波形纤维蛋白在其转基因小鼠晶状体的表达

目的 观察波形纤维蛋白（Ｖｉｍｅｎｔｉｎ）在转基因小鼠晶状体的表达,探讨波形纤维蛋白与内障形成的关系。方法 用显微注射法将鸡的１２．７ｋｂ波形纤维蛋白基因导入小鼠受精卵的雄前核内,经培育、杂交传代得到２０只波形纤维蛋白内障小鼠。取４只白内障眼球及４只正常眼球,制备石蜡切片,采用ＡＢＣ免疫组化方法,观察波形纤维蛋白在晶状体的表达。再分别正常、分部混浊和完全混浊的小鼠晶状体各１只,经ＳＤＳ－聚丙烯酰胺     

Transgenic overexpression of connexin50 induces cataracts

To examine the effects of increased expression of Cx50 in the mouse lens, transgenic mice were generated using a DNA construct containing the human Cx50 coding region and a C-terminal FLAG epitope driven by the chicken betaB1-crystallin promoter. Expression of this protein in paired Xenopus oocytes induced gap junctional currents of similar magnitude to wild type human Cx50. Three lines of transgenic mice expressing the transgenic protein were analyzed. Lenses from transgenic mice were smaller than those from non-transgenic littermates, and had cataracts that were already visible at postnatal day 1. Expression of the transgene resulted in a 3- to 13-fold increase in Cx50 protein levels above those of non-transgenic animals. Light microscopy revealed alterations in epithelial cell differentiation, fiber cell structure, interactions between fiber cells and areas of liquefaction. Scanning electron microscopy showed fiber cells of varying widths with bulging areas along single fibers. Anti-Cx50 and anti-FLAG immunoreactivities were detected at appositional membranes and in intracellular vesicles in transgenic lenses. N-cadherin, Cx46, ZO-1 and aquaporin 0 localized mainly at the plasma membrane, although some N-cadherin and aquaporin 0 was associated with the intracellular vesicles. The abundance and solubility/integrity of alphaA-, alphaB-, beta- and gamma-crystallin were unaffected. These results demonstrate that transgenic expression of Cx50 in mice leads to cataracts associated with formation of cytoplasmic vesicles containing Cx50 and decreased or slowed epithelial differentiation without major alterations in the distribution of other integral membrane or membrane-associated proteins or the integrity/solubility of crystallins.     

Lens regeneration in mice: implications in cataracts

Lens regeneration in adult mice is possible when the lens capsule is left behind after lentectomy. The lens is regenerated by the remaining adherent lens epithelial cells, which differentiate to form lens fibres within days, showing normal morphology and bow regions. Epithelial to mesenchymal cell transformation is also seen during the early stages. The mouse, therefore, can become an indispensable animal model for cataract research, surgery and therapy.     

人晶体上皮细胞的体外培养

目的:提供一种简单可行,易于掌握的体外培养人晶体上皮细胞的方法。方法:用无齿显微镊子将晶体囊膜分离出来,剪碎后直接移至细胞培养瓶中培养,当细胞长出融合后,用胰蛋白酶消化传代。结果:接种培养4天后,可见晶体上皮细胞开始长出,并以贴壁方式生长。结论:用此方法体外培养人晶体上皮细胞,具有简单、快捷、成功率高的优点。眼科学报1997;13:170—172。     

Lens Epithelial Cell Proliferation and Cell Density in Human Age—relat

Purpose: To discuss the potential effect of the lens epithelial cell proliferation inage-related cataract.Methods: In vitro cell proliferation was assayed by MTT method to evaluate the lensepithelial cell density, index, and proliferation capacity in normal lens and all kinds ofage-related cataract. Capsulotomy specimens from all kinds of patients who underwentcataract phacoemulsification extraction surgery were compared with the lens epithelialspecimens from non-cataract lenses of Eye Bank eyes.Results:Lens epithelial cell density of central anterior capsule(LECD) in female normallens was higher than that in male, LECD in nuclear cataract (> NⅢ) was higher thanthat in normal lens, but in the mature cortical cataract, LECD was lower. Mitotic indexof three kinds of age-related cataracts in vivo had no statistical difference, neither didcell proliferation capacity of cultivated cells in vitro.Conclusion: The individual difference of lens epithelial cell density and proliferationcapacity in vivo may be an     

