视网膜色素变性治疗研究进展

视网膜色素变性(retinitis pigmentosa,RP)是一组遗传性视网膜疾病,其特征是渐进性感光细胞和视网膜色素上皮(RPE)细胞功能障碍,是世界范围内常见的致盲性眼病,且缺乏有效的治疗方法。目前RP的治疗方法包括干细胞治疗、基因治疗、神经保护治疗、营养疗法、高压氧疗法、视网膜移植和中医治疗。本文综述了近年来国内外有关RP治疗的研究进展。     

神经营养因子治疗视网膜色素变性的研究进展

神经营养因子是能够促进神经元存活、生长、分化及维持其功能的多效性肽类因子的总称,可被作为有效的神经保护剂用于治疗多种神经变性类疾病.视网膜色素变性(RP)是以光感受器-视网膜色素上皮复合体损害为主的高度遗传异质性视网膜变性疾病,神经营养因子作为不针对致病基因的RP治疗策略,其疗效已在多种视网膜变性的动物模型中得到证实.以病毒为载体的转基因治疗和细胞包囊技术为神经营养因子提供了有效的给药途径,可使疗效明显提高.对神经营养因子在视网膜中的表达及其调节、受体分布特点、作用通路、疗效及副作用等方面的深入研究为神经营养因子的临床应用奠定了基础.     

神经营养因子治疗视网膜色素变性的研究进展

神经营养因子是能够促进神经元存活、生长、分化及维持其功能的多效性肽类因子的总称,可被作为有效的神经保护剂用于治疗多种神经变性类疾病.视网膜色素变性(RP)是以光感受器-视网膜色素上皮复合体损害为主的高度遗传异质性视网膜变性疾病,神经营养因子作为不针对致病基因的RP治疗策略,其疗效已在多种视网膜变性的动物模型中得到证实.以病毒为载体的转基因治疗和细胞包囊技术为神经营养因子提供了有效的给药途径,可使疗效明显提高.对神经营养因子在视网膜中的表达及其调节、受体分布特点、作用通路、疗效及副作用等方面的深入研究为神经营养因子的临床应用奠定了基础.     

碘酸钠诱发的视网膜色素变性动物模型研究进展

视网膜色素变性(RP)是一组以视网膜色素上皮(RPE)和感光细胞的功能障碍或丧失为主要特征的致盲眼病,有很大的临床和遗传异质性.碘酸钠是抗代谢药物,可以选择性破坏RPE,它引起的视网膜病变与人类RP疾病的片状RPE缺损极为相似,因此常被用来制作RP疾病的模型.碘酸钠能诱发多种实验动物的RP模型,人们多使用单次碘酸钠注射的方法建立RP动物模型.由于碘酸钠诱发的RP动物模型的发病年龄和疾病进展均可人为操控,制备简单,病变稳定,周期短,且某些动物的RPE细胞在特殊条件下有再生的潜能,极有利于RP发病机制、药物或干/前体细胞治疗及再生医学方面的研究.目前有关碘酸钠诱发动物RP模型的研究和应用已有不少报道.本文根据国内外文献将碘酸钠诱发的RP动物模型的研究进展作一综述.     

神经营养因子治疗视网膜色素变性的研究进展

目前为止发现了30余种视网膜色素变性(RP)的致病相关基因,发病机制研究提示凋亡可能是它们引起光感受器细胞萎缩的共同病理途径。神经营养因子是一类对神经系统的分化、发育,对神经元的存活,轴突再生均有重要作用的细胞因子。它们可能通过调控视网膜光感受器细胞的凋亡过程起到神经保护作用,有望成为治疗RP的有效药物。本文对近年来神经营养因子的光感受器保护作用、给药途径、治疗机制、副作用等方面的研究状况进行综述,其中基因工程改造的神经营养因子和基因修饰细胞半透膜埋植系统的研究取得了进展。     

视网膜色素上皮细胞的凋亡和预防

视网膜色素上皮细胞(retinal pigment epithelial,RPE)的凋亡在许多视网膜疾病的发生发展中起着重要作用,近年来,随着人们对细胞凋亡研究的进一步深入,对于细胞凋亡的发生机制有了更深的了解,本文阐述了RPE细胞的功能;同时对导致RPE细胞凋亡的可能机制、RPE细胞凋亡的预防作一综述.     

