角膜缘上皮细胞分化与增殖及细胞周期素表达

目的：探讨角膜缘上皮细胞分化、增殖能力与细胞周期素D1、E、A表达水平的关系。方法：应用消化培养法分别培养人角膜缘及角膜中央上皮细胞,并将角膜缘上皮细胞传至第3代,将角膜中央上皮细胞传至第1代。分别选用抗细胞周期素D1、E、A的抗体,通过免疫组织化学染色检测各代细胞表达细胞周期素D1、E、A的水平。结果：角膜缘上皮原代细胞表达细胞周期素D1、E、A的水平最高,传代培养时细胞周期素的表达水平逐代下降,至第3代已基本上检测不到细胞周期素的表达。角膜中央上皮原代细胞仅极少数表达细胞周期素D1、E、A,传至第1代时已失去表达细胞周期素的能力。各代细胞表达细胞周期素D1、E、A的水平与其增殖能力一致。结论：细胞周期素D1、E、A表达水平的高低可反映角膜缘上皮细胞增殖能力的强弱。高度表达细胞周期素D1、E、A的角膜缘上皮细胞可能就是角膜缘干细胞。细胞周期素可能成为干细胞的一种新的免疫学或生化标记。     

角膜上皮细胞Pax—6基因表达分析

目的检测体外培养角膜上皮细胞Pax-6基因的表达,探讨维持角膜缘干细胞特性的基因因素.方法地高辛标记cDNA探针进行DNA印迹杂交和蛋白质免疫印迹,检测培养的角膜上皮细胞中Pax-6基因表达状况.结果EcoRⅠ酶切角膜缘上皮细胞中9.1kbp的DNA片断与探针序列相同,其中原位和原代培养细胞、传第l代明显测出,角膜中央上皮细胞的原位和原代细胞中也能测出.Western     

体外培养不同代角膜缘干细胞特性分析

目的比较不同代角膜缘干细胞的生物学特性,为角膜缘细胞移植在临床中的氉应用提供依据。方法组织块培养法体外培养角膜缘干细胞,光镜观察、HE染色和免疫荧光染色分析原代、第1代和第2代角膜缘细胞形态学特性;流式细胞技术定量检测原代、第1代和第2代角膜缘细胞ABCG2表达;MTT比色法测定体外培养原代、第1代和第2代角膜缘干细胞的增殖活性变化。结果倒置相差显微镜下可见原代细胞培养24h有单个细胞从组织块边缘迁出,48h细胞局部可见拉网样生长趋势,72h出现大片细胞,细胞呈现多边形或多角形,96h细胞几乎铺满整个培养板。随着传代次数的增加,细胞形态变得不规则,CK3单克隆抗体表达阳性率逐渐增强;p63单克隆抗体原代和第1代细胞间表达阳性率无明显差别,第2代细胞表达量明显减弱。流式细胞检测显示原代、第1代和第2代细胞ABCG2表达阳性率分别为13.41%、22.96%和4.43%。MTT结果显示随传代次数增加细胞增殖活性逐渐下降。结论原代和第1代角膜缘干细胞可以作为制备组织工程角膜上皮细胞膜片的较理想种子细胞。     

人角膜缘干细胞体外培养的增殖与分化研究

目的了解角膜缘干细胞体外培养的增殖分化规律。方法组织块法培养细胞,测定细胞克隆形成率（ＣＦＥ）,免疫荧光染色检测干细胞表达角质蛋白Ｋ３的状况。结果原代培养２１天左右细胞生长达到饱和,传第１代７～１０天形成单层,ＣＦＥ为９．５２％±４．９７％;传第２代７天,ＣＦＥ为４．２５％±２．１０％（Ｐ＜０．０１）。正常角膜缘基底细胞不表达角质蛋白Ｋ３;原代培养的干细胞亦不表达Ｋ３,传第１代细胞有部分表达。结论人角膜干细胞位于角膜缘基底部,培养的角膜缘干细胞早期具有较高的增殖力并保持干细胞的分化特性。     