Immunohistochemical Study of Apoptosis of Lens Epithelial Cells in Human and Diabetic Rat Cataracts

Purpose: An immunohistochemical evalution of lens epithelial cell apoptosis.Methods: We performed terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays on capsulotomy specimens (68 eyes in 53 patients) from patients who had undergone cataract surgery and an epithelium of diabetic cataracts in rats (144 eyes in 72 rats). The animal model of diabetic cataracts was prepared by injection of streptozotocin in three-week old rats. The rats were also examined using the proliferating cell nuclear antigen (PCNA) immunohistochemical staining method.Results: Although some TUNEL-positive cells were detected in capsulotomy specimens, we recognized little correlation between its distribution and morphological classification of cataracts. In the animal model of diabetic cataracts, TUNEL-positive cells were seen around the region where epithelial cells had accumulated. In the accumulated region, PCNA labeled cells undergoing DNA synthesis were also detected.Conclusion: These results suggest the possibility that apoptosis occurs in human lens epithelial cells and apoptosis and proliferation may be induced by factors such as hyperglycemia.     

Transgenic expression of AQP1 in the fiber cells of AQP0 knockout mouse: Effects on lens transparency

Mutations and knockout of aquaporin 0 (AQP0) result in dominant lens cataract. To date, several functions have been proposed for AQP0; however, two functions, water permeability and cell-to-cell adhesion have been supported by several investigators and only water channel function has been readily authenticated by in vitro and ex vivo studies. Lens shifts protein expression from the more efficient AQP1 in the equatorial epithelial cells to the less efficient water channel, AQP0, in the differentiating secondary fiber cells; perhaps, AQP0 performs a distinctive function. If AQP0 has only water permeability function, can the more efficient water channel AQP1 transgenically expressed in the fiber cells compensate and restore lens transparency in the AQP0 knockout (AQP0^−/−) mouse? To investigate, we generated a transgenic wild-type mouse line expressing AQP1 in the fiber cells using αA-crystallin promoter. These transgenic mice (TgAQP1^+/+) showed increase in fiber cell membrane water permeability without any morphological, anatomical or physiological defects compared to the wild type indicating that the main purpose of the shift in expression from AQP1 to AQP0 may not be to lessen the membrane water permeability. Further, we transgenically expressed AQP1 in the lens fiber cells of AQP0 knockout mouse (TgAQP1^+/+/AQP0^−/−) to determine whether AQP1 could restore AQP0 water channel function and regain lens transparency. Fiber cells of these mice showed 2.6 times more water permeability than the wild type. Transgene AQP1 reduced the severity of lens cataract and prevented dramatic acceleration of cataractogenesis. However, lens fiber cells showed deformities and lack of compact cellular architecture. Loss of lens transparency due to the absence of AQP0 was not completely restored indicating an additional function for AQP0. In vitro studies showed that AQP0 is capable of cell-to-cell adhesion while AQP1 is not. To our knowledge, this is the first report which uses an animal model to demonstrate that AQP0 may have an additional function, possibly cell-to-cell adhesion.     

波形蛋白在老年性白内障晶状体上皮细胞中的表达及其意义

目的 :进一步明确波形蛋白的异常表达在老年性白内障形成中所起的作用。方法 :用链酶菌抗生物素蛋白 碱性磷酸酶 (streptavidin alkalinephosphatase,S P)免疫组化方法检测波形蛋白在 31例皮质性白内障、2 0例核性白内障及5例正常透明晶体上皮中的表达情况。结果 :波形蛋白在皮质性白内障晶体上皮中的表达比在核性白内障及正常透明晶状体中高。结论 :老年性皮质性白内障与老年性核性白内障的发生有不同的发病机制 ,皮质性白内障的发生与波形蛋白的过度表达有关     

