兔眼虹膜色素上皮细胞的组织培养与超微结构观察

目的 建立一种虹膜色素上皮（iris pigment epithelium,IPE)细胞培养方法。方法 使用酶消化加机械分离的方法培养IPE细胞并进行组织学观察。结果 用该方法可成功培养IPE细胞。培养的IPE细胞在原代及传代生长过程中同视网膜色素上皮（retinal pigment epithelium,RPE)细胞没有明显区别。结论 酶消化加机械分离的方法是培养IPE的理想方法。     

虹膜周边切除手术标本的色素上皮细胞体外培养与超微结构研究

目的：体外培养虹膜周边切除标本中的色素上皮细胞(iris pigment epithelium，IPE)，并进行生物学检测。方法：用酶-显微解剖-酶分离法培养IPE细胞并传代，免疫组化方法鉴定，透射电镜观察超微结构。结果：酶-显微解剖-酶分离法在小标本可成功获得较高纯度和密度的IPE细胞。其形态与免疫标志物表达与视网膜色素上皮(retinal pigment epithelium，RPE)细胞相似。结论：培养的IPE细胞为自体移植提供了较可靠的细胞来源。     

虹膜色素上皮细胞自体移植的研究

虹膜色素上皮 (iris pigment epithelium ,IPE)与视网膜色素上皮 (retinal pigment epithelium,RPE)都来源于神经外胚层 ,二者可能具有许多相似的功能 [1 - 4 ] 。国外进行了 IPE细胞移植的研究 ,但主要是异种或同种异体间的移植。自体 IPE细胞移植已应用于临床 ,然而 ,自体 IPE细胞移植后移植结果的组织学研究报道甚少。我们将兔的自体 IPE细胞移植到视网膜下腔并对其进行了光镜和电镜观察 ,现将结果报告如下。1　材料和方法1.1　IPE细胞的准备12只健康有色兔 (青岛大学医学院动物中心提供 ) ,摘除右眼 ,分别取其 IPE细胞单独培…     

虹膜色素上皮自体移植的现状及展望

随着对虹膜色素上皮(iris pigmant epithelium,IPE)细胞形态及功能研究的深入,IPE细胞有望取代视网膜色素上皮(retinal pigment epithelinm,RPE)细胞用于自体移植,从而避免了异体RPE移植的免疫排斥反应和自体RPE取材困难的问题.本文就IPE细胞生理功能和IPE细胞移植的动物试验和临床研究及进展进行了综述.     

人视网膜色素上皮细胞吞噬过程中细胞内钙离子浓度的变化

视网膜色素上皮（retina pigment epithelium，RPE）细胞具有复杂的结构和特殊的生理机能，RPE吞噬脱落的视细胞外节膜盘（ROS）是视网膜的主要功能之一，这对视细胞外节的更新及维持正常视觉功能至关重要。该功能障碍时引起ROS堆积而导致严重的视网膜疾病，目前对这类疾病尚无有效的治疗手段。RPE细胞不同的吞噬机制目前尚未阐明。本研究通过对RPE细胞不同吞噬过程中细胞内钙离子变化的测定，探讨RPE细胞吞噬时钙离子信号传导通路的作用，从而进一步了解跨膜信号传导系统在RPE细胞的特异性吞噬过程中所起的作用。     

视网膜色素上皮的胚胎发育

视网膜色素上皮(Retinal pigment epithelium简称RPE)位于视网膜最外层,由一层连续的,六棱柱形细胞组成,表面有突起包绕感光细胞的外段。相邻的细胞间有连接结构,底部细胞膜向胞体内陷入形成底部内折。细胞多为单核,并有各种细胞器以及色素颗粒和吞噬体。 RPE具有转运功能、屏障作用、吞噬活性、和遮光吸热特点,因而能够保证视网膜感光细胞处于正常的生理状态并保证眼的视觉功能。由于RPE与视网膜感光细胞和脉络膜的解剖及功能的相互关系,许多眼底疾病可导致它的改变,而它的代谢障碍也可直接影响视觉功能和邻近组织。     

眼部色素上皮培养与移植的最新进展

视网膜色素上皮 (retinal pigm entepithelium,RPE)具有重要的生理功能 ,一旦其功能或结构异常 ,将导致许多眼底病。近年来用 RPE移植已成为一个重要课题 ,但随着研究的深入 ,发现移植异体 RPE发生慢性排斥反应 ,影响手术效果或导致手术失败。鉴于虹膜色素上皮 (iris pigment epitheli-um,IPE)与 RPE具有相同的胚胎起源 ,可能具有 RPE的某些功能 ,于是人们也对 IPE进行了研究。本文对 RPE及 IPE培养与移植的最新进展进行简要综述。1　色素上皮的培养1.1　培养　由于 RPE生理状态下呈单层上皮细胞排列结构 ,具有极性 ,于是学者对…     