细胞周期依赖性激酶抑制剂P27、P21在人角膜上皮的表达研究

目的　探讨细胞周期依赖性激酶抑制剂 (CKI) P2 7、P2 1在角膜上皮细胞的表达情况。方法　应用 SP免疫组化法 ,检测 P2 7、P2 1等细胞周期依赖性激酶抑制剂和增殖细胞核抗原 (PCNA)在角膜上皮不同部位的表达情况。结果　角膜缘上皮区 PCNA呈阳性表达 ,主要位于基底细胞层 ,中央区角膜上皮内未见阳性表达 ,差异有显著意义 (P <0 .0 1) ;P2 7、P2 1在中央区角膜上皮内呈阳性表达 ,角膜缘上皮内未见阳性表达 ,差异均有显著意义(P <0 .0 1)。结论　细胞周期依赖性激酶抑制剂 P2 7、P2 1和 PCAN在角膜上皮不同部位的表达差异 ,提示在角膜缘上皮基底层内存在着一群有较高的增殖能力、处于 G1 期的细胞群 ,即干细胞     

上皮性钙黏附蛋白在人角膜上皮中的表达研究

目的 研究上皮性钙黏附蛋白（E-cadherin）在人角膜上皮中的分布。方法低钙培养基培养人角膜缘干细胞．并取人角膜中央上皮及角膜边缘上皮组织，分别使用免疫组织化学及免疫细胞化学方法检测E-cadherin在人角膜中央上皮、角膜边缘上皮及低钙培养的人角膜缘干细胞中的分布情况。结果E-cadherin在角膜边缘上皮全层细胞即处于分化阶段的短暂扩充细胞中大量表达，在抑制干细胞分化、促进增殖的低钙培养的人角膜缘干细胞中表达减少，在终末分化细胞即角膜中央上皮细胞中没有表达。结论E-cadherin在处于分化阶段的角膜上皮细胞中有大量的表达。     

改良兔角膜上皮细胞原代培养及纯化的方法

目的 改良原代培养及纯化兔角膜上皮细胞的方法,提高角膜上皮细胞原代培养的成功率.方法 采用全角膜组织培养法培养兔角膜上皮细胞,利用机械刮除和差速贴壁法进行纯化并传代,通过倒置显微镜对细胞进行形态学观察并应用免疫组织化学的方法对细胞进行鉴定.结果 兔全角膜24 h贴壁,48 h后即可见有细胞自角膜缘爬出,5d后细胞大量爬出可见成纤维细胞和角膜上皮细胞共存,界限明显;角膜上皮细胞复层生长.在界限融合前刮除成纤维细胞,角膜上皮细胞继续旺盛生长,10 d后达到融合.兔角膜上皮细胞传至第4代后细胞体积明显变大,传至第5代,细胞衰老、凋亡.兔角膜上皮细胞免疫组织化学显示PCK单克隆抗体阳性.结论 改良原代培养及纯化兔角膜上皮细胞的方法简单、经济、有效,并可获得具有良好生物学特性的角膜上皮细胞,为角膜及角膜疾病的研究奠定实验基础.     

兔角膜内皮、上皮及基质细胞体外培养扩增的研究

目的 建立角膜上皮、基质及内皮细胞体外培养扩增的简单稳定的方法，为组织工程化角膜的构建提供种子细胞。方法 内皮细胞与后弹力层在培养基中孵育后消化法获原代细胞，胰酶消化去除表层上皮后取角膜缘，组织块法培养角膜缘上皮细胞，基质细胞应用胶原酶消化法获原代培养，各细胞融合后胰酶消化依次传代培养。结果 原代内皮细胞4—5d融合成单层细胞，可连续传6—7代。上皮细胞1周左右生长融合，连续传3—4代后细胞形态改变。基质细胞接种6—7d后近融合，传代后增殖明显，可连续传10代。结论依据角膜组织特征选择合适的方法体外分离、培养角膜3种细胞成分，可获连续传代扩增的角膜细胞。     

