以纤维蛋白为基质网架的组织工程角膜上皮的构建

目的：应用组织工程技术以纤维蛋白为基质网架、角膜缘干细胞为种子细胞构建组织工程角膜上皮，为角膜上皮移植提供新的移植材料。方法：将20 g•L^-1的纤维蛋白原溶液和含有10 U•mL^-1凝血酶的催化剂溶液混合构成纤维蛋白基质网架，检测支架材料的一般结构和超微结构；取2 mm×2 mm角膜缘组织，胰蛋白酶消化后移至纤维蛋白胶上培养，观察角膜上皮干细胞的生长情况，2周后进行形态学、超微结构和免疫组织化学观察。结果：纤维蛋白基质网架柔软有伸拉性，孔径70～108 μm，平均三维空孔率为70.4%；扫描电镜下呈多层次网络状；接种36 h后，细胞从组织块边缘迁移，7 d后细胞几乎铺满整个培养板孔底，14 d左右细胞与纤维蛋白胶体形成复合植片。透射电镜下观察仍维持上皮细胞特有的超微结构特征。抗细胞角蛋白K3单克隆抗体AE5和抗p63的单克隆抗体4A4免疫组织化学染色均为阳性。 结论：纤维蛋白可作为角膜上皮干细胞移植的良好支架材料，可为组织工程角膜上皮提供新的移植材料。     

人乳头状瘤病毒16型DNA诱导的永生化人喉上皮细胞系的建立(英文)

目的建立人乳头状瘤病毒（HPv）16型DNA诱导的永生化人喉上皮细胞系，确证HPv与喉癌的发生有无关系。方法采用脂质体介导法，将pSVHPV16DNA导入原代培养的人喉上皮细胞，继续培养、传代，取20代细胞，用PCR检测细胞是否含有病毒的特异片段，用免疫组化染色检测E6、E7蛋白的表达，用倒置显微镜、生长曲线、流式细胞仪、细胞角蛋白免疫组化染色、透射电镜及软琼脂克隆形成试验检测细胞的生物学特性。结果有3株细胞已连续培养传代超过20代，细胞含有HPV16DNA的特异片段并有E6、E7蛋白的表达，细胞呈锚着依赖性、接触抑制性单层平铺生长，生长曲线呈典型的“s”型，细胞增殖指数为48％，所有细胞均表达角蛋白，胞浆含张力原纤维，软琼脂培养克隆形成试验阴性。结论成功建立了HPV16DNA诱导的永生化人喉上皮细胞系，为喉癌研究提供了新的理想模型，HPV16对人喉上皮有致癌作用，在喉癌组织标本中检测到的HPV16DNA必定在其多步癌变过程中发挥了重要作用。     

The characteristics of corneal staining in successful daily and extended disposable contact lens wearers

Purpose : The aim of this investigation was to measure levels of corneal staining in successful daily and extended wearers of disposable hydrogel contact lenses. Methods : Twenty-four subjects on a daily wear (DW) schedule and 20 subjects on an extended wear (EW) schedule were examined at six-monthly intervals over a two-year period. Subjects wore two types of disposable hydrogel lenses. Measurements were taken from the eye wearing an etafilcon A contact lens only. The cornea was divided into five equally sized zones and the extent and depth of corneal staining were assessed on a zero to four scale. Results : The overall levels of corneal staining were low with median values below or equal to grade 0.5 in all groups. There was no difference in the extent, depth or geographical distribution of corneal staining between the DW and EW groups. Staining was more frequently recorded in the superior and inferior areas of the cornea than in the central, nasal or temporal regions (Pearson's Chi-Square, p < 0.10). Conclusions : The mode of wear with disposable contact lenses is not a contributing factor to the amount of corneal staining seen in successful wearers. Low levels of corneal staining should be expected in successful DW and EW wearers.     