神经营养因子治疗视网膜色素变性的研究进展

目前为止发现了30余种视网膜色素变性（RP）的致病相关基因,发病机制研究提示凋亡可能是它们引起光感受器细胞萎缩的共同病理途径.神经营养因子是一类对神经系统的分化、发育,对神经元的存活,轴突再生均有重要作用的细胞因子.它们可能通过调控视网膜光感受器细胞的凋亡过程起到神经保护作用,有望成为治疗RP的有效药物.本文对近年来神经营养因子的光感受器保护作用、给药途径、治疗机制、副作用等方面的研究状况进行综述,其中基因工程改造的神经营养因子和基因修饰细胞半透膜埋植系统的研究取得了进展.     

骨髓间充质干细胞在视网膜色素变性大鼠视网膜下的分化

目的 研究骨髓间充质干细胞(MSCs)在视网膜色素变性(RP)大鼠体内的分化. 方法 Lewis大鼠腹腔注射3%NaIO3 100mg/kg,建立大鼠RP模型,将体外培养的MSCs植入视网膜下腔,用免疫荧光标记法对MSCs进行追踪,并观察术后第1、2、3、4、5周MSCs在该微环境中的分化.结果 术后第1周即可见MSCs位于视网膜色素上皮(RPE)层与光感受器细胞层,但全角蛋白(PCK)及Rhodopsin标记阴性,第3周开始可见MSCs在体内表达PCK及Rhodopsin.结论 MSCs植入RP模型大鼠视网膜下腔后可存活,主要分布于RPE层和视锥、视杆细胞层,并表达RPE细胞和光感受器细胞的表面标志.     

兔胚胎全层视网膜移植至大鼠视网膜下的观察

视网膜色素变性(RP)是一组能导致进行性感光细胞变性和视觉障碍的遗传病症候群，目前尚无有效治疗方法。近年来研究表明视网膜移植是最接近临床应用治疗RP的方法。当RP进展到晚期，感光细胞和视网膜色素上皮(RPE)细胞都出现变性死亡，单独移植这两种成分都没有作用，此时病变视网膜的修复需要全层视网膜的移植。我们将青紫蓝兔胚胎全层视网膜移植到正常Wistar大鼠和视网膜变性RCS大鼠的视网膜下腔，观察移植物在宿主视网膜下腔的发育状况，从而探讨异种胚胎全层视网膜移植方法的可行性。     

视网膜色素变性的眼底血流变化研究现状

视网膜色素变性(retinitis pigmentosa,RP)是一种临床常见的遗传性致盲性眼底病,多由夜盲起病,经过几十年进行性视力下降致最终失明.其病理特点为原发性感光细胞凋亡长期发展最终导致视网膜色素上皮和内层视网膜结构及功能损伤.多个研究表明RP早期出现眼底血流减少.原因可能是原发性血管功能失调和血浆内皮素-1升高.目前有多种可有效评估视网膜血流情况的方法.针对改善眼底血流的治疗能够在一定程度上延缓RP病情的进展.对于眼底血流和RP关系的认识加深有助于寻找新的有效治疗RP的方法.     

细胞因子对视网膜神经元保护和诱导作用的研究进展

近年来细胞因子在视网膜功能重建中的作用已初见端倪。研究发现,脑源性神经营养因子、睫状神经营养因子、碱性成纤维细胞生长因子及神经营养素4可以防止视网膜光感受器细胞变性的发生,增强光感受器细胞损伤后的修复,促进视网膜神经节细胞的发育,刺激神经节细胞轴突的再生。同时,晶状体上皮源性生长因子及人羊膜上皮细胞分泌的细胞因子也对神经节细胞有保护效应。而神经营养素-3在视网膜的作用性质还有待更多的研究。（中华眼科杂志,2005,41：861-864）     

Brain-derived neurotrophic factor gene delivery to muller glia preserves structure and function of light-damaged photoreceptors

