整合素与去整合素在后发性白内障方面的研究现状

晶状体摘除术后晶状体上皮细胞的增殖、黏附和迁移是后发性白内障的主要原因,整合素是一类介导C-C间以及C-ECM间的相互作用的黏附分子,在晶状体上皮细胞的增殖、黏附和迁移过程中发挥重要的作用,去整合素是一种小分子多肽,它可以特异性的被整合素识别并黏附,从而抑制整合素的生物学作用,本文就整合素的功能,整合素在晶状体上皮细胞上的分布及功能,晶状体上皮细胞与后发性白内障之间的关系以及去整合素在后发性白内障防治方面的研究进展做一综述。     

整合素连接激酶在白内障发病过程中的作用

整合素是一种在哺乳动物体内广泛表达的细胞表面受体,整合素连接激酶(ILK)是整合素信号通路的关键激酶,其与整合素结合进行细胞与细胞外基质(ECM),甚至细胞与细胞之间的信号传导.目前的研究发现,ILK及其整合素信号通路的活化可以激活磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(PI3K/AKT)和转化生长因子β/Smad蛋白(TGF-β/Smad)介导的细胞增生、黏附和迁移,引起晶状体上皮细胞异常增生和纤维化,还能激活糖原合成酶激酶3β/β-链蛋白(GSK3β/β-catenin)等信号通路,介导水通道蛋白(AQPs)调节水转运过程,最终导致晶状体内囊泡运输受限,渗透压改变,从而引起白内障.白内障是世界主要的致盲眼病之一,其主要由于老化、遗传、代谢异常、外伤、辐射、中毒和局部营养不良等引起的晶状体囊膜损坏,使其渗透性增加,丧失屏障作用,或导致晶状体代谢紊乱,使晶状体蛋白发生变性,形成混浊,但其发病机制尚未完全阐明.ILK可以通过多种信号通路介导人晶状体上皮细胞的移行、黏附、增生和凋亡,因此深入研究ILK在白内障发病中的作用对白内障的预防和治疗有重要意义.本文就近年来ILK在白内障发病中的作用进行综述.     

纤维连接蛋白对人晶状体上皮细胞系的增殖、黏附和移行的影响

目的:探讨纤维连接蛋白(fibronectin,FN)对人晶状体上皮细胞系HLE-B3的增殖、黏附和移行的影响及其受体整合素α5亚基的表达。方法:培养人晶状体上皮细胞系HLE-B3,接种于不同浓度的FN(0,10,20,40mg/L)包被的培养板中,倒置显微镜下观察HLE-B3的生物学特性,采用WST-8法检测细胞的增殖、黏附情况,划痕法观察细胞的移行并记录细胞缺损区闭合时间,免疫细胞化学法观察整合素α5亚基的表达。结果:各浓度FN包被后,增殖实验中WST-8法检测的吸光值增高不明显(P>0.05)。随FN包被浓度的增高,黏附实验中的吸光值逐渐增高,划痕实验中缺损区闭合的时间逐渐缩短,各浓度组在统计学上具有显著性差异。整合素α5亚基随FN包被浓度的增高表达增强(P<0.05)。结论:FN具有显著促进HLE-B3黏附和移行的作用,同时能上调其受体整合素α5亚基的表达。     

β1整合素过表达促进体外兔角膜上皮细胞黏附和迁移

目的:探讨β1整合素过表达对角膜上皮细胞黏附和迁移的影响机制。方法:将β1整合素-GFP融合蛋白真核细胞重组表达质粒转染兔角膜上皮细胞,观察转染细胞的融合基因表达以及细胞的黏附和迁移能力。检测β1整合素转染对角膜上皮细胞粘着斑激酶(FAK)磷酸化的影响。结果:成功将β1整合素-GFP融合蛋白转染至兔角膜上皮细胞并使其过表达;β1整合素过表达能够明显增加角膜上皮细胞的黏附和迁移能力(P<0.05)并促进FAK磷酸化(P<0.05)。结论:β1整合素过表达能够明显促进角膜上皮细胞的黏附和迁移。     