Enhanced accumulation of A2E in individuals homozygous or heterozygous for mutations in BEST1 (VMD2)

Best vitelliform macular dystrophy (BMD) is an autosomal dominant inherited macular degenerative disease caused by mutations in the gene BEST1 (formerly VMD2). Prior reports indicate that BMD is characterized histopathologically by accumulation of lipofuscin in the retinal pigment epithelium (RPE). However, this accumulation has not been quantified and the chemical composition of lipofuscin in BMD has not been examined. In this study we characterize the histopathology of a donor eye from a rare individual homozygous for a mutation (W93C) in BEST1. We find that this individual's disease was not any more severe than has been described for heterozygotes. We then used this tissue to quantify lipofuscin accumulation by enriching intracellular granules from RPE cells on sucrose gradients and counting the granules in each density fraction. Granules from the homozygous donor eye as well as a donor eye from an individual heterozygous for the mutation T6R were compared with age-matched control eyes. Interestingly, the least dense fraction, representing classical lipofuscin granules was either not present or significantly diminished in the BMD donor eyes and the autoflourescence associated with lipofuscin had shifted to denser fractions. However, a substantial enrichment for granules in fractions of higher density was also noted in the BMD samples. Inspection of granules from the homozygous donor eye by electron microscopy revealed a complex abnormal multilobular structure. Analysis of granules by HPLC indicated a approximately 1.6- and approximately fourfold overall increase in A2E in the BMD eyes versus age-matched control eyes, with a shift of A2E to more dense granules in the BMD donor eyes. Despite the increase in A2E and total intracellular granules, the RPE in the homozygous donor eyes was relatively well preserved. Based on these data we conclude that the clinical and histopathologic consequences to the homozygous donor were not any more severe than has been reported previously for individuals who are established or presumptive heterozygotes. We find that A2E is a component of the lipofuscin accumulated in BMD and that it is more abundant than in control eyes suggesting that the etiology of BMD is similar to Stargardt's disease and Stargardt-like macular dystrophy. Finally, the changes we observe in the granules suggest that the histopathology and eventual vision loss associated with BMD may be due to defects in the ability of the RPE to fully degrade phagocytosed photoreceptor outer segments.     

Multi-layered culture of primary human conjunctival epithelial cells producing MUC5AC

The purpose of our study was to establish a system for culturing normal human conjunctival epithelial (NHCE) cells under serum-free culture conditions without compromising their ability to differentiate into a mucous epithelium. To this end, small pieces of normal conjunctiva were biopsied from patients undergoing cataract surgery. Obtained NHCE cells were cultured in bronchial epithelial growth medium (BEGM) under serum free culture conditions and passage 3 cells were air-lifted. Cultured NHCE cells displayed typical epithelial morphology. Expression of cytokeratin 19 and conjunctival epithelial specific carbohydrate residue were detected. Air-lifted NHCE cells demonstrated an increase in stratification and differentiation into goblet cells up to 3weeks under air-liquid interface (ALI) culture condition. NHCE cells expressed MUC1, MUC4, MUC16, and MUC5AC mRNA, and MUC5AC production and secretion increased in a time dependent manner after culture under ALI conditions. Exposure of cells to proinflammatory cytokines (TNF-alpha or IFN-gamma) resulted in upregulation of MUC1, MUC4, MUC16, and MUC5AC gene expression. In conclusion, differentiated NHCE cells showed features of a multi-layered conjunctival epithelium, including goblet cells, and retained functional characteristics similar to those seen in vivo. This cell culture system can better facilitate investigation of conjunctival epithelial cell biology and goblet cell differentiation.     

应用二维电泳法对视网膜色素上皮细胞光损伤后蛋白质组学的初步研究

目的 应用二维电泳法观察光损伤后视网膜色素上皮细胞蛋白质组的表达。 方法 运用冷白光以（2200±300）Lx光照人视网膜色素上皮细胞(ARPE 19)6 h，建立光损伤模型；提取细胞可溶性蛋白并通过二维电泳分离和凝胶图像分析，寻找光损伤细胞的蛋白质谱变化。 结果 软件分析显示全细胞可溶性蛋白在图谱上出现（390±10）个清晰斑点,11个蛋白斑点有明显表达差异。光损伤细胞内8种蛋白表达上调,1种下调，2种蛋白表达缺失。 讨论 二维电泳实验能够找出光损伤RPE细胞与正常细胞的蛋白表达差异，为进一步研究在此改变中发挥重要作用的蛋白质奠定基础。     

Collagen gel contraction by ARPE-19 cells is mediated by a FAK-Src dependent pathway

