脱水猪角膜基质为载体体外构建猫角膜内皮组织

目的:以脱水猪角膜基质为载体体外构建猫角膜内皮组织,并观察其形态结构,以寻找体外构建角膜组织最佳载体材料及方法,最终目的是体外构建可用于移植的角膜内皮组织。方法:以脱水处理的猪角膜基质片为载体,将猫角膜内皮细胞接种于去除猪角膜内皮细胞的后弹力膜上,在添加了表皮生长因子和层粘连蛋白的培养液中培养7 d,分别观察倒置显微镜下、组织学切片及扫描电镜下内皮组织的形态结构。结果:倒置显微镜下,组织培养的猫角膜内皮细胞形成单层内皮组织,排列较规律,细胞间连接紧密;组织学观察发现,培养的猫角膜内皮细胞形成完整的内皮组织,贴附于脱水基质的后弹力膜上,与正常的角膜内皮组织结构相似;扫描电镜下,组织培养的猫角膜内皮细胞间连接紧密,细胞大小不甚一致,胞核清晰。 结论:以脱水猪角膜基质为载体,体外成功构建出猫角膜内皮组织,其形态结构近似于正常角膜内皮组织。     

以猪角膜脱细胞基质为载体培养人脐静脉内皮细胞构建实验性角膜后板层

 目的： 探讨以异种后弹力膜/基质为载体诱导人脐静脉内皮细胞向角膜内皮细胞分化的可行性及移植术后在活体的生理功能。方法：体外分离培养脐静脉内皮细胞作为诱导移植的种子细胞;取当地质检合格的猪眼球以直径6.2 mm、厚100 μm、冷冻脱水进行角膜深板层载体的制备;接种经CM-DiI荧光标记的第2~3代人脐静脉内皮细胞于载体的后弹力层面,体外培养7~10 d行细胞形态、密度、组织学和扫描电镜观察,待细胞与后弹力层面融合形成单层后,用于兔角膜移植。受眼：正常健康新西兰大白兔24只（24眼）,实验组（人脐静脉内皮细胞移植组）12眼,对照组（单纯猪角膜深板层移植组）12眼,全角膜范围去除术眼内皮细胞,实施移植手术。结果：人脐静脉内皮细胞在猪后弹力膜/基质载体上贴附生长,形成紧密连接的单层,形态近似六角形,呈铺路石状分布,具有正常兔角膜内皮细胞的超微结构。术后8周实验组角膜基本透明,周边角膜略有水肿。对照组单纯猪角膜深板层移植后,移植角膜明显水肿、混浊。结论：以异种角膜后弹力膜/基质为载体培养的人脐静脉内皮细胞,行异种异体移植后,能够在活体上成活,并能维持角膜透明,具有正常角膜内皮细胞的生物学功能。深板层角膜内皮移植术是体外培养血管内皮细胞移植的一种有效术式。     

壳聚糖-透明质酸共混膜对兔角膜基质细胞生长的影响

观察各种壳聚糖-透明质酸共混膜对角膜基质细胞生长的影响作用,研究透明质酸混入比例对共混膜与细胞相容性的影响.以共混膜为载体培养兔角膜基质细胞,通过光学和电子显微镜,观察细胞在膜上的生长情况;通过MTT法,检测细胞在共混膜上的贴附率和生长活性;通过检测培养基中乳酸脱氢酶的活性,预示壳聚糖-透明质酸共混膜与角膜基质细胞的相容性.以低于1:0.1的比例混入透明质酸.可以提高细胞在共混膜上的贴附率、生长速度,细胞在共混膜上的生长状态好于在壳聚糖膜上的生长状态.结果提示,以低于1:0.1的比例混入透明质酸,可以提高壳聚糖膜与角膜基质细胞的相容性,促进细胞生长;而以高于1:0.1的比例混入透明质酸,则不利于细胞在共混膜上的生长,降低了壳聚糖膜与角膜基质细胞的相容性.     