PURPOSE: To test the hypothesis that adenovirus (Ad)-mediated gene delivery of brain-derived neurotrophic factor (BDNF) to Müller cells can protect photoreceptors from light-induced retinal degeneration. METHODS: Adult Sprague-Dawley rats received an intraocular injection of Ad.BDNF, control Ad containing the green fluorescent protein (GFP) gene, or BDNF recombinant protein. Animals were then exposed to 5, 10, or 16 days of constant light. The effect of Ad.BDNF on photoreceptor survival was examined histologically, by measuring the outer nuclear layer (ONL) thickness, and functionally, by measuring the electroretinographic (ERG) response. RESULTS: Ad.BDNF mediated sustained expression of bioactive neurotrophin by Müller cells that lasted for at least 30 days after viral vector administration. BDNF gene delivery to Müller glia markedly increased the survival and structural integrity of light-damaged photoreceptors. For example, after 10 days of exposure to light, the average percentage of ONL preservation in the superior central retina of eyes that received Ad.BDNF was 71%, compared with 46% in eyes that received a control Ad.GFP or 15% in contralateral eyes. Of importance, retinas exposed to Ad.BDNF had more photoreceptor nuclei than retinas that received a single intraocular injection of BDNF recombinant protein. The neuroprotective effect of Ad.BDNF was accompanied by preservation of the ERG response of the treated eyes. CONCLUSIONS: These data provide proof of the concept that BDNF gene transfer into Müller cells is an effective strategy for preserving structure and function of photoreceptors in retinal degeneration.     

人视网膜前体细胞的体外视网膜组织片下移植

目的研究人视网膜前体细胞移植到体外培养的人视网膜组织片下的细胞分化。方法取无眼部发育异常的4～5个月胚胎眼球，进行视网膜前体细胞分离培养。将传代的细胞移植到体外培养的视网膜神经上皮组织片下，通过光学显微镜和免疫组织化学观察细胞分化和组织整合情况。结果人视网膜前体细胞在体外培养时形成神经球样细胞团，传代后形成子代细胞团，表达神经干细胞标志Nestin。体外培养的视网膜组织片在5d、10d均能基本维持视网膜结构。移植到视网膜组织片下的视网膜前体细胞能够与其建立细胞连接。这些视网膜前体细胞分化后能够表达胶质纤维酸性蛋白、微管相关蛋白-2和视紫红质，分别为神经胶质细胞、神经元和光感受器细胞的特异蛋白。结论人视网膜前体细胞具有神经干细胞特征，在体外移植到培养的人视网膜组织片下，能够分化成相应的终末分化细胞。     

The role of VEGF and VEGFR2/Flk1 in proliferation of retinal progenitor cells in murine retinal degeneration

PURPOSE: To analyze the role of VEGF and its receptors, VEGFR2/Flk1 and VEGFR1/Flt1, on retinal progenitor cells (RPCs) in a murine model of inherited retinal degeneration (rd1 mice). METHODS: After proliferating RPCs in the retina of rd1 mice were labeled with bromodeoxyuridine (BrdU), expressions of VEGFR2/Flk1 and VEGFR1/Flt1 were immunohistochemically analyzed. To examine its effect on the proliferation of BrdU-positive RPCs in rd1 mice, VEGF was administered into retinal culture medium with or without blocking agents against VEGFR2/Flk1 or VEGFR1/Flt1 in vitro or injected into vitreous cavity in vivo. RESULTS: BrdU-labeled RPCs in rd1 mice expressed VEGFR2/Flk1 but not VEGFR1/Flt1. These cells later expressed retinal neuronal markers such as Pax6 and rhodopsin. Exposure of the retinas from postnatal day (P) 9 rd1 mice to VEGF increased the number of proliferating RPCs by 61% in vitro. This effect was blocked by concomitant administration of VEGFR2/Flk1 kinase inhibitor. In vivo, a single intravitreal injection of VEGF in rd1 mice at P9 increased by 138% the number of RPCs and cells that developed from RPCs in the peripheral retina at P18. CONCLUSIONS: VEGF stimulates the proliferation of RPCs through VEGFR2/Flk1 in rd1 mice. The observed proliferation of RPCs that have the potential to differentiate into retinal neurons may enhance the regeneration of the degenerating retina.     

Photoreceptor differentiation and integration of retinal progenitor cells transplanted into transgenic rats