β1整合素过表达促进体外角膜上皮细胞黏附和迁移

目的:探讨β1整合素过表达对角膜上皮细胞黏附和迁移的影响机制。方法:将β1整合素-GFP融合蛋白真核细胞重组表达质粒转染兔角膜上皮细胞,观察转染细胞的融合基因表达以及细胞的黏附和迁移能力。检测β1整合素转染对角膜上皮细胞粘着斑激酶(FAK)磷酸化的影响。结果:成功将β1整合素-GFP融合蛋白转染至兔角膜上皮细胞并使其过表达;β1整合素过表达能够明显增加角膜上皮细胞的黏附和迁移能力(P<0.05)并促进FAK磷酸化(P<0.05)。结论:β1整合素过表达能够明显促进角膜上皮细胞的黏附和迁移。     

整合素在真菌与角膜上皮细胞黏附过程中的表达

目的 探讨不同真菌菌种与人角膜上皮细胞黏附过程中整合素的表达及其作用机制.方法 实验研究.体外培养人角膜上皮细胞系,建立茄病镰刀菌(CGMCC 3.1829)和烟曲霉菌(CGMCC 3.0772)与角膜上皮细胞黏附的体外模型.茄病镰刀菌或烟曲霉菌与人角膜上皮细胞共孵育后,用无菌的磷酸盐缓冲溶液冲洗掉未黏附的真菌.提取细胞的总RNA,反转录为cDNA后采用实时荧光定量聚合酶链反应(RT-PCR)检测不同时间点上不同真菌与角膜上皮细胞黏附的14种整合素分子mRNA水平的表达.各时间点之间基因表达差异的比较采用单因素方差分析.结果 在烟曲霉菌与人角膜上皮细胞黏附的过程中,随黏附时间延长,整合素家族白细胞黏附受体组成员编码基因整合素αL(ITGAL)、整合素α型(ITGAM)、整合素αX(ITGAX)及整合素β2(ITGB2)的表达显著上调.其中ITGAL的表达最高上调2倍(F=29.39,P<0.01),ITGAM的表达最高上调4倍(F=20.26,P<0.01),ITGAX的表达最高上调2.5倍(F=2.51,P<0.05),ITGB2的表达最高上调3.4倍(F=3.923,P<0.05).而在茄病镰刀菌与人角膜上皮细胞黏附的过程中,此14种整合素的表达未见显著差异.结论 整合素家族白细胞黏附受体组(β2组)成员αLβ2、αMβ2及αXβ2均参与烟曲霉菌与角膜上皮细胞的黏附;未见茄病镰刀菌与角膜上皮细胞的黏附过程中整合素表达的差异.整合素介导的黏附因菌种而异.     

整合素在角膜上皮创伤愈合中的研究进展

整合素作为一类重要的细胞黏附分子,通过影响细胞的形态,介导细胞的黏附、迁移和增生,在角膜上皮创伤愈合中发挥了重要的作用。讨论α2β1、α3β1、α5β1、αvβ3、α6β4、α9β1和αvβ6这7种整合素在角膜上皮创伤愈合中的研究进展及其临床意义。α6β4整合素为半桥粒的主要组成部分,介导角膜上皮细胞在细胞外基质上的静态黏附,损伤后该黏附就转变为α2β1、α3β1整合素介导的动态黏附,细胞在黏附-去黏附的过程中实现迁移,从而修复创面。α6β4、α3β1整合素相互协调作用,实现上皮细胞的板层状运动。研究还发现α6β4、α3β1整合素的活化还能促进细胞的增生。损伤后上皮细胞表面α5β1、αvβ3整合素的表达上调,二者与黏着斑的形成密切相关。α9β1和αvβ6为近年来新发现的与角膜上皮创伤愈合有关的整合素,其具体作用尚有待进一步的研究。     

体外培养人晶状体上皮细胞整合素的表达

目的:研究体外培养人晶状体上皮细胞整合素的表达。方法:对人晶状体上皮细胞株SRA01/04进行培养并传代,经间接免疫荧光标记法处理细胞后上流式细胞仪检测各整合素的阳性表达率。结果:整合素α2,α3,α5,β1,β2在SRA01/04的阳性表达率分别为85.3%,97.6%,74.0%,97.7%,3.65%。整合素α3与β1之间无统计学差异(P>0.05),其余两两间比较均有显著统计学差异(P<0.01)。结论:人晶状体上皮细胞株SRA01/04整合素呈阳性表达。     