细胞周期依赖性激酶抑制剂P27,P21在人角膜上皮的表达研究

目的:探讨细胞周期依赖性激酶抑制剂(CKI)P27,P21 在角膜上皮细胞的表达情况。方法:应用 SP 免疫组化法,检测 P21,P27 等细胞周期依赖性激酶抑制剂和增殖细胞核抗原(PCNA)在角膜上皮不同部位的表达情况。结果:角膜缘上皮区 PCNA 呈阳性表达,主要位于基底细胞层,中央区角膜上皮内未见阳性表达,差异有显著意义(P <0.01);P27,P21 在中央区角膜上皮内呈阳性表达,角膜缘上皮内未见阳性表达,差异均有显著意义(P <0.01)。结论:细胞周期依赖性激酶抑制剂 P27,P21 和 PCNA 在角膜上皮不同部位的表达差异,提示在角膜缘上皮基底层内存在着一群有较高的增殖能力,处于 G1 期的细胞群,即干细胞。     

人角膜缘干细胞体外培养后生物学特性的变化

目的 探讨人角膜缘干细胞体外培养后细胞形态学，抗原性和增殖能力的变化。方法 消化培养法体外培养人角膜缘干细胞。获得细胞单层并观察细胞形态学变化。利用间接免疫荧光组化检测人角膜缘组织HLA-DR抗原在培养前，后的变化，检测培养后细胞增殖核抗原和角膜上皮64KD角蛋白的表达。结果 原代细胞在培养1周形成单层细胞形态以圆柱状细胞为主，培养前角膜缘上皮下少星HLA-DR抗原分布，培养后单层细胞DR抗原表达阴性；单层细胞中多量，散在分布的增殖细胞核抗原阳性细胞，并且部分细胞表达64KD角蛋白，结论 角膜缘干细胞体外培养后分化为角膜上皮，不表达HLA-DR抗原，介仍保持较强的分裂增殖能力，可能是临床移植治疗干细胞缺乏眼表疾病患者的一种有效手段。     

Segmentation and accuracy in copying and drawing: Experts and beginners

As part of an ongoing investigation into real-world copying and drawing, I recorded the eye-hand drawing strategies of 16 subjects with drawing experiences ranging from expert to novice while they copied a line drawing of a standing nude. The experts produced accurate copies whereas all the beginners produced marked inaccuracies of overall scaling, proportion and shape. Analysis of eye and hand movements showed that the experts alone segmented the original drawing into simple line sections that were copied one at a time using a direct eye-hand strategy not requiring intermediary encoding to visual memory. The results suggest that segmentation into simple lines defines the task-specific process of accurate copying, and that this process is restricted to experts, i.e. acquired through training and practice. Additional preliminary tests also suggest that a similar process may apply to drawing a model from life.     

Proximal and accommodative convergence and age

The authors have estimated the phoria for distant and near fixation in two groups of subjects (mean age 27.5 ± 4.4 and 59.2 ± 8.2 years). Different accommodative stimuli were induced by adding minus lenses for distant fixation and plus lenses for near fixation. Statistical analysis of the experimental data indicates that, for distant fixation, the value of phoria per unit of accommodative stimulus is significantly lower in presbyopic than in nonpresbyopic subjects. Also, during near fixation, the accommodative convergence (AC/A ratio) is more reliable in the presbyopic subjects when the accommodative stimulus is progressively reduced. This varying behavior indicates in presbyopic subjects that proximal convergence is of greater relative importance in the determination of the fusion-free position. In nonpresbyopic subjects, accommodative convergence is the more important component.     

Anesthesia and preoperative and postoperative medications

Although certain methods such as retrobulbar blocks are used extensively, improvements in procedure can always be implemented. The use of ultrasound, low concentrations of anesthesia, careful monitoring, and, in the case of risk patients, anesthesia standby are all important considerations to ensure uneventful treatments. Topical anesthesia eliminates needle risk as well as risk of ptosis and bruising. Because it has been demonstrated that bacteria routinely enter the anterior chamber during uncomplicated cataract surgery, certain irrigation solutions are helpful, but still debatable. Postoperatively, diclofenac, flurbiprofen, and timolol have all been proven to be effective in reducing ocular inflammation, reducing incidence of CME, and controlling pressure increase, respectively.     

