视网膜色素变性的实验研究进展

视网膜色素变性是一种遗传性眼病,遗传方式包括常染色体显性遗传、常染色体隐性遗传及性连锁隐性遗传等,目前已知的突变位点超过3 000个,造成本病临床治疗困难。眼科学者致力于探索视网膜色素变性的治疗方式,进行了大量实验研究,主要有药物治疗、细胞移植、基因治疗等治疗方式。药物治疗包括中药、抗氧化剂、抗凋亡剂、神经营养因子等,与其它治疗方式相比,无侵入性,且方便价廉,但其作用机制尚需更深入的研究。细胞移植被认为是治疗视网膜色素变性的有效方法,但有可能引起视网膜前膜及黄斑皱褶。基因治疗虽然存在一定的局限性,但随着基因编辑技术和新型基因递送载体的发展,未来会成为视网膜色素变性最有希望的治疗方式之一。本文对近年来视网膜色素变性的实验研究进行了综述与展望。     

急性多发性视网膜色素上皮鳞状病变

急性多发性视网膜色素上皮鳞状病变(Acute Posterior multifocal Placoid Pig- ment epitheliopathy)1968年首先由Gass报告并述有如下特征:位于视网膜色素上皮或脉络膜部有多数的,散在的黄白色圆形病灶,多发于眼底后极部;伴有中心视力的急骤下降。此类活动性病灶可以很快吸收,在视网膜色素上皮留有永久性的变化。但其邻近的视网膜和脉络膜几乎没有影响而治癒;视力预后良好;原因不明,认     

与年龄相关性黄斑变性有关的细胞外沉积：基板沉积

随着年龄增长,脂蛋白来源的碎片等沉积物除了堆积在视网膜色素上皮下外,还可沉积在视网膜色素上皮基底外侧细胞膜和视网膜色素上皮基板之间,表现为视网膜色素上皮基板增厚,称为基板沉积。基板沉积也与年龄相关性黄斑变性有关。虽然大多数时候临床上不可查见位于视网膜色素上皮下的基板沉积,但视网膜色素上皮萎缩后,基板沉积可在视网膜外层和视网膜色素上皮下腔之间,或者在视网膜色素上皮下液与外层视网膜之间形成一条明显的分界,OCT有时可以发现。（眼科,2022, 31： 330-333）     

干细胞诱导分化为视网膜色素上皮细胞的途径探讨

近年来,干细胞在眼科领域的研究及应用受到高度关注,胚胎干细胞(ES)、成体干细胞能够被定向诱导分化成视网膜色素上皮细胞,由此可获得转分化的大量的视网膜色素上皮细胞源,通过体内干细胞及视网膜色素上皮细胞移植有望应用于各种视网膜退行性疾病的细胞替代治疗。本文就各种干细胞诱导分化为视网膜色素上皮细胞的途径及应用进行探讨。     

视网膜色素上皮错构瘤

视网膜色素上皮错构瘤为一种不常见肿物,其特征为视网膜色素上皮和神经胶质细胞和与视网膜色素上皮、视网膜及邻近玻璃体有关联的血管增殖。虽然此种病变几乎不会生长,但其外表与各种恶性肿瘤相似,曾有误将患眼作不必要的     

视网膜色素上皮与有关常见病

视网膜是眼球最重要的眼内组织,是视觉系统中的前哨。组织学上共分为十层,最外层为视网膜色素上皮,起源于视杯外层;其余九层为神经视网膜,由视杯内层分化而来。视网膜色素上皮(RPE)与神经视网膜(简称视网膜)相比,组织结构上看来相对简单,但功能上却十分复杂,其对视觉的影响至关紧要,必须予以重视。近代视觉研究中,视网膜色素上皮是众所关注的课题之     