纤维蛋白胶与兔角膜3种细胞构建组织工程细胞片研究

目的： 观察兔角膜3种细胞能否在体外构建的纤维蛋白胶体上良好生长，探讨纤维蛋白胶作为组织工程细胞片支架材料的可行性。方法: 将培养扩增的兔角膜3种细胞分别接种于纤维蛋白胶表面,采用倒置显微镜,HE染色和扫描电子显微镜,观察兔角膜3种细胞在纤维蛋白胶表面生长情况。结果: 制备的薄层纤维蛋白胶支架光滑、透明，随培养细胞的生长部分降解，可获得仅带少量纤维蛋白胶的细胞片。角膜3种细胞在纤维蛋白胶表面生长良好，可保持生理状态的细胞形态。角膜上皮细胞可形成单层和复层，细胞间连接紧密。角膜内皮细胞呈圆形或多角形，细胞大小一致，排列紧密。角膜基质细胞拉长生长，呈三角形或树枝状，细胞间连接明显，可形成网状连接。结论: 体外构建的纤维蛋白胶与角膜3种细胞有组织相容性,纤维蛋白胶有望作为角膜3种细胞的生长载体构建可供移植的组织工程细胞片。     

角膜体外重构异种生物载体材料植入性实验研究

目的：分析测定异种（猪）角膜基质组织免疫原性，观察其角膜层间植入后与受体角膜愈合情况，以评价该材料生物相容性，并在此载体上体外重构角膜内皮组织，探讨其作为角膜重建载体材料可行性。 方法： (1)将新鲜、脱水两种猪角膜基质植片分别植入F344大鼠角膜基质层间，术后12、90 d对外周血进行CD25和CD4/CD8双色免疫荧光标记，流式细胞仪测定分析。(2)猪角膜基质植入新西兰白兔角膜层间，定期临床观察植片愈合情况，并取受体兔角膜进行组织学观察。(3)将猫角膜内皮细胞接种于保留后弹力层的脱水猪角膜基质上，加入培养液培养7 d，组织学观察体外重构的角膜内皮组织形态结构。 结果： (1)测得新鲜组、脱水组大鼠外周血T淋巴细胞CD4＋CD25＋、CD8＋CD25＋双阳性表达率及CD4＋/CD8＋比值与同基因移植组、阴性对照组比较无显著差异（P>0.05）。(2)兔眼临床观察：全部植片存活，12只术眼未见有角膜水肿混浊、角膜新生血管、排斥反应发生，新鲜植片在2个月左右已透明，脱水植片在6个月后透明。兔角膜组织学观察：新鲜植片4个月时与兔角膜基质相融愈合，脱水植片经角膜细胞再分布、胶原纤维改建重塑于8个月后与兔角膜基质相融愈合，两组植片愈合过程中未见有淋巴细胞浸润及新生血管生成。(3)体外重构的内皮组织形态结构与正常内皮层相似。 结论： 异种（猪）角膜基质免疫原性低，具有良好的生物相容性，是目前较为理想的角膜体外重构载体材料。     

壳聚糖与硫酸软骨素共混膜性质的研究

以壳聚糖和硫酸软骨素按一定比例制备出共混膜，研究了膜片的透光性、含水量、渗透性、力学性质、表面结构、生物降解性、生物相容性等性质。结果表明该共混膜具有较好的透光性、通透性、生物降解性和生物相容性，膜表面较粗糙。以此共混膜为载体培养兔角膜基质细胞，发现细胞在此共混膜上生长良好。制备膜片随着加入CaSO4量的增加，膜的通透性也随之增加。     

