维生素C不同给药方式对早期兔角膜碱烧伤的疗效观察

目的比较结膜囊内放置维生素C药物棉片与常规点眼治疗兔角膜碱烧伤的效果。方法选取16只成年家兔制作右眼角膜碱烧伤模型并随机分为点眼组和棉片组,碱烧伤后0、12、24、36、48、60h荧光素钠染色,计算角膜上皮修复率;不同时点制作病理切片,光镜、电镜观察。结果24、36、48h棉片组角膜上皮修复率较点眼组高,差异有统计学意义(P<0.05);12h棉片组与点眼组比较,差异无统计学意义(P>0.05);60h角膜上皮均愈合。病理组织学结果显示:与点眼组比较,棉片组角膜上皮修复佳,基质层胶原纤维排列较整齐且炎症细胞浸润轻。结论结膜囊内放置维生素C药物棉片治疗角膜碱烧伤优于点眼。     

神经生长因子滴眼液对角膜碱烧伤愈合的影响

目的：研究神经生长因子滴眼液,对角膜碱烧伤愈合的影响,为临床治疗提供新思路。方法：制作兔眼碱烧伤模型。采用自身对照法,左眼均为实验组,烧伤后给予配制的神经生长因子滴眼液点眼,右眼为对照组给予平衡盐溶液（BSS）点眼。烧伤后每日观察眼前段情况。于3、10、14d行组织学检查。结果：烧伤后两组角膜上皮缺损面积差异有统计学意义（P＜0.05）。两组角膜水肿、混浊度差异具有统计学意义（P＜0.05）。组织学检查显示实验组在14d时,角膜上皮愈合完整,基质水肿较对照组轻,胶原纤维排列趋于正常。结论：神经生长因子滴眼液促进角膜碱烧伤后角膜上皮的生长,对角膜的完整性,透明性及功能的恢复具有重要意义。     

培养胎兔角膜缘干细胞移植治疗兔眼碱烧伤的实验研究

目的 探讨羊膜为载体培养的胎兔角膜缘干细胞移植治疗兔角膜缘碱烧伤的效果。方法 将胎兔角膜缘干细胞原代培养于羊膜上7d后。移植于兔角膜缘碱烧伤动物模型眼，并对治疗后的角膜进行临床及病理学检查。结果 体外培养的胎兔角膜缘干细胞可在羊膜上保持高增殖力。并分化为密集的角膜上皮细胞层。角膜缘干细胞移植术后兔角膜上皮完整、基质细胞浸润减轻、新生血管减少，与对照组和单纯羊膜移植组相比其临床评分差异有显著意义。结论 胎兔角膜缘干细胞移植术治疗角膜碱烧伤可有效重建眼表，为临床应用胎儿角膜缘干细胞进行眼表重建奠定了实验基础。     

多层羊膜移植在角膜疾病中的应用（附27例报告）

目的 探讨多层羊膜移植治疗角膜疾病的可行性。方法 采用多层羊膜对我院角膜碱烧伤、角膜热烧伤5例(5只眼)，化脓性角膜溃疡18例(18只眼)，角膜溃疡穿孔3例(3只眼)，大泡性角膜病变1例(1只眼)，共计27例(27只眼)临床住院患者，进行单纯多层羊膜移植术，分别采用嵌入法、覆盖法和填塞法三种手术方法。术后随访观察3～16个月，平均8个月。结果 27例术后临床上均未见羊膜移植片急性排斥反应。角膜烧伤的3只眼术后角膜上皮愈合，视力有不同程度提高。化脓性角膜溃疡18只眼中，术后10只眼角膜溃疡愈合；5只眼角膜溃疡无进行性加剧，经再次手术后溃疡病灶缩小；另3只眼术后病情无法控制，最终行眼球摘除术。角膜溃疡穿孔3只眼术后前房形成，溃疡渐愈合。大泡性角膜病变1只眼术后疼痛消失，上皮愈合。结论 多层羊膜移植能作为基膜，促进角膜重新上皮化，抑制炎症和新生血管的生成，抑制纤维化，是治疗角膜疾病的有效方法。     