端粒酶编码区的反义寡核苷酸对人视网膜色素上皮细胞增生的影响

目的探讨不同浓度端粒酶编码区的反义寡核苷酸对人视网膜色素上皮细胞增生活性的影响。方法用不同浓度(0μmol·L-1、5μmol·L-1、10μmol·L-1、15μmol·L-1)端粒酶编码区的反义寡核苷酸处理人视网膜色素上皮细胞24h后,使用MTT法测定视网膜色素上皮细胞增生的抑制率,使用流式细胞仪检测其对视网膜色素上皮细胞周期的影响。结果不同浓度端粒酶编码区反义寡核苷酸处理过的视网膜色素上皮细胞的抑制率随反义寡核苷酸浓度的增高而升高,并将视网膜色素上皮细胞阻滞在G0/G1期。结论端粒酶编码区的反义寡核苷酸对视网膜色素上皮细胞有抑制作用且呈浓度依赖性,这一结论可能成为增生性玻璃体视网膜疾病基因治疗的一个新靶点。     

褪黑素对体外培养的人视网膜色素上皮细胞增殖的影响

目的观察褪黑素对体外培养的人视网膜色素上皮细胞生长、增殖的影响,寻找药物以防治增殖性玻璃体视网膜病变。方法分别将不同浓度的褪黑素(0,125ng·L-1,250ng·L-1,500ng·L-1)加入体外培养的视网膜色素上皮细胞培养液,72h后用四甲基唑盐(MTT)比色法在自动酶标测定仪测540nm处的吸光值A,并计算出不同浓度条件下的细胞增殖抑制率。结果褪黑素在不同浓度下抑制视网膜色素上皮细胞增殖,并存在剂量-效应关系,实验组125ng·L-1和500ng·L-1组与对照组比较有显著性差异(P<0.001),但实验组250ng·L-1组与对照组之间的差异无显著性。结论褪黑素能抑制体外培养的人视网膜色素上皮细胞增殖,可作为防治增生性玻璃体视网膜疾病的侯选药物作进一步研究。     

早期衰老大鼠视网膜色素上皮-光感受器细胞复合体超微结构的变化

目的：观察早期衰老大鼠视网膜色素上皮-光感受器细胞复合体超微结构的变化。

方法：取3月龄及12月龄大鼠视网膜,制成半薄切片,透射电镜下观察视网膜色素上皮-光感受器细胞复合体超微结构的变化。

结果：12月龄大鼠视网膜中出现视网膜色素上皮细胞(retinal pigment epithelium, RPE)凋亡和RPE-光感受器细胞复合体的退行性改变。

结论：视网膜色素上皮-光感受器细胞复合体的退行性改变是视网膜衰老的早期表现。     

视网膜色素变性

视网膜色素变性(Retinitis Pigmentosa;RP)是一种遗传型式多样的视网膜色素性萎缩,累及全世界人口的0.5%。临床上可通过眼底的特殊改变以及心理物理学和电诊断学的检查来识别。本病也可与其他几种遗传性疾病并存。基础研究指出,尽管本病尚无特异疗法,但可采取某     

Hydrogen peroxide causes significant mitochondrial DNA damage in human RPE cells.

Retinal pigment epithelial cell dysfunction mediated by reactive oxygen intermediates has been suggested as a possible cause of age-related macular degeneration. To test the hypothesis that retinal pigment cells are susceptible to genetic damage mediated by reactive oxygen intermediates, retinal pigment epithelial cells were treated with 50 micrometers-200 micrometers of hydrogen peroxide in vitro. Damage to mitochondrial DNA and three nuclear loci were assessed using quantitative polymerase chain reaction. Hydrogen peroxide treatment of retinal pigment epithelial cells resulted in significantly increased mitochondrial DNA damage. Significant mitochondrial DNA damage occurred rapidly and was not completely repaired within 3 hr post-treatment. By contrast, no DNA damage was observed in three different nuclear loci (beta-globin gene cluster, hprt, and beta- polymerase genes). Hydrogen peroxide treatment of retinal pigment epithelial cells also resulted in decreased mitochondrial redox function compared to controls, consistent with increased mitochondrial DNA damage. Consequently, retinal pigment epithelial cell mitochondrial DNA appears susceptible to hydrogen peroxide mediated damage in vitro, and thus, may serve as a catalyst in the initial events leading to retinal pigment epithelial cell dysfunction in vivo.     