成人虹膜色素上皮细胞的体外培养与超微结构观察

目的建立成人虹膜色素上皮(iris pigment epithelium,IPE)细胞体外分离培养方法.方法利用酶辅助显微分离-酶消化法分离成人IPE细胞后体外培养,应用免疫学方法鉴定,电镜观察超微结构.结果体外培养出成人IPE细胞,其形态呈多角形或不规则圆形,色素含量随传代次数增加而减少,角蛋白抗体免疫学染色阳性,电镜显示其具有正常的细胞器结构以及色素颗粒.结论酶辅助显微分离-酶消化法是IPE细胞分离与培养的理想方法.     

高级糖基化终末产物与糖尿病视网膜病变的关系

高级糖基化终末产物(advanced glycation end products,AGE)的堆积是糖尿病视网膜病变(diabetic retinopathy,DR)主要的致病因素,其可促进视网膜色素上皮(retinal pigment epithelium,RPE)细胞AGE受体(receptor of AGE,RAGE)的表达、血管内皮生长因子(vascular endothelial growth factor,VEGF)的分泌及细胞凋亡损伤.RPE细胞参与构成视网膜的外屏障,具有屏障、滤过、吞噬和分泌等作用,在维持视网膜正常生理功能方面具有重要作用,RPE细胞的功能损害会导致多种视网膜疾病.通过研究AGE对RPE细胞的损伤,探讨AGE在DR发生发展中的作用,有利于寻找有效防治DR的新途径.     

Development and cellular functions of the iris pigment epithelium

A number of studies have shown that transplantation of retinal pigment epithelial (RPE) cells to the subretinal space offers a promising treatment modality for retinal degenerative diseases. However, it is necessary to transplant autologous cells to avoid rejection; unfortunately, obtaining autologous RPE cells necessitates such traumatic surgical intervention as to make this approach irrelevant. It has been hypothesized that iris pigment epithelial (IPE) cells may be a possible substitute for RPE cells for transplantation into the subretinal space. The iris pigment epithelium, which has the same embryonic origin as retinal pigment epithelium, has not received much attention from visual scientists. Even though it forms a highly specialized tissue, it is not clear whether the iris pigment epithelium contributes critical functions to the health of the visual system. In vivo the IPE does not appear to have any of the functions characteristic of RPE; however, in vitro cultured IPE cells do acquire functions, such as specific phagocytosis of rod outer segments, that are characteristic of RPE cells, and have been shown to have the potential to carry out many functions characteristic of RPE cells, e.g., retinol metabolism. This review outlines the development and cellular functions of the IPE with special emphasis on the modulation of those functions that can allow the IPE cells to be transplanted to the subretinal space where they appear to acquire differentiated properties of retinal pigment epithelium (RPE).     

Rescue effects of IPE transplants in RCS rats: short-term results.

PURPOSE: The aim of this study was to investigate the possible rescue effect of subretinal iris pigment epithelial (IPE) cell transplantation in Royal College of Surgeons (RCS) rats by light and electron microscopic histology. METHODS: IPE cells were harvested from 20- to 26-day-old Long-Evans rats and were directly trans planted transsclerally into the subretinal space of 32 16- to 20-day-old RCS rats using a 32-gauge Hamilton syringe. Specimens of transplanted eyes were embedded for electron microscopy after 8 weeks. Specimens from the iris and retinal pigment epithelium (RPE) of Long-Evans rats and RPE from RCS rats without surgical treatment were also embedded. Sham surgery was also performed in 8 eyes. RESULTS: The IPE cells transplanted into the subretinal space were localized between host RPE and retina, had round cell shapes without polar organization, and contained phagosomes resulting from rod outer segment (ROS) uptake. The underlying host RPE cells were heavily pigmented. RPE cells from RCS rats revealed fragmentation of endoplasmic reticulum, which distinguishes them ultrastructurally from pigment epithelial cells of Long-Evans rats. Ultrastructural alterations were observed in the cytoplasm of transplanted cells. Melanin granules in the IPE cells were found in large vacuoles, which also contained phagosomes originating from ROS uptake. In 13 eyes, 1 to 4 rows and 5 to 8 rows of saved photoreceptors were detected facing transplanted IPE cells in 6 (46%) and 4 (31%) eyes, respectively, 2 months after surgery. However, in 10 (53%) and 7 (37%) of 19 eyes, 1 to 4 rows and 5 to 8 rows, respectively, were also found at sites without IPE cells in the plane of section. ROS directed toward transplanted IPE cells were seen in one case, but these rods were shortened and disorganized. At most sites between transplanted cells and inner segments of photoreceptors, outer segments and cellular debris were absent. In eyes without transplanted cells no photoreceptor cells were alive at the age of 2 months. After sham surgery 6 (75%) eyes had 1 to 4 rows and 2 (25%) 5 to 8 rows of photoreceptors. CONCLUSIONS: Transplanted IPE cells can take up and degrade ROS in vivo in RCS rats. Uptake of ROS alters the morphology of pigment granules in transplanted IPE cells. Pigmentation is an uncertain marker for identifying transplanted pigment cells. IPE transplants are not as good as RPE transplants in rescuing photoreceptors. However, there is a significant difference between transplanted eyes and nontreated eyes. The rescue effect of IPE cells was not significantly different from that of sham surgery.     