32例爆炸致非穿透性角膜外伤临床治疗分析

目的探讨不同损伤程度的爆炸性角膜外伤治疗的临床疗效。方法回顾性分析病例32例爆炸性角膜外伤,8眼角膜裂伤<3 mm者,行角膜浅层异物剔除等保守治疗;30眼角膜裂伤>3 mm且伤口不规整伤口者行角膜异物剔除、角膜裂伤缝合、羊膜遮盖的综合治疗。对角膜上皮修复、角膜裂伤愈合、角膜新生血管生长情况进行观察分析。结果 8眼角膜裂伤<3 mm者术后1周角膜上皮愈合,遗留浅层角膜薄翳。30眼角膜裂伤>3 mm者术后3～4周羊膜溶解后观察到角膜上皮愈合、角膜裂伤瘢痕混浊;随访1～6个月,84.21%(32/38眼)无角膜新生血管生长。结论对不同损伤的爆炸性角膜外伤采用不同治疗方法是切实有效的,羊膜遮盖术在促进角膜上皮愈合、减轻瘢痕混浊和抑制新生血管生长方面疗效显著。     

重组人角质细胞生长因子异构体对兔角膜碱烧伤的疗效观察

目的:观察重组人角质细胞生长因子异构体(简称K102)对实验性兔角膜碱烧伤的治疗作用。方法:60只新西兰大耳白兔,均选左眼为实验眼,随机分成5组,各组均以1mol·L-1的氢氧化钠溶液建立碱烧伤模型。其中,A、B、C组为治疗组(浓度分别为0.5、0.25、0.125mg·mL-1K102滴眼液),D组为阳性对照组(贝复舒滴眼液),E组为阴性对照组(K102滴眼液的溶媒)。镜下观察各组角膜上皮愈合速度(率)及血管新生(CNV)发生情况。结果:在碱烧伤24h内,A、B、C组的角膜上皮生长速率分别为1.52、1.57、1.46mm2·h-1,与E组(0.98mm2·h-1)比较具有显著性差异(P<0.01);各组上皮愈合率各时间点比较治疗组均优于阴性对照组并有显著性差异(P<0.01或<0.05)。治疗组低浓度可抑制CNV,高浓度可促进CNV。结论:K102滴眼液不同浓度均能够显著促进兔角膜碱烧伤后上皮愈合,抑制角膜CNV生长,改善角膜碱烧伤预后。     

羊膜匀浆提取液与羊膜移植对大鼠碱烧伤后新生血管抑制作用的比较

目的 比较羊膜匀浆提取液和羊膜移植对大鼠角膜碱烧伤后新生血管的影响.方法 SD大鼠30只随机分为三组,每组10只.使用3 mm直径圆形滤纸片浸入1 mol·L-1,NaOH溶液中60 s后放置于大鼠角膜中央建立右眼角膜碱烧伤模型.A组行PBS（磷酸缓冲盐溶液）液点眼,B组行羊膜移植术,C组行AM（羊膜匀浆提取液）点眼.通过裂隙灯显微镜观察三组的角膜新生血管情况.结果 碱烧伤后11 d,三组的角膜新生血管面积分别为（49.12±2.05）、（35.17±1.95）、（34.04±1.37）mm2.B、C两组的新生血管面积比A组显著减少（qA、B=15.59,P A、B〈0.001,qA、C=19.340 6,PA、C〈0.001）,而B、C组之间没有显著性差异（q=1.499 4,P〉0.05）.结论 新鲜人羊膜匀浆提取液与羊膜移植均能有效减少角膜新生血管形成.     