Collagen gel contraction by cells associated with proliferative vitreoretinopathy.

The capacities of porcine choroidal fibroblasts, retinal glial cells, and retinal pigment epithelial cells to contract collagen gels in vitro were compared. Experiments with varied cell numbers indicated that glial cells are the most effective, followed by choroidal fibroblasts and retinal pigment epithelial cells. Analysis of the secretory products from cultures of these cell types revealed that retinal pigment epithelial cells synthesize and secrete peptides that promote fibroblast contraction of collagen gels in vitro. The mechanism of action of the retinal pigment epithelial cell-secreted contraction promoter was compared with that found in serum (type A) and secreted by cultured endothelial cells (type B). Like the serum factor, the retinal pigment epithelial cell-secreted factor was not dependent on active protein synthesis by the target cell and must be present continuously to promote contraction.     

Photosensitization of retinal pigment epithelium by protoporphyrin IX

Background: Clinical evidence of injury to the retinal pigment epithelium is an important feature of age-related macular degeneration, but the mechanism of this injury is unknown. Blue-light-dependent activation of the blood-borne photosensitizer protoporphyrin IX is known to produce free radicals which may damage cells and tissues. This study was undertaken to determine the effect of blue light and protoporphyrin IX on retinal pigment epithelial cells in vitro. • Methods: Third-passage porcine retinal pigment epithelial cells were plated in six-well culture plates at 100 000 cells/well and grown to confluence. Retinal pigment epithelial cells were then incubated in culture media with and without 35 μg/dl protoporphyrin IX and exposed to low intensity (118 μW/cm²) blue, blue-free, or full-spectrum white light in an irradiating incubator for 16 h on/8 h off cycles for 7 days. Some of the wells were shielded from light (dark controls). Retinal pigment epithelial cells were examined by light microscopy and were trypsinized and counted after 7 days. • Results: White light with and without protoporphyrin IX and protoporphyrin IX in dark conditions did not decrease the retinal pigment epithelial cell count significantly. Blue light alone and blue light with protoporphyrin IX decreased the cell count by 22 ± 4% and 35 ± 3% compared to the controls, respectively. • Conclusion: Blue wavelength light without exogenous protoporphyrin IX has a cytotoxic effect on confluent cultures of retinal pigment epithelium, suggesting that endogenous photosensitizers may be present in retinal pigment epithelial cells. Protoporphyrin IX has an additive cytotoxic effect in the presence of blue light, suggesting that this photosensitizer is capable of mediating blue-light-induced retinal pigment epithelial damage. Since protoporphyrin IX is present in blood and tissue fluids, and the retina is chronically exposed to light, protoporphyrin IX-mediated free radical formation may occur in vivo and may play a role in retinal pigment epithelial changes that occur early in the pathogenesis of age-related macular degeneration. Received: 12 February 1997 Revised version received: 26 May 1997 Accepted: 31 May 1997     

A new isolation procedure for retinal pigment epithelium

A new isolation procedure for bovine retinal pigment epithelial cells has been developed. It is based on perfusion of the whole bovine eye via the central ophthalmic artery with a cold, buffered isotonic salt solution free of divalent cations for 15 min. The perfusion both weakens the association of the pigment epithelial cells with Bruch's membrane and the adhesion between retina and pigment epithelium. The retina then is removed carefully, after which the pigment epithelial cells are detached from the Bruch's membrane by gentle jets of buffer solution. The perfusion technique provides a high yield of intact retinal pigment epithelial cells, which show good viability in subsequent cell culture. Hence, cells isolated in this way are not only very well suited for long-term cell culture but also for direct biochemical analysis and short-term incubation studies.     

