上皮间充质转化在增生性玻璃体视网膜病变发病机制中的潜在作用

增生性玻璃体视网膜病变（proliferative vitreoretinopathy,PVR）是一种以纤维化为特点具有潜在致盲性的眼科并发症,其病理改变是产生视网膜前膜（epiretinal membranes,ERMs）。ERMs是一种纤维增殖膜,在视网膜前后表面及玻璃体内形成,当其收缩后导致PVR的发生,最终形成视网膜皱褶和牵引视网膜脱离（tractional retinal detachment,TRD）。多种细胞类型在ERMs中已被确定,视网膜色素上皮（retinal pigment epithelium,RPE）在PVR的病理生理学中至关重要。大量的临床和实验证据研究表明,RPE细胞采用了成纤维细胞表型发生了上皮间充质转化（epithelial-mesenchymal transition,EMT）,并且还涉及大量的细胞因子、转录因子。EMT的特点是细胞间黏附和顶面-底侧细胞极性的缺失,这增加了细胞的移行性。由此产生的细胞能够通过细胞外基质迁移和转移。细胞间黏附由连接复合体维系着,它们在维护RPE细胞表型方面起重要作用,其通过激活一些信号通路及转录因子破坏这些连接复合体并导致EMT的发生。经过EMT过程后,转化生长因子-β使RPE细胞进一步分化为肌成纤维细胞。源自RPE细胞的成纤维细胞和肌成纤维细胞可以通过细胞迁移、增殖,导致ERMs的形成,这在PVR的病程发展中起着关键性作用。     

生长因子与增生性玻璃体视网膜病变相关性的研究进展

增生性玻璃体视网膜病变(PVR)是眼球穿通伤及孔源性视网膜脱离手术后的常见并发症。该病的发病机制仍未明确,但研究表明,视网膜色素上皮(RPE)细胞具有自分泌细胞因子的能力,许多生长因子在PVR患者的玻璃体或者视网膜下积液中过度表达,这些生长因子及其受体在PVR的发生发展中扮演了重要角色,血-视网膜屏障被破坏后,生长因子的生理平衡即被打破,RPE细胞受到生长因子的刺激发生上皮-间质转化(EMT)、迁移及增殖,进而与其他细胞及细胞外基质形成视网膜前膜,牵拉视网膜造成视网膜脱离。近年来学者们对生长因子在PVR的形成中所涉及的信号通路、促EMT进程及细胞增殖做了大量研究,本文将对近年来促PVR发展的生长因子及拮抗生长因子治疗PVR的研究结果作一综述。     

血管内皮生长因子及上皮-间充质转化在增生性玻璃体视网膜病变发病中的作用

增生性玻璃体视网膜病变(PVR)是一种发生在孔源性视网膜脱离(RRD)自然病程中或复位手术后的严重并发症,常常导致患者视力丧失。目前,临床缺乏有效的治疗方法。PVR病理特征是多种细胞在细胞因子的作用下发生的过度炎症反应和异常增生,最终在视网膜表面形成增殖膜及进一步的牵拉性视网膜脱离(TRD)。对PVR发病机制的深入研究将有助于为其治疗寻找有前景的分子靶标。近年研究发现,血管内皮生长因子(VEGF)及视网膜色素上皮(RPE)细胞的上皮-间充质转化(EMT)在PVR发病中发挥着重要作用。本文就VEGF及RPE细胞EMT在PVR发病中的作用,以及二者的相互联动机制进行了总结,以期为PVR的治疗和临床研究提供新的思路。     

RPE细胞发生上皮间充质转化机制及PVR的治疗研究进展

增殖性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)是眼外伤、糖尿病性视网膜病变、血管性视网膜病变和炎症性视网膜病变等眼部疾病的严重并发症,也是孔源性视网膜脱离手术失败的最重要原因,对视功能的危害较大。大量研究已证明PVR发生的主要危险因素是视网膜损伤后血视网膜屏障受损,视网膜色素上皮(retinal pigment epithelial,RPE)细胞受到玻璃体腔内细胞因子的刺激,RPE细胞发生上皮间充质转化(epithelial-mesenchymal transition,EMT),转分化为成纤维样细胞,细胞的形态发生了变化,细胞间的紧密连接消失,细胞极性丧失,增殖、迁移、侵袭能力增强。在视网膜前表面或视网膜下形成具有收缩性的纤维增殖膜,形成的纤维增殖膜会使视网膜形成皱褶,牵拉视网膜导致视网膜脱离,最终会导致患者视力下降甚至失明。国内外对预防和治疗PVR进行了大量的研究,本文对RPE细胞发生上皮间充质转化相关信号通路及PVR的治疗进行简要综述。     