整合素-α5及其配体在大鼠角膜创伤愈合中的表达

整合素属细胞黏附分子，是由α和β亚单位通过非共价键形式结合成为跨膜糖蛋白，主要介导细胞与细胞和细胞与细胞外基质间的黏附，并在角膜创伤愈合中起重要作用。α5主要与β1结合。文献报道，配体纤维连接蛋白(fibronectin，FN)是以二聚体形式存在的大分子糖蛋白，经典受体为整合素-α5，大鼠角膜移植术后角膜创缘上皮细胞、迁移上皮细胞膜基底面及基质切口边缘α5及FN蛋白表达量明显增加；当创伤愈合后，其α5及FN蛋白表达消失。为此我们对大鼠角膜创伤愈合中整合素-α5及FN蛋白表达量进行检测，探讨其在角膜创伤愈合中的作用，现将结果报告如下。     

β1整合素过表达抑制角膜上皮细胞凋亡的实验研究(英文)

目的:探讨β1整合素功能性过表达对角膜上皮细胞凋亡的影响及机制,为角膜细胞移植提供理论依据。方法:构建β1整合素-绿色荧光蛋白(GFP)融合基因并转染兔角膜上皮细胞。观察融合基因在角膜上皮细胞的表达及对各细胞外基质蛋白的黏附能力。检测β1整合素功能性转染对角膜上皮细胞凋亡及丝裂素活化蛋白激酶(mitogen-activated protein kinase,MAP激酶)磷酸化的影响。结果:β1整合素-GFP融合基因成功转染至兔角膜上皮细胞并过表达;β1整合素过表达能够明显增加角膜上皮细胞对各细胞外基质蛋白的黏附力并抑制角膜上皮细胞凋亡及促使MAP激酶磷酸化。结论:β1整合素过表达能有效抑制角膜上皮细胞凋亡,MAP激酶磷酸化可能在这一过程中起重要作用。     

Differential responses of human lens epithelial cells to intraocular lenses in vitro: hydrophobic acrylic versus PMMA or silicone discs

Background The purpose of this study was to determine the influence of different materials of intraocular lenses (IOLs) on human lens epithelial cell behavior, including adhesion, migration, proliferation, apoptosis, and epithelial-mesenchymal transdifferentiation (EMT) in vitro. Methods Human lens epithelial cells (SRA 01/04) were grown on hydrophobic acrylic (Acrysof), polymethylmethacrylate (PMMA), and silicone IOLs. Cellular adhesion, migration, proliferation, and apoptotic assays were performed to assess cell behavior. The expression of EMT markers (fibronectin and type I collagen) produced by cells on IOLs was determined by immunoblotting and immunocytochemistry. Results Human lens epithelial cells exhibited preferred adhesion and reduced apoptosis when cultured on acrylic IOLs, in comparison to PMMA and silicone IOLs. Cells grown on acrylic lenses formed a confluent epithelial monolayer. Migration of lens epithelial cells under the acrylic lens was substantially blocked in an in vitro assay. In contrast, cells grown on PMMA and silicone lenses displayed a spindle-shaped, myofibroblast-like morphology, increased apoptosis, reduced adhesion, and enhanced production of EMT proteins such as fibronectin and type I collagen. The migration of lens epithelial cells under PMMA and silicone IOLs was substantial in the in vitro assay. Conclusion This report demonstrates that hydrophobic acrylic lenses are more capsular biocompatible than PMMA and silicone lenses. The in vitro assays are reliable measurements for evaluating the responses of human lens epithelial cells to different IOL materials, and could advance our understanding of the preferential capsular opacification conferred by different IOL materials.     