三种角膜内皮细胞载体膜片的性质研究

以壳聚糖分别与硫酸软骨素、羧甲基壳聚糖、透明质酸按一定比例制备出共混膜，研究了膜片的透光性、吸水率、渗透性、力学性质、表面结构、红外图谱等物理性质。以共混膜为载体培养兔角膜内皮细胞，从而筛选出了透明度高，亲水性好，机械性能优异，最适合内皮细胞贴附生长的壳聚糖．硫酸软骨素共混膜片，并对其生物降解性、生物相容性等性质进行了研究。为了提高壳聚糖．硫酸软骨素共混膜片的通透性，在膜片制备过程中加入不同剂量的醋酸盐作为扩孔剂，结果表明当加入一定量的醋酸盐为扩孔剂时，膜片的通透性提高。     

壳聚糖相对分子量对壳聚糖膜与角膜基质细胞相容性的影响

以脱乙酰度为95％，相对分子量分别为130KDa、220KDa、300KDa和500KDa的壳聚糖制备不同的壳聚糖膜。以各种壳聚糖膜作为基质，体外培养兔角膜基质细胞，通过观察角膜基质细胞在不同壳聚糖膜上的生长状态、贴附情况、生长曲线以及乳酸脱氢酶的活性，研究壳聚糖相对分子量对壳聚糖膜与角膜基质细胞生物相容性的影响。实验结果表明壳聚糖相对分子量对壳聚糖膜与角膜基质细胞的相容性具有重要的影响，相对分子量过高或过低的情况下，壳聚糖膜与角膜基质细胞相容性较差，对细胞损伤程度较大，细胞在膜上的贴附、生长能力较差；以相对分子量在200KDa～300KDa之间的壳聚糖制备出的膜与角膜细胞具有较好的相容性，细胞可在膜上长成密集单层，适合作为角膜培养的支架材料。     

不同脱乙酰度对壳聚糖膜与角膜基质细胞相容性的影响

以分子量为30万．脱乙酰度分别为63．3％、73．7％、83％和97％的壳聚糖制备不同的壳聚糖膜，在不同脱乙酰度的壳聚糖膜上培养兔角膜基质细胞．通过观察角膜基质细胞在不同壳聚糖膜上的生长状态、贴附情况、生长曲线以及乳酸脱氢酶的活性，研究壳聚糖分子脱乙酰度对壳聚糖膜与角膜基质细胞生物相容性的影响。实验结果表明壳聚糖脱乙酰度越高。壳聚糖膜对细胞的损伤越小。越有利于细胞在膜上的生长和贴附，反之．低脱乙酰度的壳聚糖膜与角膜细胞的相容性较差。     

超声乳化术中不同灌注液对角膜内皮细胞的影响

目的:观察超声乳化术中使用不同灌注液(世可和平衡盐液)对兔眼角膜内皮细胞结构及功能的影响。方法:3.5月龄纯种日本大耳白兔,随机分为正常对照组、空白对照组、世可组和平衡盐液组,采用接触性角膜内皮显微镜进行角膜内皮细胞形态学定量测定。锥兰-茜素红活性染色,观察各组角膜内皮细胞微细结构的变化。结果:术后6h,各组较正常对照组细胞密度和六边形细胞百分率降低;细胞面积和六边形细胞百分率均高于正常对照组;光镜显示空白对照组角膜内皮细胞形态改变明显,而世可组和平衡盐液组角膜内皮细胞改变轻微,无细胞坏死。结论:在超声乳化术中,灌注液对角膜内皮的损伤为化学性损伤,在相同手术条件下,世可对角膜内皮细胞具有良好的保护作用。     

Ocular cell monolayers cultured on biodegradable substrates.

