视网膜色素上皮细胞损伤机制研究进展

近年来许多研究表明,视网膜色素上皮(retinal pigment epithelium,RPE)细胞的损伤或增殖与多种视网膜疾病的发生发展有关。本文通过对RPE细胞损伤现状的阐述、以及它在视网膜病变过程中所起到的作用,期望从RPE细胞损伤机制寻找预防和治疗这些疾病的新途径。     

视网膜色素上皮细胞凋亡机理的探讨

了解诱导视网膜色素上皮细胞在体外发生凋亡的以推测该细胞凋亡在体内发生的可能机理。方法培养的人ＲＰＥ细胞分别给予低氧，肿瘤坏死因子，蛋白激酶抑制剂及抗Ｆａｓ抗体处理２４－７２ｈ，同时也观察了纤维母细胞生长因子对体外凋亡诱导因子所致ＲＰＥ细胞凋亡的保护作用。     

可见光对体外培养兔视网膜色素上皮细胞凋亡的实验研究

目的研究可见光对体外培养的兔视网膜色素上皮(retinal pigmen tepithelium,RPE)细胞凋亡的影响。方法用两步酶法消化分离、培养并鉴定RPE细胞。RPE细胞分为对照组及实验组,实验组用(3652±213)Lux的白色荧光灯照射6h。分别于光照前及光照后3h、12h、24h、72h终止培养,取细胞行Bcl-2mRNA原位杂交检测,观察细胞Bcl-2基因表达情况。选取部分光照后24h组细胞进行透射电镜的观察。结果对照组各时间点Bcl-2mRNA原位杂交阳性细胞率无统计学差异(P>0.05)。实验组光照后3h阳性细胞率为(82.29±5.65)%,有轻度增高,但无统计学意义(P>0.05);光照后72h阳性细胞率为(44.07±7.11)%,光照后12h、24h、72h阳性细胞逐渐减少,均有统计学意义(P<0.05)。RPE细胞光照后透射电镜观察到染色质碎裂、凋亡小体等细胞凋亡的典型形态学变化。结论一定强度的可见光照射可引起体外RPE的凋亡。光照后一定时间内可以刺激并启动细胞自身的保护机制。     

三氧化二砷对兔视网膜色素上皮细胞DNA损伤的研究

目的探讨三氧化二砷（As2O3）对视网膜色素上皮（RPE）细胞的损伤作用。方法按照Singh的方法从色素兔眼分离RPE细胞并进行体外培养,培养的细胞用细胞角蛋白进行免疫组织化学鉴定。按照对培养细胞的干预方式不同分为阴性对照组（培养基中加入PBS）、阳性对照组（254nm紫外线照射10min）及实验组。实验组分别在培养基中加入终浓度为50．00、5．00、2．00、1．00、0．50、0．25μmol／L的As2O3作用1h。采用锥虫蓝染色法检测各组细胞的活性。采用彗星实验的方法测定细胞的损伤程度。结果90％以上的培养细胞对细胞角蛋白产生阳性的免疫反应。锥虫蓝染色表明各组细胞的形态和细胞活力均无明显差别。不同组中RPE细胞的尾部DNA含量和尾距的差异均有统计学意义（F=88．548,P=0．000;F=129．704,P=0．000）。与对照组比较,不同浓度的As2O3实验组RPE细胞的尾部DNA含量和尾距明显增加,差异均有统计学意义（P〈0．05）,其中50μmol／L组最明显,而0．5μmol／L、0．25μmol／L组与对照组间差异则无统计学意义（P〈0．05）。结论As2O3能够诱导RPE细胞的DNA损伤,损伤程度与As2O3的浓度有关。     

视网膜色素上皮细胞凋亡相关疾病的治疗现状及展望

细胞凋亡是生理或病条件下的一种由遗传基因控制的基础主动死亡形式,对维持机体的正常发育以及内环境的稳定起重要作用。细胞凋亡平衡的破坏可导致多种疾病的发生发展。现介绍基因治疗、细胞因子或生长因子、视网膜移植对视网膜色素上皮（retinal pigment epithelial,RPE)细胞凋亡所致疾病的治疗现状及前景。     