Previous studies evaluating neural stem cells transplanted into the mature retina have demonstrated limited levels of graft-host integration and photoreceptor differentiation. The purpose of this investigation is to enhance photoreceptor cell differentiation and integration of retinal progenitor cells (RPC) following subretinal transplantation into retinal degenerate rats by optimization of isolation, expansion, and transplantation procedures. RPCs were isolated from human placental alkaline phosphatase (hPAP)-positive embryonic day 17 (E17) rat retina and expanded in serum-free defined media. RPCs at passage 2 underwent in vitro induction with all trans retinoic acid or were transplanted into the subretinal space of post-natal day (P) 17 S334ter-3 and S334ter-5 transgenic rats. Animals were examined post-operatively by ophthalmoscopy and optical coherence tomography (OCT) at weeks 1 and 4. Differentiation profiles of RPCs, both in vitro and in vivo were analysed microscopically by immunohistochemistry for various retinal cell specific markers. Our results demonstrated that the majority of passage 2 RPCs differentiated into retina-specific neurons expressing rhodopsin after in vitro induction. Following subretinal transplantation, grafted cells formed a multi-layer cellular sheet in the subretinal space in both S334ter-3 and S334ter-5 rats. Prominent retina-specific neuronal differentiation was observed in both rat lines as evidenced by recoverin or rhodopsin staining in 80% of grafted cells. Less than 5% of the grafted cells expressed glial fibrillary acidic protein. Synapsin-1 (label for nerve terminals) positive neural processes were present at the graft-host interface. Expression profiles of the grafted RPCs were similar to those of RPCs induced to differentiate in vitro using all-trans retinoic acid. In contrast to our previous study, grafted RPCs can demonstrate extensive rhodopsin expression, organize into layers, and show some features of apparent integration with the host retina following subretinal transplantation in slow and fast retinal degenerate rats. The similarity of the in vitro and in vivo RPC differentiation profiles suggests that intrinsic signals may have a significant contribution to RPC cell fate determination.     

Revisiting nestin expression in retinal progenitor cells in vitro and after transplantation in vivo

The purpose of this study is to characterize the co-expression of nestin--a neuroectodermal stem cell and a reactive glial marker-with various mature retinal cell markers in retinal progenitor cells (RPCs) expanded in vitro, followed either by in vitro induction or subretinal transplantation. Rat RPCs derived from embryonic day (E) 17 rat retina were expanded in serum free defined culture, and induced to differentiate by all-trans retinoic acid (RA). Following induction, cells were stained for nestin in combination with retinal neuronal and glial markers. Cultured cells were collected for quantitative RT-PCR gene expression analysis prior to and after induction. In a second series, passage 2 RPCs were transplanted into the subretinal space of S334ter-3 retinal degeneration rats at postnatal day 28. After 1-4 weeks, sections through the transplant were double immunostained for nestin and various retinal specific neuronal markers. The cultured RPCs treated with RA exhibited nestin co-expression with various retinal specific markers, including protein kinase C alpha (PKC), neurofilament 200 (NF200), cellular retinaldehyde binding protein (CRALBP), and rhodopsin. Following RA induction, quantitative RT-PCR analysis demonstrated downregulation of nestin, PAX-6, thy1.1, and PKCalpha, and upregulation of rhodopsin, glial fibrillary acidic protein (GFAP), and CrX. No nestin coexpression was observed with any of the retinal specific neuronal markers in RPC transplants in vivo except for some nestin-immunoreactivity overlapping with GFAP positive cells in the host retina. The role of nestin as a unique neural stem/progenitor cell marker should be reconsidered. Nestin expression during RPC maturation appears to be different in vitro versus in vivo.     

Overlapping spatiotemporal patterns of regulatory gene expression are required for neuronal progenitors to specify retinal ganglion cell fate

Retinal progenitor cells (RPCs) are programmed early in development to acquire the competence for specifying the seven retinal cell types. Acquiring competence is a complex spatiotemporal process that is still only vaguely understood. Here, our objective was to more fully understand the mechanisms by which RPCs become competent for specifying a retinal ganglion cell (RGC) fate. RGCs are the first retinal cell type to differentiate and their abnormal development leads to apoptosis and optic nerve degeneration. Previous work demonstrated that the paired domain factor Pax6 and the bHLH factor Atoh7 are required for RPCs to specify RGCs. RGC commitment is marked by the expression of the Pou domain factor Pou4f2 and the Lim domain factor Isl1. We show that three RPC subpopulations can specify RGCs: Atoh7-expressing RPCs, Neurod1-expressing RPCs, and Atoh7-Neurod1-expressing RPCs. All three RPC subpopulations were highly interspersed throughout retinal development, although each subpopulation maintained a distinct temporal pattern. Most, but not all, RPCs from each subpopulation were postmitotic. Atoh7-Neurod1 double knockout mice were generated and double-mutant retinas revealed an unexpected role for Neurod1 in specifying RGC fate. We conclude that RPCs have a complex regulatory gene expression program in which they acquire competence using highly integrated mechanisms.     