上皮-间充质转化在增生性玻璃体视网膜病变发病机制中的作用

增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)是孔源性视网膜脱离及玻璃体视网膜手术后的常见并发症,其主要特征是在视网膜前形成无血管的纤维细胞膜.超微结构和免疫病理研究表明,多种细胞参与PVR的发生与发展,如视网膜色素上皮细胞、神经胶质细胞、巨噬细胞、玻璃体细胞、成纤维细胞和肌成纤维细胞等.其中,肌成纤维细胞是引起PVR增生膜收缩导致视网膜复位手术失败的主要细胞,主要来自于视网膜色素上皮细胞的上皮-间充质转化(epithelial-mesenchymal transition,EMT),在PVR的发生发展中具有重要作用.因此,阻断视网膜色素上皮细胞转分化为肌成纤维细胞可能成为预防和治疗PVR的一种有效的方法.     

视网膜下液中转化生长因子-β1水平及受体细胞的检测

孔源性视网膜脱离的视网膜下液中的细胞以视网膜色素上皮(retinal pigment epithelium,RPE)细胞为主。有认为,RPE细胞对生长因子的特殊敏感性与增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)的发生有关。孔源性视网膜脱离患者其视网膜下液中碱性成纤维生长因子(basic fibroblast growth factor,bFGF)呈现高水平表达,本文将进一步研究PVR患者的视网膜下液中的转化生长因子-β1(transforming growth factor—beta 1,TGFβ1)水平和受体表达细胞。     

增生性玻璃体视网膜病变相关细胞因子研究进展

增生性玻璃体视网膜病变(PVR)是一种常见的致盲眼病,表现为受损的玻璃体及视网膜的异常修复.PVR的形成涉及多种细胞因子,包括血小板衍生生长因子(PDGF)、血管内皮生长因子、肝细胞生长因子、转化生长因子β、表皮生长因子、肿瘤坏死因子α、结缔组织生长因子等.视网膜色素上皮(RPE)细胞涉及Jagged-1/Notch信号通路和RhoA/ROCK信号通路发生细胞上皮-间充质形态改变.RPE细胞的增生、迁移与PKC/ERK信号通路和Akt/mTORCl信号通路相关.非PDGF间接激活PDGF受体α是PVR形成的主要通路.对PVR的治疗开展实验研究,联合抑制多种细胞因子及应用抗氧化剂N-乙酰半胱氨酸均取得一定疗效.本文对相关细胞因子在PVR形成中的作用机制、信号通路及治疗相关的实验研究进行综述.     

TGF-β参与黄斑前膜形成的研究进展

黄斑前膜(ERM)是一种以发生于视网膜内表面的纤维细胞膜为特征的视网膜疾病。现有的临床指南及文献资料对ERM的诊断与治疗已基本达成共识，但对其机制的阐述仍众说纷纭。转化生长因子-β(TGF-β)是一种高度多效性的细胞因子，在伤口愈合、血管生成、免疫调节、癌症、炎症及纤维化疾病中起着重要作用，越来越多的研究表明，ERM是玻璃体后脱离(PVD)引起视网膜发生炎性损伤以及视网膜色素上皮层细胞等细胞经历上皮间质转化(EMT)所致纤维化的一类病理改变，而多种细胞因子通过参与非经典的TGF-β-Snail途径与经典的TGF-β-Smad途径调控TGF-β介导的EMT进程。目前已有部分针对上述途径相关细胞因子的药物进入研发阶段，对提供ERM临床治疗及预防新思路具有重要意义。本文就TGF-β在ERM形成中相关细胞因子的研究进展作一综述。     