The aim of this study was to culture retinal pigment epithelial (RPE) and corneal endothelial cells on biodegradable substrates for future use in monolayer transplantation in the eye. The biodegradable polymers, poly-l-lactic (PLLA) and poly-dl-lactic-co-glycolic acid (85:15) (PLGA) (both of molecular weight 105 kd) were the biomaterials used. All materials were seeded with either pig/human retinal pigment epithelial cells or rabbit corneal endothelial cells and were maintained in tissue culture conditions. Upon confluency, the cell density was calculated and cell viability determined. All monolayers were stained with phalloidin-rhodamine for F-actin and antibodies to the tight junction (zonula occludens) protein, ZO1, to demonstrate the presence of tight junctions. The final cell density of human RPE monolayers on PLLA films was 2950 cells/mm(2) (+/-185). The final cell density of pig RPE on PLLA and PLGA film was 2350 cells/mm(2) (+/-152 and 178, respectively). Rabbit corneal endothelial cells had a final cell density of 2650 cells/mm(2) (+/-164). F-actin staining revealed a circumferential ring of actin filaments in all of the cells grown on substrates. ZO(1) immunohistochemistry demonstrated staining along the lateral cell borders of all cell types. The successful culture of retinal pigment epithelial and corneal endothelial monolayers on these substrates may have potential for transplanting cell monolayers in the eye to improve vision.     

The mucinous layer of corneal endothelial cells

PURPOSE: The goal of this study was to characterize the morphology of the mucinous layer on rabbit, bovine, owl, and human corneal endothelial cells. METHODS: Corneoscleral buttons were fixed using cetylpyridinium chloride to stabilize "mucus" and the tissue was prepared for transmission electron microscopy. Photomicrographs were measured to determine the thickness of the endothelial and epithelial mucinous layer in the central cornea. RESULTS: The endothelial mucinous layer was seen as a nearly uniform electrodense region on the apical aspect of the endothelium. It was found to be 0.9 microm, 0.9 microm, 0.9 microm, and 0.5 microm thick in rabbit, bovine, owl, and human, respectively. The owl endothelium had an additional less electrodense layer with a granular appearance and a thickness of about 200 microm. The mucinous layer on the epithelium was similar in appearance to that on the endothelium and across species. CONCLUSIONS: The morphologic similarity of the endothelial and epithelial mucinous layers is a serendipitous finding that should prove valuable in experimental design. Ultimately, it is hoped that studies of the posterior corneal surface will deepen our knowledge of endothelial protection.     

Functional reconstruction of corneal endothelium using nanotopography for tissue-engineering applications

Dysfunction in the corneal endothelium, which controls the hydration and transparency of the cornea, is one of the common reasons for transplantation. A tissue-engineered corneal endothelium is of interest for corneal regeneration and for in vitro testing of ocular drugs. In the native environment, corneal endothelial cells interact with the nanotopography of the underlying Descemet's membrane. This study showed that nanotopography enhanced bovine corneal endothelial cell (BCEC) responses, creating a monolayer which resembled the healthy corneal endothelium. Topographies of different geometries were first tested to identify those that would elicit the most significant responses. A BCEC monolayer was then generated on both micro- and nanoscale pillars and wells. The BCEC monolayer cultured on topographies exhibited polygonal geometries with well-developed tight junction proteins. Scanning electron microscopy revealed that cells on pillars showed a higher density of microvilli, which was similar to native corneal endothelium. BCECs on nanopillars displayed a lower coefficient of variation of area (0.31) that was within the range of healthy corneal endothelium. More importantly, a BCEC monolayer cultured on nanopillars also had an enhanced Na(+)/K(+)-ATPase immunofluorescence expression, mRNA upregulation and a higher Na(+)/K(+)-ATPase activity. These results suggest that nanopillar substrate topography may provide relevant topographical cues, which could significantly enhance the formation and function of corneal endothelium.     