羊膜移植治疗对眼表烧伤患者视力及角膜恢复的影响

目的 探讨羊膜移植治疗对眼表烧伤患者视力及角膜恢复的影响.方法 选择眼表烧伤患者30例(38眼),采取羊膜移植术治疗,术后随访3个月,记录患者的视力及角膜恢复情况.结果 术后随访3个月,所有患者的视力均得到不同程度的提高.38眼中,术后最佳视力达到1.0,视力＞0.3者17眼(50.00％).患者角膜恢复透明13眼(34.21％),出现角膜云翳14眼,出现角膜斑翳7眼,出现角膜白斑4眼.术后2～3周,陆续有5眼出现角膜新生血管,且向瞳孔区蔓延.术后＜21 d,有30眼(78.95％)的角膜上皮得到全部愈合;术后21d～28 d,有6眼的角膜上皮得到全部愈合;术后32 d,严重碱烧伤2眼角膜上皮得以愈合.结论 羊膜移植术治疗眼表烧伤的疗效显著,术后患者的视力及角膜恢复良好,临床应值得推广实施.     

保存人羊膜移植治疗眼表疾病

目的：探讨保存人羊膜治疗难治性眼表疾病的有效性及实用性。方法：采用保存人羊膜移植术治疗陈旧性眼化学烧伤（2例,3眼）及角膜溃疡（8例,8眼）。结果：术后随访3－6个月,11眼中9眼角膜透明或半透明,术后视力不同程度提高,仅一蚕蚀性角膜溃疡病人术后13天溃疡复发。结论：保存人羊膜能有效修复角膜基质及上皮的缺损,是眼表重建的理想材料,羊膜移植术能有效治疗眼表疾病。     

新鲜羊膜联合自体角膜缘干细胞移植治疗翼状胬肉的临床观察

目的：探讨新鲜羊膜联合自体角膜缘干细胞移植治疗翼状胬肉的临床疗效。方法：对30例（33眼）原发性和复发性翼状胬肉施行显微镜下切除术加新鲜羊膜联合自体角膜缘干细胞移植，术后随访3-12个月，平均7个月。结果：33眼无1例复发．术后短期内未见植片溶解，及急性排斥反应，角膜缘移植片及羊膜移植片均愈合良好，羊膜移植创面完全结膜上皮化，角膜组织上皮稳定。结论：新鲜羊膜联合自体角膜缘干细胞移植是治疗翼状胬肉的有效方法。     

兔角膜碱烧伤后角膜、房水和晶状体中NO与MDA含量的实验探讨

目的:探讨碱烧伤后角膜、房水、晶状体组织中一氧化氮(NO)与丙二醛(MDA)含量改变及其意义.方法:选择健康新西兰大白兔20只(40眼),经麻醉后,用直径9mm圆形滤纸浸透2mmol/L氢氧化钠,贴附于兔右眼角膜中央区,制作碱烧伤模型.72h后处死,取角膜、房水、晶状体,角膜及晶状体做组织匀浆,用比色法测量其NO和MDA的含量,左眼为正常对照组.结果:碱烧伤72h后角膜、房水、晶状体中NO和MDA含量较对照组显著升高(P＜0.01), MDA及NO各组中的含量经过方差分析均不相同,对角膜、房水、晶状体中MDA及NO进行相关性分析,两者为正相关关系.结论:NO、MDA均参与了碱烧伤后眼部损伤的病理生理过程.     

Monocarboxylate transport in human corneal epithelium and cell lines

Monocarboxylate transporters (MCTs) are transmembrane proteins capable of transferring lactate and other endogenous and exogenous monocarboxylates across the cell membrane. The aim of the present study was to assess the expression and transporter role of human MCT1, MCT3 and MCT4 in the corneal epithelium, corneal epithelial cell lines (primary HCEpiC and immortalized HCE cells) and isolated rabbit corneas. MCT1 and MCT4 were expressed in the human corneal epithelium and the cell lines at mRNA and protein levels. Cellular uptake studies showed saturable and pH-dependent l-lactic acid transport, which was inhibited by various monocarboxylates like diclofenac and flurbiprofen. The permeability of benzoic acid across the rabbit cornea was higher in absorptive direction and this directionality was diminished in the presence of monocarboxylate drug valproic acid. Monocarboxylate transport was functional in the human corneal epithelial cells and rabbit cornea and it may play a role in the ocular drug absorption.     