抑制视网膜色素上皮细胞发生上皮-间充质转化的研究进展

视网膜色素上皮细胞(retinal pigment epithelium,RPE)的上皮间充质转化(epithelial-mesenchymal transition, EMT)是增殖性玻璃体视网膜疾病(proliferative vitreoretinopathy,PVR)的主要发病机制,也是治疗脉络膜新生血管(choroidal neovascularization, CNV)时,造成抗血管内皮生长因子(anti-vascular endothelial growth factor, anti-VEGF)疗效降低的重要因素。除此之外,随着细胞替代治疗作为年龄相关性黄斑变性(age-related macular degeneration,ARMD)的新型治疗方式而兴起,人类胚胎干细胞(hESC-RPE)、体细胞诱导多能干细胞(iPSC-RPE)定向诱导分化的RPE细胞以及来自于流产胎儿的胎儿视网膜色素上皮细胞(fetal RPE,fRPE)逐渐受到了研究者们的关注。为了发挥最好的治疗效果,保证治疗用细胞的稳定增殖及高效分化是极其重要的。但是在传代扩增的过程中,由于上皮间充质转化的存在,导致这些细胞出现增殖困难以及再分化能力下降,从而影响治疗效果并阻碍了治疗人群的普及,使细胞替代治疗难以推广。所以,鉴于上皮间充质转化在视网膜疾病的发生和治疗中都造成了重要影响,抑制视网膜色素上皮细胞发生上皮间充质转化就显得尤为重要,为此,我们将阻止上皮间充质转化研究的最新进展综述如下。     

转化生长因子β与增生性玻璃体视网膜病变的关系

增生性玻璃体视网膜病变 (proliferativevitreo retinopathy ,PVR)在临床上具有反复发生 ,难以控制的特点。视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞是PVR最重要的细胞成分 ,功能十分复杂 ,许多生长因子在PVR发生发展过程中的影响皆与病理状态下RPE细胞的功能异常有关。我们从RPE细胞入手 ,重点对转化生长因子 β与PVR的关系作一综述 ,从一个侧面揭示PVR形成机制和发展方向 ,以期对PVR的生物性治疗起一些提示作用。     

The role of gremlin, a BMP antagonist, and epithelial-to-mesenchymal transition in proliferative vitreoretinopathy

PURPOSE: Proliferative vitreoretinopathy (PVR), a major reason for failure of retinal detachment surgery, is characterized by the formation of scarlike tissue that contains transdifferentiated retinal pigment epithelial (RPE) cells. The scar tissue occurs in response to growth factors such as transforming growth factor (TGF)-beta and epidermal growth factor (EGF). The authors postulate that transdifferentiation of RPE cells may arise via epithelial-to-mesenchymal transition (EMT). Bone morphogenetic proteins (BMPs) are expressed in the retina and have an antiproliferative role. Gremlin is expressed in the outer retina and is a BMP antagonist. The study was conducted to establish a model of PVR by inducing EMT in the human RPE cell line ARPE-19, using TGF-beta and EGF and to establish the contribution of gremlin to EMT. METHODS: ARPE-19 cells were cultured and stimulated with TGF-beta1, EGF, and gremlin. The expression of alpha-smooth muscle actin (alpha-SMA), vimentin, and zona occludens (ZO)-1 were examined via PCR, Western blot analysis, and immunofluorescence. Zymography was performed for matrix metalloproteinase (MMP) activity. Scratch assays were performed to assess migration. RESULTS: A model of EMT was established in the ARPE-19 cell line. The characteristics of EMT include gain of alpha-SMA, loss of ZO-1, upregulation of MMP activity and enhanced migration. Gremlin plays an important role in this process, contributing to the gain of alpha-SMA, loss of ZO-1, and upregulation of MMP activity. CONCLUSIONS: EMT occurs in vitro in the ARPE-19 cell line in response to the growth factors TGF-beta1 and EGF. EMT is also induced by Gremlin.     