Modifications of the Water-insoluble Human Lens α-Crystallins

Since the water-insoluble crystallins of the lens may be the precursors of cataract, identifying the modifications that differentiate the water-insoluble from the water-soluble crystallins may provide the basis for understanding the chemistry leading to cataract. This investigation of the α-crystallins of the water-insoluble urea-soluble portion of 45-year-old normal clear lenses, isolated using gel filtration, ion exchange and reversed phase chromatography, has employed state-of-the-art mass spectrometric techniques to identify and locate the modifications of the water-insoluble α-crystallins. Modifications present in the isolated α-crystallins were identified by the molecular weights of the modified proteins, by the molecular weights of peptides produced by enzymatic digestion of the proteins, and by the fragmentation patterns produced by collisional activation of the peptides. Modifications that are either unique to the water-insoluble α-crystallins or are more prevalent in the water-soluble portion than in the water-soluble part include complete oxidation of the two Cys residues of αA-crystallin to form an intra-molecular disulfide bond, partial truncation at both the C-termini andN-termini of αA- and αB-crystallins, partial oxidation of Met residues to methionine sulfoxide, partial deamidation of several Asn and Gln residues, and evidence of peptide bond cleavage at some of the deamidated residues. Although many reactions have been proposed to contribute to the insolubility of crystallins, this compilation of in-vivo post-translational modifications of water-insoluble α-crystallins delineates products that are actually present at levels of 5% or more. From these results, it is hypothesized that α-crystallin becomes water-insoluble following deamidation of various Asn and Gln residues which cause conformational changes leading to formation of an intra-molecular disulfide bond between the Cys residues of αA-crystallin.     

The Biological Study of the Cultured Human Lens Epithelial Cells in Vitro

The human lens epithelial cells (HLE) cultured in vitro was established in normal and cataractous lenses. The biological feature, histological characteristics and the ultrastructure of the cultured HLE cells were investigated. The results reveal that the proliferative capacity of the culutured HLE cells is reversely proportional to the donour age; the cultured HLE cells has the limited proliferative capacity in vitro. The relieve of the contact inhibition is the effective trigger of the HLE cell proliferation. This research work is possible to make a basis for the further study of the regeneration of the lens and the roles of the lens epithelial cells in the development of the cataract. Eye Science 1994; 10:27-31.     

Rncat先天性白内障小鼠的晶状体形态学研究

目的　研究Crygs基因发生突变的rncat先天性白内障小鼠的晶状体组织形态学改变。方法　使用裂隙灯观察rncat白内障小鼠自出生后晶状体混浊的形态改变。并取白内障小鼠的晶状体进行光镜、透射电镜和扫描电镜的检查 ,同时以正常昆明小鼠为对照。结果　发现rncat小鼠白内障是双眼逐渐形成的核心性混浊。病理检查发现晶状体上皮细胞的异常增殖 ,去核纤维化的过程受阻。电镜检查发现晶状体纤维的异常排列和细胞间连接结构的异常。结论　Crygs基因突变可导致rncat白内障小鼠晶状体上皮细胞的变性和晶状体纤维的排列紊乱和细胞间连接的结构改变。     

Distribution of Human Lens Crystallins and Their Sulphydryl Contents of Different Age in Two—dimension Electrophoresis

Purpose: To analyze water-soluble(WS) human lens proteins of fetus, adult and age-related cataract by two-dimensional IEF/SDSPAGE electrophoresis. Methods: DACM [ N- (7-Dimethylamino-4-methyl-3-coumarinyl) maleimide ] was used to determine the lens proteins sulphydryl (SH) content.Result: Protein SH contents in WS lens proteins have no significant difference among fetus, adult and age-related cataract lens. This is different from the relative published results obtained in lens proteins of animal cataract model using similar SH detecting methods.Conclusions: IEF/SDS-PAGE electrophoresis demonstrated that there were much more fragmentation of crystallins during lens development and cataractogenic process. It is suggested that this phenomenon is likely to be due to further conformational changes in the fragmented cyrstallins during aging and cataractogenic process. Eye Science 1999; 15:32-35.     

Effects of Calcium on Human Lens Epithelial Cells In Vitro

Purpose To analyze the effect of calcium on human lens epithelial cells (LECs) in vitro.Methods Human LECs were obtained from the anterior lens capsule during a cataract operation, and were cultured in minimum essential medium (MEM) containing 12% fetal bovine serum for a week at 37°C, 5% CO₂. The LECs were then isolated with trypsin and placed in culture dishes. To analyze the effects of calcium, the LECs were incubated in MEM with calcium concentrations of 0, 2, 10, or 20mM. After H&E staining for 72h, the LECs were analyzed with the Scion imaging program.Results The LECs proliferated rapidly and their shapes were uniform in 2-mM-calcium MEM. The LECs proliferated more slowly and had irregular shapes in MEM with lower and higher concentrations of calcium.Conclusions Calcium affects both the proliferation and the shape of human LECs in culture. Abnormal (hyper- or hypo-) calcium concentrations in the lens and aqueous humor may change the homeostasis of LECs, resulting in cataracts and secondary posterior capsular opacification. Jpn J Ophthalmol 2004;48:97–100 © Japanese Ophthalmological Society 2004     