视网膜色素上皮细胞凋亡的研究进展

细胞凋亡是由遗传基因调控的细胞主动死亡形式,具有不同于细胞坏死的典型特征,越来越多的证据表明,视网膜色素上皮（RPE）细胞凋亡参与了许多眼底疾病的发生发展。阐述了RPE细胞凋亡的形态学特点,生物化学特征、遗传基因调控以及活性氧自由基在其凋亡过程中所起的作用。     

手机光照刺激对视网膜色素上皮细胞的影响

目的：观察手机光照刺激体外培养人视网膜色素上皮细胞(retinal pigment epithelium; RPE)后形态及B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl2-Associated X(Bax)、半胱天冬酶-3(Caspase-3)的mRNA表达变化。

方法：将体外培养的人RPE细胞随机分成4组,根据RPE细胞光照时间长短分为照射3h光照组、6h光照组、12h光照组和0h光照组。在特制的培养箱内,将智能手机屏幕开到最大亮度(200±20Lx)做为光源,持续静音情况下循环播放彩色图片。然后应用苏木精-伊红(HE)染色法,末端脱氧核糖核酸转移酶介导的原位缺口末端标记(TUNEL)染色,四甲基偶氮唑盐微量酶反应比色(MTT)法观察各实验组RPE细胞形态学改变,并运用聚合酶链反应(PCR)技术检测各组细胞中Bcl-2、Bax、Caspase-3的mRNA表达变化。

结果：用HE染色、TUNEL染色、MTT法观察发现各光照组RPE细胞形态学无明显改变,差异均无统计学意义(P>0.05)。应用PCR技术检测发现RPE细胞在光照刺激3h光照组和6h光照组,细胞内的Bcl-2、Bax、Caspase-3的mRNA表达与0h光照组的差异均无统计学意义(P>0.05)。但随着光照时间持续延长(12h),细胞内的Bcl-2表达下调,Bax、Caspase-3表达水平均上升,差异均有统计学意义(P<0.05)。

结论：手机屏幕等作为人造光源在长期持续高亮度照射下会引起体外培养人视网膜色素上皮细胞的损伤。     

视网膜色素上皮与氧化损伤

视网膜色素上皮与氧化损伤白禹诗喻晓兵综述吴景天审校中国医科大学院附属第一医院眼科（１１０００１）视网膜色素上皮（ＲＰＥ）是位于神经视网膜和Ｂｒｕｃｈ＇ｓ膜之间排成整齐单层的六角形细胞。ＲＰＥ生理功能的正常发挥可维护和保证光感细胞对光信号的接收和传导，...     

视网膜色素上皮细胞凋亡的研究现状

细胞凋亡是一种由基因控制的程序化的细胞死亡过程，不同的基因参与凋控该过程。目前的研究表明，视网膜色素上皮细胞参与多种玻璃体视网膜病变的发生与发展。阐述了视网膜色素上皮细胞凋亡的概况、相关基因以及在玻璃体视网膜病变过程中所起到的作用，探索通过视网膜色素上皮细胞凋亡预防和治疗这些疾病的新途径。     

自体虹膜色素上皮细胞移植治疗视网膜色素上皮细胞变性研究

目的:研究自体虹膜色素上皮细胞(iris pigment epithelium,IPE)移植治疗视网膜色素上皮细胞变性的效果。方法:采用酶-机械分离-酶消化法获取高纯度、高活力的IPE细胞用于移植,采用外路法将IPE细胞悬液移植入视网膜下腔。结果:IPE移植术后患者眼底移植区色素沉着减少,未见渗出,出血,视网膜平,视力入院时的0.15,术后2wk提高至0.2。多焦ERG显示移植区P1波平均反应密度上升。结论:自体虹膜色素上皮细胞移植有望成为视网膜色素上皮细胞变性疾病治疗的一新方法。     