Extent of corneal injury as a biomarker for hazard assessment and the development of alternative models to the Draize rabbit eye test

We have characterized 22 ocular irritants differing in type (surfactants, acid, alkali, bleaches, alcohol, aldehyde, acetone) and severity (slight to severe) by using the low-volume rabbit eye test. Ocular irritation was evaluated by 1) light microscopy to assess pathological changes, 2) in vivo confocal microscopy (CM) to quantify 4-dimensionally (x, y, z, and t) initial corneal injury and later responses in the same eye, and 3) laser scanning CM to quantify initial cell death. These studies revealed that regardless of the processes leading to injury, slight irritants injure the corneal epithelium, mild irritants injure the corneal epithelium and the superficial stroma, and moderate/severe irritants injure the epithelium, deep stroma, and at times the corneal endothelium. Furthermore, extent of initial corneal injury was shown to predict subsequent responses and final outcomes. These findings suggest that extent of corneal injury may be used as a basis for the development of alternative ocular irritation tests. To test the validity of this approach, we have used an ex vivo, rabbit cornea culture model to measure extent of corneal injury following exposure to ocular irritants. Data indicate that the extent of ex vivo corneal injury significantly correlate with the extent of initial injury measured previously in live animals. Overall, these findings indicate that extent of initial corneal injury can be used as a new "gold standard" for the continued refinement and ultimate replacement of the Draize rabbit eye Ocular Irritation Test.     

Quantitative measurement of acute corneal injury in rabbits with surfactants of different type and irritancy.

We have hypothesized that differences in ocular irritancy are related to differences in extent of initial injury and that, regardless of the processes leading to tissue damage, extent of injury is the primary factor that determines the final outcome of ocular irritation. In previous in vivo confocal microscopic (CM) studies we identified quantifiable differences in the extent of corneal injury occurring with four surfactants (three anionic, one cationic) known to cause different levels of ocular irritation and demonstrated that extent of initial corneal injury was related to the magnitude of cell death. The purpose of this study was to assess the applicability of this hypothesis to a broad sampling of surfactants. Specifically, initial corneal changes induced by seven different surfactants (one anionic, three cationic, three nonionic) were measured by in vivo CM and cell death was measured by an ex vivo live/dead assay. The right eye of each rabbit was treated by placing 10 microl of a surfactant directly on the cornea. Eyes were examined macroscopically and scored for irritation at 3 h and 1 day. At 3 h and 1 day, in vivo CM was used to examine the corneas and quantitate epithelial cell size, epithelial thickness, corneal thickness, and depth of stromal injury. At 3 h and/or at 1 day, corneas were removed and excised regions were placed in culture media containing 2 microM calcein AM and 4 microM ethidium homodimer. Using laser scanning CM, the number of dead epithelial and/or stromal cells in a 300 x 300 x 170-microm3 (xyz) volume of the cornea was determined. In vivo CM and live/dead assay findings revealed three surfactants to affect only the epithelium, three surfactants to affect the epithelium and superficial stroma, and one surfactant to affect the epithelium and deep stroma. Extent of initial corneal injury reflected level of ocular irritation, and magnitude of cell death was related to the extent of initial corneal injury. These findings are consistent with those for known slight, mild, and moderate to severe irritants, respectively. They suggest that our hypothesis is broadly applicable to surfactants. Additionally, we believe these surfactants should be included as part of a new "gold standard" for use in developing and validating in vitro tests to replace the use of animals in ocular irritancy testing.     