Isolation and cultivation of human iris pigment epithelium.

There have been very few attempts to isolate and culture human iris pigment epithelium (IPE). Earlier efforts that used whole iris explant methods did not achieve pure cultures of IPE. We have developed methods for separating the IPE from the iris stroma of post-mortem eyes that avoid contamination by other cell types. Three different isolation methods were studied: direct dissection, enzyme digestion, and enzyme-assisted microdissection. The latter method yielded the best results. After treatment with enzyme solution, the IPE was easily separated from the stroma under the stereomicroscope and subsequently cultured with supplemented F12 medium. With this method, approximately 2.3 x 10(5) cells were isolated from each iris with an average viability of 90.2%. IPE cells isolated from 19 of 24 eyes grew to confluence in primary culture. The IPE could be maintained in pure culture for many generations over several months with up to 20 population doublings. Cultured IPE demonstrated cytokeratin and S-100 protein by immunocytochemistry. Some of these cells also displayed desmin, indicating origin from the anterior IPE. Cultured IPE cells retained most of the characteristics of IPE in vivo, such as apical/basal polarization, microvilli, and many cell junctions. Gradual dilution of pigment occurred in the dividing IPE cells, suggesting an inability to produce melanin in vitro. A subpopulation of the IPE cells contained myofilaments by electron microscopy, also indicating a anterior IPE origin. This method provides a source for large numbers of human IPE cells and could be useful in studies of the biology of IPE and the role of IPE in pathogenesis of several eye diseases, most notably exfoliation syndrome and its associated glaucomas.     

Adeno-associated Virus Mediated LacZ Gene Transfect to Cultured Human Iris Pigment Epithelium Cells

Purpose: To study the feasibility of adeno-associated virus mediated gene transfection tocultured human iris pigment epithelium (IPE) cells in vitro.Methods: Recombinant replication deficient adeno-associated viruses (AAV) expressingLacZ gene were produced without helper virus. The LacZ gene was transduced into culturedhuman IPE cells.Results: Cultured human IPE cells stained positively anticytokeratin, The titer ofrAAV-LacZ was 2. 1×108 virus particles/ml, 42% cultured human IPE cells expressedβ-galactosidase 7 days after transfection and 67% after 14 days.Conclusions: Recombined AAV produced without helper virus can transfer a foreign geneinto human IPE cells with high efficiency in vitro. Eye Science, 2003; 19: 49 - 53.     

Autologous subretinal transplantation of cultivated porcine iris pigment epithelial cells (IPE)

BACKGROUND: Subretina transplantation of epithelium may be a therapeutic option for surgical treatment of age-related macular degeration (AMD). Various experimental data have demonstrated that homologous transplantation of retinal pigment epithelium (RPE) can prevent photoreceptor deterioration. However, most investigators experienced immunogenic graft rejection when using homologous pigmented cells for grafting. Autologous cells were soon considered as an alternative for subretinal grafting. Particularly iris pigment epithelium (IPE) appeared suitable to replace homologous RPE for it embryogenetic similarity and its simple availability. Recent studies have shown, that IPE is capable of taking over functions of RPE in maintaining retinal metabolism. the purpose of this study was to evaluate if autologous IPE cells would survive when being transplanted subretinally. In addition, immunogenic reponses to the presence of "foreign" iris pigment cells needed to be excluded. MATERIALS AND METHODS: Iris tissue was obtained by peripheral iridectomy in the anesthetized pig. Sheets of pigment iris epithelium were separated from the specimens and transferred into tissue culture. After the cells had been grown to confluency, cell suspensions were injected into the subretinal space of the donor animal's fellow eye. After 4 weeks, the grafted eye was enucleated and examined histologically.. RESULTS: The histological exam revealed that the graft cells had survived in the subretinal space. No evidence of immunogenic rejection was observed. CONCLUSION: Autologous IPE-cells can survive in the host's sub-retinal space without creating inflammatory reactions. Transplanted IPE appears to interact with photoreceptor outer segments.     