Anesthesia and preoperative and postoperative medications

Retrobulbar blocks, although widely used, still have potentially serious complications. Topical anesthesia presents less risk of injury to the globe and less pain but requires careful usage and an experienced surgeon. New techniques, however, allow for an increase in the percentage of patients able to have topical anesthesia. Preoperatively, 2.5% phenylephrine is found to be just as effective as 10% phenylephrine, and, when compared with wound closure and surgeon's experience, the effect of prophylactic medications was found to be negated. Postoperatively, diclofenac is found to be as effective an anti-inflammatory agent as prednisolone. Also, the addition of 10% phenylephrine to 4% pilocarpine drops enhances the effectiveness of pharmacologic treatment of postoperative iridocorneal adhesions. In addition, ophthalmologists should be aware of emerging antibiotic resistance.     

Paraneoplastic and non-paraneoplastic retinopathy and optic neuropathy: Evaluation and management

Paraneoplastic syndromes involving the visual system are a heterogeneous group of disorders occurring in the setting of systemic malignancy. Timely recognition of one of these entities can facilitate early detection and treatment of an unsuspected, underlying malignancy, sometimes months before it would have otherwise presented, and gives the patient an increased chance at survival. We outline the clinical features, pathogenesis, and treatment strategies for the retinal- and optic nerve–based paraneoplastic syndromes: cancer-associated retinopathy; melanoma-associated retinopathy; paraneoplastic vitelliform maculopathy; bilateral diffuse uveal melanocytic proliferation; paraneoplastic optic neuropathy; and polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome. Distinguishing these disorders from their non-paraneoplastic counterparts (e.g., autoimmune-related retinopathy and optic neuropathy, and acute zonal occult outer retinopathy) and determining appropriate systemic evaluation for the responsible tumor can be challenging. In addition, we discuss the utility and interpretation of autoantibody testing.     

Sensitivity to first- and second-order motion and form in children and adults

We compared the sensitivity of adults and children aged 3-10 years to first- and second-order motion and form. For first-order stimuli, at all ages sensitivity was better for motion than form, and motion thresholds were better at 6 Hz than at 1.5 Hz. For second-order stimuli, at all ages sensitivity was better for form than motion, and motion thresholds were better at 0.25 cyc/deg than at 1 cyc/deg. Thresholds became adult-like later for motion than for form and later for first-order than second-order stimuli. For first-order stimuli, the changes with age were larger and more protracted.     

Nodes and nodal points and lines in eyes and other optical systems

The typical stigmatic optical system has two nodal points: an incident nodal point and an emergent nodal point. A ray through the incident nodal point emerges from the system through the emergent nodal point with its direction unchanged. In the presence of astigmatism nodal points are not possible in most cases. Instead there are structures, called nodes in this paper, of which nodal points are special cases. Because of astigmatism most eyes do not have nodal points a fact with obvious implications for concepts, such as the visual axis, which are based on nodal points. In order to gain insight into the issues this paper develops a general theory of nodes which holds for optical systems in general, including eyes, and makes particular allowance for astigmatism and relative decentration of refracting elements in the system. Key concepts are the incident and emergent nodal characteristics of the optical system. They are represented by 2 × 2 matrices whose eigenstructures define the nature and longitudinal position of the nodes. If a system's nodal characteristic is a scalar matrix then the node is a nodal point. Otherwise there are several possibilities: Firstly, a node may take the form of a single nodal line. Second, a node may consist of two separated nodal lines reminiscent of the familiar interval of Sturm although the nodal lines are not necessarily orthogonal. Third, a node may have no obvious nodal line or point. In the second and third of these classes one can define mid-nodal ellipses. Astigmatic systems exist with nodal points and stigmatic systems exist with no nodal points. The nodal centre may serve as an approximation for a nodal point if the node is not a point. Examples in the Appendix , including a model eye, illustrate the several possibilities.     

Corneal and external disorders and refractive surgery

A review of disorders of the anterior segment of the eye may show new avenues of research as well as clinical perspectives arising from recently accumulated data. It will have to select new theoretical and clinical findings, which may most probably be apt to survive the test of time, thus providing a long-standing stimulus in science. This body of scientific work in corneal and external disorders may be divided into infectious and noninfectious entities as well as refractive and tectonic or structural problems. In this review, we will focus on the inflammatory implications of anterior segment ocular disease, and mention some evolving diagnostic features and new treatment modalities, as well as opinions on refractive surgery complicated by inflammation.