Effectively Intervening Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells With a Combination of ROCK and TGF-β Signaling Inhibitors

PurposeEpithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a key pathological event in proliferative retinal diseases such as proliferative vitreoretinopathy (PVR). This study aimed to explore a new method to reverse EMT in RPE cells to develop an improved therapy for proliferative retinal diseases.MethodsIn vitro, human embryonic stem cell–derived RPE cells were passaged and cultured at low density for an extended period of time to establish an EMT model. At different stages of EMT after treatment with known molecules or combinations of molecules, the morphology was examined, transepithelial electrical resistance (TER) was measured, and expression of RPE- and EMT-related genes were examined with RT-PCR, Western blotting, and immunofluorescence. In vivo, a rat model of EMT in RPE cells was established via subretinal injection of dispase. Retinal function was examined by electroretinography (ERG), and retinal morphology was examined.ResultsEMT of RPE cells was effectively induced by prolonged low-density culture. After EMT occurred, only the combination of the Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor Y27632 and the TGF-β receptor inhibitor RepSox (RY treatment) effectively suppressed and reversed the EMT process, even in cells in an intermediate state of EMT. In dispase-treated Sprague-Dawley rats, RY treatment maintained the morphology of RPE cells and the retina and preserved retinal function.ConclusionsRY treatment might promote mesenchymal-epithelial transition (MET), the inverse process of EMT, to maintain the epithelial-like morphology and function of RPE cells. This combined RY therapy could be a new strategy for treating proliferative retinal diseases, especially those involving EMT of RPE cells.     

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。     

Induction of proliferative vitreoretinopathy by a unique line of human retinal pigment epithelial cells

BACKGROUND: The most widely used models of proliferative vitreoretinopathy (PVR) rely on injection of cells into the vitreous of animals. Using retinal pigment epithelial (RPE) cells from human PVR membranes may produce a more accurate model of human PVR. We performed a study to determine whether human RPE cells derived from a single epiretinal membrane (ERM) are capable of inducing the same disease in the rabbit eye, and whether the induced ERMs had cellular components similar to those of human PVR membranes. METHODS: Cells were harvested from a human ERM obtained at surgery for PVR. RPE cells were cultured from the membrane and injected into the right eye of 24 New Zealand albino rabbits. The left eyes served as controls. The eyes were examined by indirect ophthalmoscopy over 4 weeks. The enucleated eyes were then examined by means of microscopy and histochemical analysis. RESULTS: By day 7, PVR had developed in all but 1 of the 24 experimental eyes, with 8 progressing to localized tractional retinal detachment. By day 21, localized tractional retinal detachment had developed in 17 eyes; 1 eye progressed to extensive tractional retinal detachment by day 28. Immunostaining showed that mostly RPE cells, but also myofibroblasts, glial cells and collagen, were present in the newly formed rabbit PVR membranes. INTERPRETATION: Human RPE cells cultured from a PVR membrane appear to be capable of inducing PVR in rabbits. The resultant ERMs are similar to those formed in human PVR and consist mainly of RPE cells.     

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.     

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)     

Modulation of growth in retina-derived cells by extracellular matrices

Intravitreal membranes from patients with proliferative vitreoretinopathy (PVR) consist partly of retinal glial (RG) and retinal pigment epithelial (RPE) cells surrounded by varying amounts of extracellular matrix (ECM). The contribution of the ECM to the growth of PVR membranes is unknown. This study was undertaken to determine if proliferation in cultured RPE and RG cells is affected by different substrates, including some ECM materials which have been identified in PVR membranes. Substrates tested included type I collagen, basement membrane Matrigel, and poly-D-lysine, as well as uncharacterized cell type-specific matrices deposited by cultured RPE and RG cells. Proliferation was quantified by 3H-thymidine incorporation and radioautography 24 hours after plating and by cell counts after 14 days in the presence of serum. Relative to uncoated culture plastic, growth of RPE cells was inhibited by Matrigel, enhanced by poly-D-lysine, and unaffected by type I collagen. In contrast, growth in RG cells was inhibited by type I collagen and unaffected by the other substrates. Analysis of the timing of DNA synthesis after plating suggested that the substrates which affected RPE growth did so by altering the fraction of cycling cells rather than the cell cycle time. For the cell-derived matrices, heterotypic matrix (matrix produced by the other retinal cell type) enhanced the growth of both RPE and RG. The results suggest that the ECM may modify the growth of cells contributing to PVR membranes. Of note is that the cell-derived matrices reciprocally stimulated growth of RG and RPE cells, cell types which may interact in PVR membranes.