The effect of plasma-induced graft copolymerization of PHEMA on silicone rubber towards improving corneal epithelial cells growth

A PHEMA grafted polymer film was prepared by plasma induced graft copolymerization onto an elastic material, silicone rubber. The control, Ar plasma-treated, and PHEMA-grafted silicone rubber surfaces were characterized by ESCA, FTIR-ATR, and SEM techniques. ESCA verified the respective chemical shift of control and Ar plasma-treated films. The presence of the grafted PHEMA was also verified by ESCA. The amounts of grafted PHEMA did not monotonously increase with the plasma exposure conditions, but decreased after passing a maximum. The introduction of PHEMA onto a hydrophobic support provided an adequate surface for rabbit corneal epithelium cell attachment and growth. Cell attachment and growth onto these surfaces were examined by light microscopy. Cell attachment onto the control and Ar plasma-treated surface was negligible, while improved attachment and growth of rabbit corneal epithelium cells was demonstrated on the PHEMA-grafted polymer surface. The PHEMA-grafted silicone rubber surface demonstrated a confluent cell layer after 72 h.     

房水中转化生长因子水平与角膜内皮损伤及修复的关联分析

目的 探讨房水中转化生长因子水平与角膜内皮损伤及修复的关联.方法 研究选取本院就诊患者102例,检测患者房水中总TGF-β2和活化TGF-β2的水平,监测分析角膜内皮细胞密度水平、角膜内皮六角形细胞的比例水平以及中央角膜厚度.分析讨论房水中TGF-β2水平与角膜内皮损伤及修复的关联.使用SPSS软件处理数据.结果 对治疗前总TGF-β2水平与表征角膜内皮细胞损伤状态的四项指标水平进行相关分析结果提示,治疗前研究样本总TGF-β2水平与角膜内皮细胞密度之间的相关系数为-0.123、于角膜内皮六角细胞水平之间的相关系数为-0.198,另外治疗前总TGF-β2水平与中央角膜厚度水平之间的相关系数为0.254,且均P＜0.05;治疗前活化TGF-β2水平与表征角膜内皮细胞损伤状态的指标水平相关分析结果也提示了类似结果,除了活化TGF-β2水平与内皮细胞密度的r为-0.145,未见统计学意义之外,活化TGF-β2水平与其他指标的相关系数均有统计学意义.治疗后总TGF-β2水平以及活化TGF-β2水平与四项指标的相关系数也发现了类似规律,且均P＜0.05.结论 总TGF-β2水平以及活化TGF-β2水平增高与角膜内皮损伤有关,与内皮细胞密度、角膜内皮六角细胞比例下降,中央角膜厚度则会增加密切相关.     

Localization of hyaluronic acid, chondroitin sulfate, and CD44 in rabbit cornea.

To demonstrate the localization of hyaluronic acid (HA) in rabbit cornea, the biotinylated HA-binding region, which specifically binds to the HA molecule, was applied to the tissue. Localization of chondroitin sulfate (CS) and CD44, a possible cell surface receptor for HA, were also examined by immunohistochemistry. The stainability of HA changed depending on the fixatives used. Reaction products for HA were distinctly detected in epithelial cells and stromal keratocytes, but faintly in the extracellular matrix of the stroma when unfixed cryosections were applied. No positive reaction was found in the endothelium, except that the positive deposits formed a continuous layer on the apical surface of the endothelium. Electron microscopy using samples fixed with 2% paraformaldehyde revealed gold particles indicating HA labeling the intercellular space of the epithelium and stromal extracellular matrix. No intracellular deposition was detected in epithelial cells, whereas the gold labeling was seen in vacuolar structures of stromal keratocytes. Immunodeposits for CS were intensely localized in the epithelium and stroma, and weakly in the endothelium. Immunoreactivity for CD44 was found in the epithelial, endothelial and stromal cells. In particular, immuno-deposits for CD44 were detected in basal parts of epithelial cells, while they were localized in the apical surface of endothelial cells. These results suggest that HA is synthesized in and secreted from epithelial and stromal cells of rabbit cornea, while the localization of HA in the apical surface of the endothelium is closely associated with that of CD44. Moreover, the presence of CS in corneal tissue may play a role in its transparency, as has been suggested for keratan sulfate and dermatan sulfate.