眼部碱烧伤126例综合治疗临床分析

目的：探讨眼部碱烧伤综合治疗的效果。方法回顾性分析解放军白求恩国际和平医院2009年1月—2014年12月收治的采用综合疗法治疗的126例(179只眼)眼部碱烧伤患者的临床资料。结果眼部碱烧伤患者角膜上皮修复时间Ⅱ度<Ⅲ度<Ⅳ度。Ⅱ度眼部碱烧伤患者全部脱残,预后良好,无严重并发症发生。Ⅲ度眼部碱烧伤患者视力恢复情况优于Ⅳ度患者,并发症发生率低于Ⅳ度患者。术后3个月随访,45例早期行羊膜移植术者19例出现角膜血管化,其中2例半年后再次住院行穿透性角膜移植术,术后视力恢复良好。结论综合疗法治疗眼部碱烧伤效果满意,疗效及预后与患者眼部碱烧伤程度相关。     

兔口腔黏膜上皮干细胞分化为角膜上皮样细胞的体外研究

目的以羊膜做载体将兔口腔黏膜上皮干细胞诱导为角膜上皮样细胞,探讨口腔黏膜上皮细胞作为种子细胞体外培养构建组织工程角膜的技术方法,为眼表重建提供材料。方法用Ⅳ型胶原黏附法分离纯化口腔黏膜上皮干细胞,对体外培养的口腔黏膜干细胞进行免疫表型鉴定。将筛选后获得的口腔黏膜上皮干细胞种植在去上皮羊膜表面体外培养,待细胞融合成单层后置入插入式培养皿中进行气液界面培养,促进细胞分化形成复层。培养数日后进行苏木素-伊红(HE)染色和免疫组织化学、光镜及透射电镜检测,观察羊膜-复层上皮组织结构,免疫荧光检测干细胞特异性标志物P63以及角膜上皮细胞标志物角蛋白3(K3)并与角膜上皮组织比较。结果体外诱导培养数天后,羊膜载体的口腔黏膜上皮细胞形成复层,在组织形态及生物学特性上与角膜上皮相似。并且组织细胞P63、K3表达明显,角膜特异性生物学标志物免疫组织化学染色呈阳性。结论在本实验的培养条件下,获得的口腔黏膜上皮干细胞可在体外培养扩增,呈克隆性生长,具有很强的增殖能力。口腔黏膜上皮干细胞体外培养可构建类角膜上皮。     

Polyethylenimine-conjugated gold nanoparticles: Gene transfer potential and low toxicity in the cornea

This study examined the gene transfer efficiency and toxicity of 2-kDa polyethylenimine conjugated to gold nanoparticles (PEI2-GNPs) in the human cornea in vitro and rabbit cornea in vivo. PEI2-GNPs with nitrogen-to-phosphorus ratios of up to 180 exhibited significant transgene delivery in the human cornea without altering the viability or phenotype of these cells. Similarly, PEI2-GNPs applied to corneal tissues collected after 12 hours, 72 hours, or 7 days exhibited appreciable gold uptake throughout the rabbit stroma with gradual clearance of GNPs over time. Transmission electron microscopy detected GNPs in the keratocytes and the extracellular matrix of the rabbit corneas. Additionally, slit-lamp biomicroscopy in live animals even 7 days after topical PEI2-GNP application to the cornea detected no inflammation, redness, or edema in rabbit eyes in vivo, with only moderate cell death and immune reactions. These results suggest that PEI2-GNPs are safe for the cornea and can potentially be useful for corneal gene therapy in vivo. FROM THE CLINICAL EDITOR: This study examined the gene transfer efficiency and toxicity of 2-kDa polyethylenimine conjugated to gold nanoparticles in the human cornea in vitro and rabbit cornea in vivo. The results suggest that PEI2-GNPs are safe for the cornea and can potentially be useful for corneal gene therapy in vivo.