视网膜色素上皮细胞直接铺片法与剥离铺片法比较

目的 介绍2种视网膜色素上皮(RPE)细胞铺片实验技术,比较其实验操作的难易程度及对RPE细胞形态和铺片完整性的影响.方法 实验研究.直接铺片法:大鼠眼球取出后用4％多聚甲醛固定30 min,解剖眼球,去除眼前段部分和眼内容物,小心剥去视网膜,将RPE贴附的眼后段部分剪成花瓣形并平整贴附在载玻片上,继续固定过夜.剥离铺片法:大鼠眼球取出后用4％多聚甲醛固定过夜,解剖眼球,去除眼前段部分和眼内容物,小心剥去视网膜,用镊子将RPE-Bruch膜复合物从眼球上分离,保存在PBS液中.结果 剥离铺片法能得到完整RPE细胞铺片,RPE细胞形态完整;直接铺片法中RPE细胞部分丢失,铺片边缘的RPE细胞被部分破坏.结论 直接铺片法操作简便,难度小,但容易破坏边缘的RPE细胞.剥离铺片法操作难度大,但对RPE细胞破坏程度小.     

RPE transplantation and its role in retinal disease

Retinal pigment epithelial (RPE) transplantation aims to restore the subretinal anatomy and re-establish the critical interaction between the RPE and the photoreceptor, which is fundamental to sight. The field has developed over the past 20 years with advances coming from a large body of animal work and more recently a considerable number of human trials. Enormous progress has been made with the potential for at least partial restoration of visual function in both animal and human clinical work. Diseases that have been treated with RPE transplantation demonstrating partial reversal of vision loss include primary RPE dystrophies such as the merTK dystrophy in the Royal College of Surgeons (RCS) rat and in humans, photoreceptor dystrophies as well as complex retinal diseases such as atrophic and neovascular age-related macular degeneration (AMD). Unfortunately, in the human trials the visual recovery has been limited at best and full visual recovery has not been demonstrated. Autologous full-thickness transplants have been used most commonly and effectively in human disease but the search for a cell source to replace autologous RPE such as embryonic stem cells, marrow-derived stem cells, umbilical cord-derived cells as well as immortalised cell lines continues. The combination of cell transplantation with other modalities of treatment such as gene transfer remains an exciting future prospect. RPE transplantation has already been shown to be capable of restoring the subretinal anatomy and improving photoreceptor function in a variety of retinal diseases. In the near future, refinements of current techniques are likely to allow RPE transplantation to enter the mainstream of retinal therapy at a time when the treatment of previously blinding retinal diseases is finally becoming a reality.     

Photooxidation of RPE lipofuscin bisretinoids enhances fluorescence intensity

Light-related cycling of chemically reactive vitamin A aldehyde leads to the formation autofluorescent bis-retinoid pigments that accumulate as lipofuscin in retinal pigment epithelial cells. The amassing of these diretinoid compounds is implicated in the pathogenesis of age-related macular degeneration and in some inherited forms of retinal degeneration. For all of these fluorophores, extended conjugation systems confer absorbance maxima in the visible spectrum. We report that an increase in fluorescence emission can accompany photoxidation of the bisretinoids A2E and all-trans-retinal dimer. These findings are relevant to the quantification of RPE lipofuscin based on inference from fluorescence intensity.     