Autologous iris pigment epithelial cell transplantation in monkey subretinal region

PURPOSE: To establish autologous iris pigment epithelial (IPE) cell transplantation in monkey eyes. METHODS: Autologous IPE cells from three monkeys were obtained by peripheral iridectomy, and cell culture was performed with autologous serum. The cultured cells were labeled with DiI, and transplantation was performed by transvitreal approach to the submacular region of each monkey. Fundus examination, photography, and fluorescein angiography were performed monthly. Histochemical analysis and electron microscopy were also performed six months after transplantation. RESULTS: Auto IPE cells grew well in auto serum. No fluorescein leakage or retinal thickening was observed after submacular transplantation. We demonstrated the presence of autologous IPE cells in the region 6 months after transplantation by histologic and electron microscopic examination. Electron microscopy also demonstrated that the transplanted cells appeared to be less pigmented and to have less mitochondria than did the host retinal pigment epithelial cells. However, some transplanted cells looked as if they embrace the photoreceptor outer segments. DISCUSSION: Autologous IPE cell culture of the monkey was established using auto serum. Six months after transplantation of the cultured IPE cells, we could still observe the cells in the region. This method may have potential use in human disease that requires autologous cell transplantation to prevent host-graft rejection, and to provide an alternative substance that functions like retinal pigment epithelium.     

兔虹膜色素上皮细胞和视网膜色素上皮细胞血管内皮生长因子及其受体FLK-1 mRNA表达的对比研究

目的　研究血管内皮生长因子 (VEGF)及其受体 (FLK 1)mRNA在健康兔眼虹膜色素上皮 (IPE)细胞和视网膜色素上皮 (RPE)细胞中的表达特征 ,探讨IPE细胞代替RPE细胞用于自体移植治疗年龄相关性黄斑变性 (ARMD)及RPE异常相关疾病的可行性。方法　分离成年健康有色家兔的IPE细胞和RPE细胞 ,采用逆转录聚合酶链反应 (RT PCR)检测其VEGF及其受体FLK 1mRNA的表达情况。结果　兔眼IPE细胞和RPE细胞均可表达VEGF和FLK 1mRNA。IPE细胞的VEGF和FLK 1mRNA含量低于RPE细胞。IPE细胞的VEGFmRNA的表达量约为RPE细胞的 4 4 6 2 % ,FLK 1mRNA的表达量约为RPE细胞的 5 2 36 % ,差异均有显著意义 (P <0 0 0 1)。结论IPE细胞VEGF及其受体FLK 1mRNA的表达水平比RPE细胞低 ,IPE细胞代替RPE进行细胞移植可能更为有利。     

Retinal pigment epithelial cells from Royal College of Surgeons dystrophic rats can take up melanin granules

· Background: Many successful pigment epithelium transplantation studies involving pink-eyed Royal College of Surgeons (RCS) dystrophic rats showed highly pigmented transplanted cells forming a double layer with slightly pigmented cells, attached to Bruch’s membrane. Since it is not clear whether transplanted pigmented cells can displace retinal pigment epithelial (RPE) host cells from Bruch’s membrane, we suggested that RPE cells of RCS dystrophic rats can phagocytize melanin granules, possibly derived from perished transplanted cells. · Methods: In a series of three experiments, RPE cells of nine pink-eyed, 2-month-old RCS dystrophic rats were isolated by trypsinization and mechanical dissection and cultivated in Dulbecco’s modified Eagles’ medium. These cells were then fed with melanin granules, isolated from bovine RPE cells, double-trypsinized after phagocytosis and viewed by light and electron microscopy. We also transplanted iris pigment epithelial (IPE) cells of 20-day-old Long-Evans rats into the subretinal space of pink-eyed RCS dystrophic rats of the same age, shown in light-microscopic photography after 42 days. · Results: Living RPE cells were heavily pigmented after feeding with isolated melanin granules in all three experiments as viewed by light microscopy. In addition, we identified melanin granules phagocytized by dystrophic RPE cells in electron microscopy. After transplantation of pigmented IPE cells into the subretinal space of pink-eyed RCS dystrophic rats’ eyes, a layer of slightly pigmented cells was seen on Bruch’s membrane below the transplanted IPE cells, shown in light microscopy. · Conclusion: We have shown by phagocytosis assay that dystrophic RPE cells can take up melanin granules in vitro. Our results assume that pigmented cells in transplantation studies, found as a monolayer, attached to Bruch’s membrane, cannot automatically be identified as transplanted cells. Instead, the possibility of perished transplanted cells serving as melanin donors for RPE host cells must be taken into consideration. Received: 11 March 1998 Revised version received: 5 May 1998 Accepted: 26 May 1998