Proliferative vitreoretinopathy (PVR) may result in part from de-differentiation of retinal pigment epithelium (RPE) in an aberrant wound-healing strategy. An in vitro model of PVR, collagen gel contraction by RPE, likely requires integrin engagement and activation as an important initial step. The purpose of this study was to identify the important associated integrins and signal transduction pathway. The retinal pigment epithelial cell line ARPE-19 was used in these studies. Cell surface integrin expression was assessed using flow cytometry. An in vitro contraction assay was performed and the percent contraction quantified at specific time intervals using image capture (Gel Doc) and NIH Image software. Cells were pretreated with either small molecule inhibitors of signal transduction pathways or monoclonal antibodies with specificity for specific integrin isoforms. Transient transfections with a FAK siRNA were used to decrease FAK expression. ARPE-19 cells express alpha1, alpha2, and alpha3 integrin, isoforms involved in collagen ligation. Cell surface integrin blockade using anti-integrin alpha2 (P=0.02), alpha3 (P=0.01), or a combination of alpha1, alpha2, and alpha3 (P=0.001) antibodies significantly reduced collagen gel contraction. Inhibition of the FAK-Src complex, but not MEK or PI3K, significantly decreased contraction (P=0.0001). FAK siRNA transient transfection significantly reduced FAK protein expression by 71% (P=0.02) and concordantly decreased gel contraction (P=0.0001). RPE-mediated collagen gel contraction is a multi-step process. Integrin ligation and FAK-Src activation is necessary for collagen gel contraction produced by the ARPE-19 cell line. Validation of these observations in primary RPE cells may suggest new targets for therapeutic intervention in PVR.     

Effects of subretinal implant materials on the viability,apoptosis and barrier function of cultured RPE cells

Background Subretinal microphotodiode array (MPDA) is a type of visual prosthesis used for the implantation in the subretinal space of patients with progressive photoreceptor cell loss. The present study aimed to evaluate the effect of materials for MPDA on the viability, apoptosis and barrier function of cultured pig retinal pigment epithelium (RPE) cells.Methods Primary culture of pig RPE cells was performed and 24 pig eyes were used to start RPE culture. The third passage of the cultures was plated on different materials for MPDA and MPDAs. The tetrazolium dye-reduction assay (MTT) was used to determine RPE cell viability. Flow cytometry was measured to indicate the apoptosis rates of RPE cells on different materials. RPE cells were also cultured on microporous filters, and the transepithelial resistance and permeability of the experimental molecule were measured to determine the barrier function.Results The data from all the methods indicated no significant difference between the materials groups and the control group, and the materials tested showed good biocompatibility.Conclusions The materials for MPDA used in the present study had no direct toxicity to the RPE cells and did not release harmful soluble factors that affected the barrier function of RPE in vitro.This research is supported by National Basic Research Program of China under the project: 2005CB724307     

多西环素对体外培养的人视网膜色素上皮细胞移行的抑制作用

目的探讨基质金属蛋白酶抑制剂多西环素对体外培养并经转化生长因子（TGF）-β1刺激的人视网膜色素上皮（RPE）细胞迁移的影响。方法对3～6代培养的人RPE细胞,用不同浓度（0．01、0,10、1．00、10．00μg／L）TGF-β1处理36h,采用明胶酶谱分析法检测人RPE细胞上清液中明胶酶的活性,采用Boyden室迁移测定法评估RPE细胞经TGF-β1刺激后的迁移情况。结果TGF-β1能促进人RPE细胞中基质金属蛋白酶一2的分泌,并具有浓度依赖性。在无多西环素的环境下,TGF-β1刺激RPE细胞后,能明显促进RPE细胞迁移,迁移的RPE细胞数约增加27％,与对照组相比差异有统计学意义（P〈0．01）。在多西环素的环境中,TGF-β1刺激人RPE细胞后迁移受到明显抑制,随着多西环素浓度的增加,RPE细胞穿透微小多孔膜发生迁移的细胞数相应减少,迁移的RPE细胞数约减少50％～70％,与对照组相比差异有统计学意义（P〈0．01）。结论多西环素能抑制TGF-β1作用所致的RPE细胞迁移,TGF-β1介导的基质金属蛋白酶-2活性的增加在RPE细胞的迁移中起重要作用。