生长因子对培养人眼视网膜色素上皮细胞增殖的调控作用

目的探讨生长因子对人眼视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ，ＲＰＥ）细胞增殖的调控作用。方法建立人眼ＲＰＥ细胞培养体系，利用３Ｈ－ＴｄＲ掺入法和细胞计数观察表皮生长因子（ｅｐｉｄｅｒｍａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＥＧＦ）、碱性成纤维细胞生长因子（ｂａｓｉｃ－ｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ，ｂ－ＦＧＦ）、胰岛素样生长因子（ｉｎｓｕｌｉｎ－ｌｉｋｅｇｒｏｗｔｈｆａｃｔｏｒ，ＩＧＦ－Ｉ）对培养ＲＰＥ细胞的作用。结果（１）ＥＧＦ，ｂ－ＦＧＦ，ＩＧＦ－Ⅰ与对照组比较，可明显增强人眼ＲＰＥ细胞ＤＮＡ合成６．６～１２．２倍，增强能力为ＥＧＦ＞ｂ－ＦＧＦ＞ＩＧＦ－Ⅰ。（２）当生长因子联合作用时，可数倍明显增强３Ｈ－ＴｄＲ掺入，能较单一因子更有效地刺激人眼ＲＰＥ增殖。结论结果提示，生长因子的协同作用可能是增殖性视网膜病变发生的重要机理之一。     

Regulated secretion of complement factor H by RPE and its role in RPE migration

Background  Variants in the gene for complement factor H (CFH) have been implicated as a major risk factor for the development of age-related macular degeneration (AMD). Little is known, however, about the factors regulating local expression and secretion of CFH by retinal pigment epithelial cells (RPE). Methods  Cultured human early passage RPE cells, highly differentiated, polarized human RPE cultures, and bovine RPE explants were incubated in the presence or absence of recombinant human or bovine interferon-γ (IFN-γ; 25 ng/ml). CFH expression in cell lysates, and secretion into culture supernatants were examined by Western blot. CHF expression and localization was analyzed by confocal microscopy. Migration assay was performed in a modified Boyden chamber with early passage human RPE cells after stimulation with recombinant CFH protein (1–100 ng/ml). Results  CFH was expressed in the cell lysates of RPE cells, and this expression was significantly upregulated by IFN-γ. Immunoreactivity for CFH was detected in RPE cells of bovine explants and highly differentiated human RPE monolayers, and the level of immunoreactivity increased after IFN-γ stimulation. Confocal microscopy revealed that CFH was predominantly localized in the apical cytoplasm of polarized human RPE. Western blot confirmed that IFN-γ increased CFH secretion into RPE supernatants. Dose-dependent RPE cell chemotactic migration was induced by CFH. Conclusion  IFN-γ promotes CFH expression in the apical compartment of RPE cells and increases secretion of CFH into RPE culture supernatants. Furthermore, CFH promotes chemotactic migration of RPE. This study suggests that interactions between CFH and IFN-γ have the potential to play a role in the pathogenesis of AMD. Shikun He contributed equally to the work, and therefore should be considered equivalent first author     

兔虹膜和视网膜色素上皮细胞的体外培养比较

目的 观察比较有色素兔虹膜色素上皮(IPE)及视网膜色素上皮(RPE)细胞的体外生长状况。方法 采用酶消化法及酶辅助机械分离法分别分离IPE和RPE细胞。观察培养的两种细胞的生长状况，并对其进行免疫组化鉴定。结果 原代IPE及RPE细胞大多呈圆形或六边形，细胞内充满了黑色素颗粒。随着传代的增加，呈梭形或纤维细胞样生长的细胞增多，细胞中的色素颗粒也逐渐减少。细胞角蛋白免疫组化染色显示IPE及RPE细胞绝大多数呈棕黄色阳性反应。Desmin兔疫组化染色显示IPE细胞中阳性细胞约占5％。结论 分离培养的IPE和RPE细胞纯度很高。IPE细胞中绝大多数为后层IPE细胞。IPE和RPE细胞的形态及体外生长特点类似。     

视网膜色素上皮移植细胞超微结构的观察

目的 观察视网膜色素上皮移植细胞的超微结构,为临床应用提供实验基础。方法 取有色兔眼球后段,去除视网膜的神经上皮层,获得视网膜色素上皮细胞作为移植的供体细胞。以１０只无色兔为移植的受体,内路法将供体色素上皮细胞移植到视网膜下腔,电镜观察移植细胞的超微结构。结果 移植的１０只眼,细胞成功移植到视网膜下腔,可见移植细胞粘附于受体的Ｂｒｕｃｈ膜并形成基底内褶,细胞间紧密连接,细胞内可见吞噬体。结论 内路     