An in vitro study of human lens epithelial cell adhesion to intraocular lenses with and without a fibronectin coating

PURPOSE: To demonstrate differences in human lens epithelial cell adhesion to different intraocular lens biomaterials in vitro and to determine whether these differences can be influenced by coating the intraocular lens surface with commercially available fibronectin. METHODS: A prospective laboratory-based study comparing human lens epithelial cell adhesion to silicone (n=18), polymethylmethacrylate (PMMA; n=18), and acrylic (n=18) intraocular lenses in vitro. The three types of intraocular lenses were then coated with fibronectin: silicone (n=6), PMMA (n=6), and acrylic (n=6). The main outcome measure was the mean number of lens epithelial cells attached to each lens type after 24 hours of incubation. RESULTS: In the uncoated lens group, there was a significantly higher number of lens epithelial cells attached to the acrylic than to the silicone or PMMA lenses (P<0.001). Coating the lenses with fibronectin caused a significant increase in attachment of lens epithelial cells for all three lens types. CONCLUSIONS: There was a significant difference in the degree of lens epithelial cell attachment to the various types of intraocular lenses in vitro. Cell attachment was more prominent in the acrylic lenses, but the fibronectin coating negated differences in lens type and caused a significant increase in cell attachment across all groups.     

Light scattering caused by cells on the intraocular lens

The anterior surface of a posterior chamber intraocular lens (IOL) was covered with cultured rat lens epithelial cells and light scattering was measured densitophotometrically. The light scattering intensity was maximum at a 30-degree scattering angle and was proportional to the number of cells per area on the IOL. Results suggests that the adhesion of lens epithelial cells on the IOL may influence its optical function.     

Lovastatin-induced cytoskeletal reorganization in lens epithelial cells: role of Rho GTPases

PURPOSE: To understand the involvement of isoprenylated small guanosine triphosphatases (GTPases) in lovastatin-induced cataractogenesis, Rho- and Rac-mediated cell adhesion and actin cytoskeletal reorganization were investigated in lovastatin-treated lens epithelial cells. METHODS: The effects of lovastatin on F-actin reorganization (phalloidin staining), focal adhesion formation (paxillin or vinculin), cell-cell adhesions (cadherin and beta-catenin), and protein tyrosine phosphorylation were evaluated in human and porcine lens epithelial cells by immunocytochemical staining with specific antibodies. To explore the involvement of the Rho and Rac GTPases in lovastatin-mediated effects, changes in distribution of Rho and Rac GTPases were analyzed by Western blot analysis, and the effects of C3-exoenzyme on lovastatin-induced cytoskeletal changes were evaluated by immunocytochemical analysis. RESULTS: Lovastatin induced drastic changes in cell shape in both human and porcine lens epithelial cells, including a profound loss of actin stress fibers, focal adhesions, protein phosphotyrosine, and cell-cell adhesions. Lovastatin treatment also led to the accumulation of nonisoprenylated Rho and Rac GTPases in cytosolic fraction. Supplementation of culture media with geranylgeranyl pyrophosphate dramatically reversed the lovastatin-induced morphologic and cytoskeletal changes, whereas farnesyl pyrophosphate was ineffective. Treatment of cells with C3-exoenzyme (a Rho GTPase-specific inhibitor), however, abolished the geranylgeranyl-supplementation-induced recovery from the morphologic and cytoskeletal effects of lovastatin. CONCLUSIONS: This study demonstrates that inhibition of protein prenylation by lovastatin leads to disruption of actin cytoskeletal organization, and to loss of integrin-mediated focal adhesions and cadherin-mediated cell-cell adhesions in lens epithelial cells. Based on isoprenoid supplementation studies, it could be concluded that impairment of geranylgeranylated Rho and Rac GTPase function is most likely responsible for lovastatin-induced cytoskeletal changes in lens epithelial cells.     

Reduced epithelial adhesion after extended contact lens wear correlates with reduced hemidesmosome density in cat cornea.

Reduced epithelial adhesion in cat corneas after continuous wear of thick hydrogel contact lenses has been reported previously. To investigate the mechanism(s) underlying this observed loss of epithelial adhesion further, the corneas of both eyes of cats that had worn low-oxygen-transmissible thick parallel-design hydrogel contact lenses only in one eye for 8-121 days were examined using both light and transmission electron microscopy (TEM). Contact lens wear induced many changes in the epithelium, including a decrease in the number of cell layers and appearance of cuboidal rather than columnar basal cell shapes. In addition, TEM revealed that the number of hemidesmosomes (HDs) per micrometer of basement membrane was reduced significantly after contact lens wear. Anchoring fibrils in lens-wearing corneas appeared normal, and the reduction in epithelial adhesion occurred without obvious epithelial edema. Decreased epithelial adhesion after contact lens wear appears to be directly related to the reduced numbers of HDs. Possible reasons for decreased HD density, such as loss of basal cell shape and chronic epithelial hypoxia after contact lens wear, are discussed.     