Cataracts in the aging rat lens. Morphology and acid phosphatase histochemistry of incipient forms

The present investigation deals with morphological and certain histochemical changes that precede and accompany cataracts associated with aging in the rat. The cataracts were of two types: supranuclear (SNC) and posterior subcapsular (PSC). The initial biomicroscopie lesions, which were associated with the auterior and posterior lens suture systems, respectively, were attributed to cytological alterations of the ends of the superficial fiber cells. Cortical alterations common to both types of cataract included segmental cellular swelling, and the formation of assorted globular and multilamellar inclusions. With SNC, large multilamellar bodies corresponded to anterior sutural vacuolar opacities; while swelling and dissociation of the fiber cell bodies were associated with the formation of lamellar separations, lesions which eventually appeared in the peripheral cortex. Posterior subcapsular intercellular vacuoles, many of which developed a lamellated outer structure, were characteristic of the early form of PSC.An acid phosphataso which served as a marker for lysosomal hydrolases was demonstrated in the intercellular spaces of lenses with early forms of degeneration. The major source of the extracellular enzyme appeared to be the Golgi/lysosomal system within the epithelium and cortex, although the cortical smooth endoplasmic reticulum was not ruled out.Our studies support the concept that the swelling and degeneration of cortical fibers occurring in the initial stages of the cataracts might have been caused by intercellularly located hydrolases. The value of the aged rat lens as a model for human senile cataract was reinforced by the many similarities between the murine and human forms of the disease.     

A New Method for Studying Protein Changes in the Human Lens During Ageing and Cataract Formation

ABSTRACT: Human lenses can be separated into concentric layers by dissolution and the fates of various lens constituents in such layers can be studied with appropriately sensitive techniques. These techniques have been applied in a study of the ageing of lens proteins. It was found that insoluble protein increases with progression from periphery to lens centre. This increase is more marked in older lenses. Analysis of soluble protein using High Performance Liquid Chromatography shows that the proportion of α-crystallin decreases towards the centre of the lens, and that this decrease becomes greater with age. β-Crystallins maintain a constant proportion except in inner layers of older lenses. γ-Crystallins show a slight decrease in content from periphery to centre. With cataract formation ageing changes are exaggerated.     

青岛开发区沿海社区50 岁以上人群白内障流行病学调查

目的：通过对青岛开发区沿海社区50 岁以上人群白内障的患病及治疗情况进行调查,了解该地区白内障流行病学状况。方法：调查研究。在2013 年2 月到2015 年1 月期间采用整群随机抽样方法,调查青岛开发区沿海社区9 028 例50 岁以上居民年龄相关性白内障患病情况,通过制订统一的检查方法和诊断标准,固定普查人员深入到社区集中检查。采用χ2 检验对结果进行统计学分析。结果：9 028例接受调查,共发现白内障患者2 008例,患病率为22.24%。70岁以上人群白内障患病率（60.03%）与70岁以下人群（12.70%）相比明显较高,差异有统计学意义（χ2=1 882,P <0.001）;女性患病率（27.11%）明显高于男性（17.05%）（ χ2=132,P <0.01）。糖尿病患者白内障患病率为66.94%,高于非糖尿病者（21.01%）（ χ2=154,P <0.01）。本次调查中发现白内障盲人166例,其中114例已经接受手术,白内障盲人手术覆盖率为68.67%。白内障盲人社会负担率为1.84%,不同年龄段社会负担率不同,随着年龄增长,白内障盲人社会负担率增加（χ2=154,P <0.01）,优势比为5.83。结论：白内障目前仍然是致盲的主要原因,社会上还有大量的白内障盲人得不到及时有效的治疗,给社会和家庭带来较重的负担,白内障复明工作任重而道远。     

