quantify fibronectin, 10 and 40% human serum significantly increased the total fibronectin (P < 0.01; n = 4), whereas FBS did not affect fibronectin expression. CONCLUSIONS: The results show that human serum contains substances stimulating fibronectin synthesis in human RPE cells.     

Epithelial-mesenchymal transition in proliferative vitreoretinopathy: intermediate filament protein expression in retinal pigment epithelial cells.

PURPOSE: To improve our understanding of how retinal pigment epithelial (RPE) cells behave in vivo and to establish similarities with dedifferentiation and adaptive events observed in RPE cells cultured under simulated intraocular pathologic conditions. At the same time, to examine the origin of epithelioid-shaped and fibroblast/fusiform-shaped cells in epiretinal membranes (ERM) from proliferative vitreoretinopathy (PVR). METHODS: Cells of ERM were studied by electron-immunocytochemical techniques, using simple, double, and triple immunostaining for cytokeratins (CK), vimentin (Vim), and glial fibrillary acidic protein (GFAP). Ultrastructural morphology analysis was also carried out. Adult human RPE cells were obtained and cultured with normal and pathologic vitreous from proliferative vitreoretinal disorders, subretinal fluid aspirates from retinal detachment, and normal human serum. Their cytoskeleton was fractionated at 7 (early cultures) and 24 (late cultures) days of culture, electrophoresed, immunoblotted for intermediate filament proteins, and quantified by densitometric analysis for each condition. Changes in phenotype characteristics were also evaluated. RESULTS: Epithelioid-shaped and fibroblast/fusiform-shaped cells, resembling RPE cells, expressed CK-Vim-GFAP simultaneously as intermediate filament proteins in their cytoskeleton. RPE cells in culture also expressed CK-Vim-GFAP and changed from an epithelial shape to a migratory fibroblast/fusiform-shaped phenotype in the presence of subretinal fluid aspirates and pathologic vitreous from proliferative intraocular disorders. In simulated cultures of proliferative intraocular disorders, cells decreased or retained their CK7, CK8, and CK18, retained Vim, and increased CK19 and GFAP, while their mesenchymal morphology became clearer over time. CONCLUSIONS: Studies of intermediate filament proteins in vivo suggest that dedifferentiation occurs in RPE cells in ERM. Dedifferentiated RPE cells may be responsible for epithelioid-like and fibroblast/fusiform-like cells. Furthermore, changes in intermediate filament protein levels were observed in RPE cells in simulated cultures of proliferative intraocular disorders. These changes were linked to cells acquiring a mesenchymal migratory, phenotype. Results indicate that the dedifferentiation of RPE cells occurs both in vivo and in vitro and that it can be explained as an epithelial-mesenchymal transition.     

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。     

增生性玻璃体视网膜病变增生膜再塑型机制的研究

目的 观察不同病程增生性玻璃体视网膜病变（PVR）增生膜中不同细胞成分、细胞外基质（ECM）、基质金属蛋白酶（MMPs）及其抑制剂（TIMPs）随病程变化的规律，探讨PVR增生膜的再塑型机制。 方法 病程2个月至8年的孔源性视网膜脱离伴PVR患者的增生膜手术标本16例，用免疫组织化学方法标记增生膜中视网膜色素上皮（RPE）细胞、胶质细胞等不同的细胞成分，纤维连接蛋白（FN）、层粘连蛋白（l aminin）、Ⅰ～Ⅳ型胶原等不同ECM成分，以及MMPs（MMP2、MMP9）和TIMP1，分析不同病程PVR增生膜中各标记成分的变化以及与病程的相关性。 结果 随PVR病程延长，增生膜中RPE细胞、MMP2、FN表达逐渐减少(P=0.014，P=0.001，P=0.008)， 胶质细胞、Ⅰ、Ⅲ型胶原逐渐增多（P=0.022，P=0.001，P=0.008)，层粘连蛋白和Ⅱ、Ⅳ型胶原均有表达，但不随病程变化。RPE细胞、MMP2、纤维连接蛋白的表达与病程呈负相关，胶质细胞、Ⅰ、Ⅲ型胶原的表达与病程呈正相关；MMP2与FN变化呈正相关。MMP9、TIMP1始终都有表达，但不随病程变化。 结论 在PVR增生膜形成和发展的过程中，增生膜中的RPE细胞、胶质细胞、FN、Ⅰ、Ⅲ型胶原、MMP2参与了PVR的再塑型过程。 (中华眼底病杂志， 2006， 22： 308-312）     

结缔组织生长因子在兔视网膜增生膜中的表达

目的观察结缔组织生长因子（CTGF）在实验性增生性玻璃体视网膜病变（PVR）增生膜中的表达,探讨CTGF在PVR视网膜增生膜形成过程中的作用。方法采用Nobuyo的方法从兔视网膜中分离视网膜色素上皮（RPE）细胞并进行体外培养和传代。第3代RPE细胞制备密度为1.1×10^5/mL的细胞悬液。32只日本大耳白兔,取8只作为正常对照组,其余24只采用玻璃体腔内注入RPE细胞悬液的方法建立PVR动物模型。动物分别于造模后7、30、60d处死并摘除眼球,每次处死8只动物。光学显微镜下观察视网膜增生膜的组织学改变,免疫组织化学法检测PVR视网膜增生膜中CTGF的表达。结果造模后5d检眼镜下可见视网膜增生组织开始形成,增生膜随时间的推移逐渐增厚。组织学检查显示,造模后7d兔视网膜表面可见红染的条索状和网状胶原纤维,并可见大量的增生细胞分布其中。光学显微镜下可见视网膜内界膜变厚、粗糙、断裂或结构不清,视网膜各层结构欠清。免疫组织化学法检测表明,正常对照组在玻璃体及视网膜内未见CTGF的特异性染色。造模后7d,CTGF主要表达于视网膜表面增生细胞;造模后30d,CTGF主要表达于增生的细胞及增生的胶原纤维组织中;造模后60d,CTGF主要表达于增生的胶原纤维组织中。结论 CTGF在实验性PVR动物模型中呈高表达,提示CTGF参与了PVR增生膜的形成。     

增殖性视网膜病变玻璃体切割物的细胞学及免疫组化研究

目的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（ｃｙｔｏｋｅｒａｔｉｎ，ＣＫ），抗胶质纤维酸性蛋白（ｇｌｉａｌｆｉｂｒｉｌａｒｙａｃｉｄｉｃｐｒｏｔｅｉｎ，ＧＦＡＰ），抗肌动蛋白（Ａｃｔｉｎ）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。结果增殖性玻璃体视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅｖｉｔｒｅｏｒｅｔｉｎｏｐａｔｈｙ，ＰＶＲ）的增殖特征以胶质细胞和视网膜色素上皮细胞（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ，ＲＰＥ）为主，二者在ＰＶＲ发病中作用不同。增殖性糖尿病视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅｄｉａｂｅｔｉｃｒｅｔｉｎａｏｐａｔｈｙ，ＰＤＲ）玻璃体内出现ＲＰＥ细胞可加剧增殖膜收缩。增殖膜血管壁上肌动蛋白含量增多可能对周细胞脱落起促进作用。结论玻璃体内细胞增殖与生长因子水平升高可能是增殖性病变日益加剧的重要机制和原因之一。