Indocyanine green effect on cultured human retinal pigment epithelial cells: implication for macular hole surgery

PURPOSE: To evaluate potential toxic effects of indocyanine green dye on cultured human retinal pigment epithelial cells. METHODS: Controlled laboratory experiment. Cultured human retinal pigment epithelial cells were exposed to balanced saline solution, balanced saline solution with endoillumination, indocyanine green or indocyanine green with endoillumination. Cells were evaluated by light microscopy, electron microscopy, and a mitochondrial dehydrogenase assay. RESULTS: Retinal pigment epithelial cells exposed to indocyanine green showed no histologic or ultrastructural changes. Those exposed to indocyanine green alone or indocyanine green plus light demonstrated a significant decrease in mitochondrial enzyme activity (P = 0.0002 and 0.005, respectively). CONCLUSION: Brief exposure of cultured human retinal pigment epithelial cells to indocyanine green results in decreased mitochondrial enzyme activity but does not appear to influence cellular morphology or ultrastructure.     

蓝光对人视网膜色素上皮细胞增殖的影响和机制研究

目的:研究蓝光对人视网膜色素上皮细胞增殖能力的影响,并初步探讨其可能机制.方法:利用35W白色冷光灯加用蓝色滤光片建立蓝光损伤体外培养的RPE细胞模型,蓝光控制波长在470~520nm,光照强度控制为2000Lx左右,光照时间控制为24~96h,利用CCK-8法检测RPE细胞的增殖能力,利用real-time PCR技术检测RPE细胞中miR-103的含量.结果:与对照组相比,蓝光照射组的RPE细胞的增殖能力减弱;蓝光照射组的RPE细胞内的miR-103含量较对照组增加;miR-103过表达时,RPE细胞的增殖能力减退,降低miR-103的表达时,细胞增殖能力增强;降低miR-103的表达能够减弱蓝光对RPE细胞增殖的抑制作用.结论:蓝光通过上调miR-103抑制RPE细胞的增殖,miR-103可能成为治疗年龄相关性黄斑变性等视网膜变性疾病治疗的新靶点.     

脂褐素与年龄相关性黄斑变性

年龄相关性黄斑变性(ARMD)是引起中老年人视力丧失的首要原因,其确切病因尚未明了,故治疗效果不佳。脂褐素随着年龄增长在视网膜色素上皮的积累是眼睛老化的标志,通过光化学作用等影响正常视网膜色素上皮细胞的功能,与ARMD的发生发展有一定联系。本文就脂褐素及其与ARMD的关系进行综述。     

Clinicopathologic correlation of lipidization and detachment of the retinal pigment epithelium

A 49-year-old woman had a few small drusen-like lesions and one larger area of depigmentation in the right fundus. The pin-point lesions corresponded to single enlarged retinal pigment epithelial cells with lipid accumulation and the larger area represented a small, localized retinal pigment epithelial detachment (soft druse). Lipoidal degeneration of individual retinal pigment epithelial cells may be a mechanism of nodular drusen formation. The soft druse was associated with thickening of the basement membrane of the retinal pigment epithelium.     

Hydrogen peroxide stimulates apoptosis in cultured human retinal pigment epithelial cells.

PURPOSE: To determine whether hydrogen peroxide (H2O2), a physiological mediator of oxidative stress induces apoptosis in retinal pigment epithelial (RPE) cells. METHODS: To demonstrate that oxidatively stressed retinal pigment epithelial cells undergo apoptosis consequential to mitochondrial dysfunction, biochemical parameters of apoptosis were determined in cultured cells after treatment with 50-200 mM H2O2 for different times. Caspase-3 protease activity was determined from hydrolysis of DEVD-rho-nitroanilide. Expression of the anti-apoptotic protein, bcl-2 and the pro-apoptotic proteins p53 and p21 were analyzed by western blotting. RESULTS: Caspase-3 activity significantly increased in cells exposed to H2O2. Also, the expression of bcl-2 in cells treated with 200 microM H2O2 was diminished, whereas expression of p53 and p21waf-1 was increased compared to the controls. CONCLUSIONS: Exposure of retinal pigment epithelial cells to concentrations of H2O2 that cause in vitro mitochondrial DNA damage also promotes apoptosis.