激光共聚焦显微镜研究人胚胎视网膜发育过程中波形纤维蛋白表达的差异

目的研究波形纤维蛋白在人胚胎视网膜发育过程中表达的动态变化。方法收集25例8~39孕周胎龄的胎儿眼球标本,5例正常成人眼球标本。冰冻连续切片,波形纤维蛋白抗体孵育,普通光镜及激光共聚焦显微镜观察视网膜。结果25~28周波形纤维蛋白在人胚视网膜M櫣ller细胞上的表达上调。25周在视网膜神经节细胞层、内丛状层和内核层出现了长丝状阳性表达;28周附着于内界膜的锥状突起在神经节细胞层部位,与长丝状的阳性表达突起交错排列。部分阳性突起由附着点的内界膜直达外界膜,贯穿视网膜大部分区域,外界膜亦呈阳性反应。结论波形纤维蛋白为观察M櫣ller细胞在视网膜发育过程中分化和迁移的一个良好标记物。人视网膜M櫣ller细胞基本发育成熟的时间为胚胎25~28周。     

Tau蛋白和磷酸化Tau蛋白在C57BL/6小鼠和转基因鼠的表达

目的　探讨总Ｔａｕ蛋白（Ｔ-ｔａｕ）和磷酸化Ｔａｕ蛋白（Ｐ-ｔａｕ）在慢性青光眼模型小鼠视神经中的表达以及意义。方法　选取Ｃ５７ＢＬ／６雄性小鼠以及ＡＰＰ＋转基因鼠各４０只,每只小鼠右眼为实验眼,左眼为对照眼,通过烙闭实验眼巩膜上静脉建立慢性青光眼模型,采用免疫组织化学方法,检测视神经Ｔ-ｔａｕ以及Ｐ-ｔａｕ在不同青光眼模型中的表达情况。结果　Ｃ５７ＢＬ／６小鼠以及ＡＰＰ＋转基因鼠实验眼术后眼压均高于术前（均为Ｐ＜０．０５）,而两组术后眼压差异无统计学意义（Ｐ＞０．０５）。Ｃ５７ＢＬ／６小鼠以及ＡＰＰ＋转基因鼠实验眼Ｔ-ｔａｕＩＯＤ值均低于对照眼（均为Ｐ＜０．０５）,而Ｐ-ｔａｕＩＯＤ值均高于对照眼（均为Ｐ＜０．０５）;Ｃ５７ＢＬ／６小鼠实验眼Ｔ-ｔａｕ和Ｐ-ｔａｕＩＯＤ值分别为１０２．２４±１２．１２和１７２．１２±９．０１,均小于ＡＰＰ＋转基因鼠（均为Ｐ＜０．０５）。ＡＰＰ＋转基因鼠实验眼视神经细胞凋亡率为（９．７２±１．０８）％,明显高于Ｃ５７ＢＬ／６小鼠实验眼（７．２４±１．２２）％,差异有统计学意义（均为Ｐ＜０．０５）,且两者实验眼均高于对照眼（均为Ｐ＜０．０５）。结论　慢性青光眼小鼠视网膜Ｔ-ｔａｕ蛋白表达量降低,Ｔａｕ蛋白磷酸化水平增加,视神经细胞凋亡率上升,在ＡＰＰ＋转基因小鼠中尤为明显。     

视网膜计对白内障术后视力预测可靠性评价及相关影响因素分析

目的为了客观评价视网膜计对白内障患者潜视力预测的准确率、找出影响准确率的相关因素和指导其临床应用。方法对拟手术患者600例白内障患者在手术前用视网膜计对患者的潜在视力(手术后可能达到的视力)进行预测,手术后对其视力复查。并以第2周时做验光矫正后所达到的最佳视力为准。结果判定标准:相符:手术后2周所达视力与手术前预测一致或相差一行对数视力者;基本相符:手术后视力与手术前预测相差2行者;不符:手术后视力与手术前预测相差2行以上者。结果600眼中其中手术前预测与手术后实际最佳矫正视力相符者85眼占14.17%,基本相符者339眼占56.50%,不符合者176眼,占29.33%,总体符合率为70.67%。其中107眼超度高近视眼中符合与基本符合相加所占比例高达93.46%。结论视网膜计具有预测方法简便,检查时间短,结果符合率较高,有较高的参考价值,可以作为白内障手术前常规检查项目。影响其准确率的因素主要有:成熟期、IV级以上硬度核白内障因光栅的透过性差而直接影响患者的分辨能力和测试准确度。高龄、弱智、不合作者也是相关因素。