A new method for continuous intraocular drug delivery

A novel device for the continuous intraocular delivery of drugs is described. The active intraocular component of the device is made from hollow haemodialysis fibres. During operation, the drug to be administered diffuses from a small extraocular reservoir to the intraocular fibres, where it passes through the semipermeable walls and into the vitreous body. The delivery of gentamicin by the device into the vitreous body of the cat is compared with direct intraocular injection techniques. Characteristics of the process of drug delivery were influenced by the geometry and design of the device. A reservoir concentration of 10 g/L gentamicin sulphate in a device with four fibres, each 5 mm long, resulted in stable vitreal gentamicin levels of 104 to 128 mg/L at 10 hours. Intravitreal injection of 0.1 ml of a 10 g/L solution of gentamicin caused localised peaks of intravitreal gentamicin concentration in the range 1180 to 3296 mg/L, before falling to 356 to 665 mg/L at 10 hours. The device provides the opportunity of a more controlled and continuous drug delivery, thus avoiding the high localised concentrations that may occur with direct injection techniques in which the required dose is delivered as a bolus. Refinement of a device of this type may result in a clinically useful method of intraocular drug delivery for drugs which present a risk of localised retinal toxicity or require repetitive administration with conventional intraocular injection techniques.     

A new method for continuous intraocular drug delivery

A novel device for the continuous intraocular delivery of drugs is described. The active intraocular component of the device is made from hollow haemodialysis fibres. During operation, the drug to be administered diffuses from a small extraocular reservoir to the intraocular fibres, where it passes through the semipermeable walls and into the vitreous body. The delivery of gentamicin by the device into the vitreous body of the cat is compared with direct intraocular injection techniques. Characteristics of the process of drug delivery were influenced by the geometry and design of the device. A reservoir concentration of 10 g/L gentamicin sulphate in a device with four fibres, each 5 mm long, resulted in stable vitreal gentamicin levels of 104 to 128 mg/L at 10 hours. Intravitreal injection of 0.1 ml of a 10 g/L solution of gentamicin caused localised peaks of intravitreal gentamicin concentration in the range 1180 to 3296 mg/L, before falling to 356 to 665 mg/L at 10 hours. The device provides the opportunity of a more controlled and continuous drug delivery, thus avoiding the high localised concentrations that may occur with direct injection techniques in which the required dose is delivered as a bolus. Refinement of a device of this type may result in a clinically useful method of intraocular drug delivery for drugs which present a risk of localised retinal toxicity or require repetitive administration with conventional intraocular injection techniques.     

BDNF-treated retinal progenitor sheets transplanted to degenerate rats: improved restoration of visual function

The aim of this study was to evaluate the functional efficacy of retinal progenitor cell (RPC) containing sheets with BDNF microspheres following subretinal transplantation in a rat model of retinal degeneration. Sheets of E19 RPCs derived from human placental alkaline phosphatase (hPAP) expressing transgenic rats were coated with poly-lactide-co-glycolide (PLGA) microspheres containing brain-derived neurotrophic factor (BDNF) and transplanted into the subretinal space of S334ter line 3 rhodopsin retinal degenerate rats. Controls received transplants without BDNF or BDNF microspheres alone. Visual function was monitored using optokinetic head-tracking behavior. Visually evoked responses to varying light intensities were recorded from the superior colliculus (SC) by electrophysiology at 60days after surgery. Frozen sections were studied by immunohistochemistry for photoreceptor and synaptic markers. Visual head tracking was significantly improved in rats that received BDNF-coated RPC sheets. Relatively more BDNF-treated transplanted rats (80%) compared to non-BDNF transplants (57%) responded to a "low light" intensity of 1cd/m2 in a confined SC area. With bright light, the onset latency of SC responses was restored to a nearly normal level in BDNF-treated transplants. No significant improvement was observed in the BDNF-only and no surgery transgenic control rats. The bipolar synaptic markers mGluR6 and PSD-95 showed normal distribution in transplants and abnormal distribution of the host retina, both with or without BDNF treatment. Red-green cones were significantly reduced in the host retina overlying the transplant in the BDNF-treated group. In summary, BDNF coating improved the functional efficacy of RPC grafts. The mechanism of the BDNF effects--either promoting functional integration between the transplant and the host retina and/or synergistic action with other putative humoral factors released by the RPCs--still needs to be elucidated.