Human lens cells in culture. I. Isolation of adult lens epithelial cells from lens capsule preparations and reactivation of nucleus-containing fiber cells

BACKGROUND: Bovine lens epithelial cells in culture revealed a high sensitivity against micromolar concentrations of linoleic acid. To prove the assumption that unsaturated free fatty acids are risk factors for cataractogenesis, human lens cell lines are needed. Furthermore, the reactivation of nucleus-containing fiber cells to mitotic growth may hint at their role in after cataract genesis. MATERIAL AND METHODS: Epithelium-capsule-preparations obtained by capsulorhexis were cultured in serum containing medium. Subculturing of these adult human lens epithelial cells was done by trypsinization. Fiber cell bundles from the equator region of a fetal human lens were transferred into culture medium. Aggregates of nucleus containing fiber cells were isolated from floating fiber cell bundles by trypsinization. Subculturing and cryoconservation of suitable cell lines. RESULTS: Primary culture of epithelium-capsule-preparations results in flattening, migration and proliferation of adult human lens epithelial cells. Nucleus containing fiber cells were reactivated to mitotic growth after adhesion to a suitable substratum. Established cell lines were received from adult human lens epithelial cells and fetal human fiber cells after repeated subculturing. CONCLUSIONS: Lens-capsule-preparations available from cataract surgery are well suited for the isolation of human lens cell lines, which were needed for testing cytotoxicity of drugs and for tracing of cataractogenic risk factors. The finding that nucleus containing fiber cells from the equator of human lenses can be reactivated to proliferating cells let us suppose, that these cells, which can not be removed easily from the posterior lens capsule, contribute to the after cataract formation.     

The effects of drug delivery via hydrophilic acrylic (hydrogel) intraocular lens systems on the epithelial cells in culture.

BACKGROUND AND OBJECTIVE: Secondary posterior subcapsular opacification is still among the most important complications after phacoemulsification. MATERIALS AND METHODS: This study was designed to assess the inhibitory effects of drugs delivered via hydrophilic acrylic (hydrogel) intraocular lens (IOL) systems in vitro. Lens epithelial cells were collected from albino rabbits. The following seven groups of hydrogel IOLs were prepared: untreated IOLs and IOLs infiltrated with diclofenac sodium, tranilast, mitomycin C, colchicines, 5-fluorouracil, and ethylenediaminetetraacetic acid. The IOLs were fixed to a Cell Culture Insert; they were then bathed and incubated in minimum essential medium containing cultured lens epithelial cells. Subsequently, a comparative analysis of the cells adhering to the collagen membrane and the lens surfaces was conducted. RESULTS: Adhesion of lens epithelial cells to the lens surfaces and the collagen membrane was observed in the control group. However, only slight cellular adhesion was found on the surfaces of the IOLs and on the collagen membrane in the treated IOL groups. CONCLUSION: Use of hydrogel IOLs infiltrated with drugs was associated with inhibition of posterior subcapsular opacification in vitro.     

Spatiotemporal distribution of zonulae adherens and associated actin bundles in both epithelium and fiber cells during chicken lens development