兔眼虹膜和视网膜色素上皮细胞移植的形态学观察

目的 比较有色兔虹膜色素上皮及视网膜色素上皮细胞在白兔视网膜下腔的生长状况。 方法 分离培养有色兔的IPE及RPE细胞。采用内路法在8只白兔16眼中进行了IPE和RPE细胞悬液的移植,其中左眼移植IPE细胞,右眼移植来自同一供体兔的RPE细胞。8只兔分别在2周(n=3)、4周(n=3)和6周(n=2)时处死。对眼球壁做光镜和电镜检查。 结果 IPE和RPE细胞在异体视网膜下腔存活,绝大多数贴附在Bruch’s膜上,并具有极性,细胞基底部有大量的皱褶,细胞顶部有一些微绒毛。6周时未见移植细胞周围有炎性细胞浸润。 结论 移植的IPE和RPE细胞可以在视网膜下腔存活,它们的形态均和原先RPE细胞类似。IPE细胞有望替代RPE细胞用于移植。     

HTRA1基因过表达对视网膜色素上皮细胞增殖及迁移影响

目的 研究HTRA1基因过表达对人视网膜色素上皮细胞(RPE)的增殖和迁移能力的影响,探讨HTRA1基因在老年黄斑变性(AMD)发病中的作用.方法 用携带人HTRA1基因的慢病毒感染体外培养的人RPE细胞,荧光显微镜下观察细胞的感染效率,RT-PCR和Westem Blot检测HTRAlmRNA和蛋白的表达,MTT比色法检测感染细胞毒性及细胞增殖能力,TransweH小室检测细胞迁移能力变化.结果 与空载体慢病毒感染RPE细胞相比,HTRA1基因慢病毒感染的RPE细胞HTRA1mRNA和蛋白的表达量明显增加,细胞增殖能力及迁移能力均明显下降,差异有统计学意义(P＜0.01).结论 过表达HTRA1基因能抑制人视网膜色素上皮细胞的增殖及迁移,THRA1基因可能通过影响RPE细胞功能参与AMD的进程.     

Reduction of iatrogenic RPE lesions in AMD patients: evidence for wound healing?

Purpose Our purpose was to study retinal pigment epithelium (RPE) wound healing in patients with age-related macular degeneration (AMD). Patients and methods Abrasive debridement of nasal RPE was performed with a metal cannula during pars plana vitrectomy for foveal choroidal neovascularization (CNV) membrane excision combined with simultaneous autologous RPE transplantation. Fundus autofluorescence, fluorescein angiography images, and red-free pictures were taken initially within 1–2 weeks postoperatively, subsequently in 2-week intervals until 3 months, monthly until 6 months, and every 3 months thereafter. The borders of these lesions were measured; areas were calculated and compared using ArchiCad Software. Fourteen eyes of 14 patients suffering from AMD were included (nine women and four men, mean age 75.6 years ±6.6 years). Results Six of 14 (42.9 %) patients showed a reduction of the RPE debrided area. The size of these lesions reduced 5.6–20% within 2 postoperative months compared with their size at first examination (from a mean of 13.7 mm2 ± 7.2 at baseline to a mean of 12.8 mm2 ± 6.7 at 2 months postoperatively). No further reduction of the lesions was seen after the 2 months. In eight cases, borders of the RPE debrided areas stayed stable during observation time. Conclusions Wound healing of abrasively debrided RPE monolayer defects in patients with AMD occurs to a certain extent in nearly half of the cases. This process seems to stop after 2 months. Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, FL, USA, 25–29 April 2004. Supported by unrestricted research grant from the L. Boltzmann Institute to SB.