Zonulae adherens and associated actin bundles (ZA/AB) are believed to play a major role in epithelial folding and invagination during morphogenesis of neural tube and other vesicular structures. The lens morphogenesis is associated with the formation of the lens vesicle in which ZA/AB would be needed during the formation process. However, the existence of ZA/AB in the lens has never been established. In this study we report for the first time the existence of ZA/AB in both lens epithelium and fiber cells during embryonic development of chicken lens from E4 to E20. Light microscopy revealed contacts between the lens epithelium and primary fiber cells, and between the lens epithelium and secondary fiber cells at E4 and E11, respectively. Thin-section electron microscopy consistently revealed ZA/AB near both the apical ends of lens epithelial cells and primary fiber cells at E4. This arrangement manifests as a parallel pair of belt-like ZA/AB along the epithelium-fiber interface. In semi-tangential sections, a continuous belt-like ZA/AB was also evidenced in individual epithelial cells and fiber cells. Furthermore, the same ZA/AB arrangement was observed near both the apical ends of epithelial cells and secondary fiber cells at E11. Besides ZA/AB, macular-type fasciae adherens were distributed regularly between epithelial cells, between primary fibers, between secondary fibers, and between epithelium and both primary and secondary fibers. Immunofluorescence strongly and preferentially labeled N-cadherin at both the apical ends of lens epithelium and primary or secondary fibers at the corresponding ages, suggesting a direct association with the zonulae adherens. Also punctate N-cadherin labeling was commonly seen along various regions of primary and secondary fiber cells at different ages, and to a larger extent in the mature fibers of older lenses. This study suggests that: (1) ZA/AB located at the apices of lens epithelial cells may play a crucial role in the early stages of lens morphogenesis (e.g. lens vesicle formation); (2) ZA/AB of primary and secondary fiber cells originate from the epithelial cells during their elongation and differentiation; (3) owing to the restricted distribution of ZA/AB, abundant fasciae adherens are needed to maintain the structural stability of the epithelium and fiber cells during development and maturation; and (4) N-cadherin is the principle adhesion protein for both the zonulae adherens and fasciae adherens in the lens.     

Disruption of the Sparc locus in mice alters the differentiation of lenticular epithelial cells and leads to cataract formation

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein that regulates cellular adhesion and proliferation. In this report, we show that SPARC protein is restricted to epithelial cells of the murine lens and ends abruptly at the equatorial bow region where lens fiber differentiation begins. SPARC protein was not detected in the lens capsule or in differentiated lens fibers. SPARC-null mice developed cataracts at approximately 3-4 months after birth, at which time posterior subcapsular opacities were observed by slit lamp ophthalmoscopy. Histological analyses of ocular sections from 3-month old animals revealed several microscopic abnormalities present in the SPARC-null mice but absent from the wild-type animals. Fiber cell elongation was incomplete posteriorly and resulted in displacement of the lenticular nucleus to the posterior of the lens. Nuclear debris was present in the posterior subcapsular region of the lens, an indication of the abnormal migration and elongation of either fetal or anterior epithelial cells, and the bow region was disrupted and vacuolated. In the anterior lens, the capsule appeared to be thickened and was lined by atypical, plump cuboidal epithelium. Moreover, anterior cortical fibers were swollen. Polyacrylamide gel electrophoresis of the epithelial, cortical and nuclear fractions of wild-type and SPARC-null lenses indicated no significant differences among the alpha-, beta-, and gamma-crystallins. Expression of alphaB-crystallin appeared similar in fiber cells of wild-type and SPARC-null lenses, although the distribution of alphaB-crystallin was asymmetric in SPARC-null lenses as a result of abnormal lens fiber differentiation. No evidence of atypical extracellular matrix deposition in areas other than the capsule was detected in wild-type or SPARC-null lens at 3 months of age. We conclude that the disruption of the Sparc locus in mice results in the alteration of two fundamental processes of lens development: differentiation of epithelial cells and maturation of fiber cells.     

Results of 87 cases of intraocular lens implantation at the front of the whole capsular bag

To prevent or delay Elschnig pearl formation, we performed an unusual surgical technique on 87 patients: intraocular lens implantation at the front of the whole capsular bag. We tried to obtain tight adhesion between the anterior and posterior capsules so the lens epithelial cells could not proliferate and form Elschnig pearls. In fact, the patients exhibited no Elschnig pearls or Soemmering's ring, but developed large anterior capsular folds and a multilayer cell membrane between both capsules after two (three cases), six (19 cases), and 12 months (14 cases). This led to a decrease in visual acuity, and a Nd:YAG laser anterior and posterior capsulotomy was required in most cases (83%). Several months later, these eyes developed enormous Elschnig pearls emanating centrifugally from the laser opening, strongly suggesting that lens epithelial cells require aqueous humor for Elschnig pearl transformation. Since it is not yet possible to obtain perfect adhesion between anterior and posterior capsules, our results confirm the need to remove the anterior capsule after extracapsular cataract extraction.