纤维连接蛋白在家兔角膜碱烧伤中的治疗作用及其测定意义

本实验应用纤维连接蛋白滴眼剂治疗兔角膜碱烧伤,测定烧伤前后不同时期血和房水中纤维连接蛋白含量。结果表明:纤维连接蛋白能促进早期角膜上皮愈合并防止晚期上皮脱落。烧伤早期,房水纤维连接蛋白比未烧伤前明显升高,说明血-房水屏障的破坏;晚期血浆纤维连接蛋白的逐渐下降,表明组织修复对其消耗和利用的增加。本文探讨了纤维连接蛋白在角膜碱烧伤中的治疗作用及血和房水中含量变化及意义。     

兔角膜碱烧伤后房水及血浆纤维连接蛋白含量变化的观察

采用单向琼脂免疫扩散法,对实验性兔角膜碱烧伤后房水及血浆纤维连接蛋白(简称FN)含量进行了观察。表明房水FN含量增加发生于房水pH增高之后,60分钟时达高峰。此间房水FN与房水总蛋白含量的增高呈正相关,与总蛋白的百分比高于血浆。此后房水FN含量呈降低趋势,与总蛋白的百分比低于血浆。讨论了房水和血浆FN含量变化的意义。     

实验性角膜损伤愈合过程中房水纤维连接蛋白的变化

用酶联免疫吸附试验方法对家兔角膜切伤愈合过程中,房水纤维连接蛋白水平的动态检测。发现在损伤后第1天,房水纤维连接蛋白即比损伤前高1倍以上。1周后又下降,在第9天时,约为损伤前水平的70％,随后又逐渐回升,3周后接近损伤前水平。     

角膜碱烧伤新鲜羊膜移植治疗对自由基反应的影响

目的 探讨新鲜羊膜移植在早期角膜碱烧伤治疗中与自由基反应的关系。方法 48只兔制作角膜碱烧伤模型,分为新鲜羊膜移植组、保存羊膜移植及对照组,烧伤后24小时分别实施手术。术后14天及30天处死实验兔取材房水及角膜组织,检测其超氧化物歧化酶活力及丙二醛含量。结果 新鲜羊膜移植组及保存羊膜移植组的丙二醛含量均低于对照组,超氧化物歧化酶活力均高于对照组。术后14天新鲜羊膜移植组角膜超氧化物歧化酶活力高于保存羊膜组。术后30天新鲜羊膜组角膜及房水丙二醛含量均低于保存羊膜组。结论 新鲜羊膜移植在早期角膜碱烧伤治疗中能减轻自由基质应对照组织的损伤,其效果优于保存羊膜移植。     

准分子激光术后角膜、房水中SOD、MDA的变化

目的 探讨氧自由基对准分子激光屈光性角膜切削术(PRK)术后角膜及房水的影响。方法 将16只兔右眼行-5.0DPRK。于术后1、3、7、14d测定角膜组织及房水中超氧化物歧化酶(SOD),丙二醛(MDA)的变化,并观察角膜组织病理变化。结果 在术后1、3d角膜SOD活性明显小于对照组(P<0.01),MDA的水平明显高于对照组(P<0.01);但术后房水中SOD、MDA与对照组比较无明显变化(P>0.05)。结论 PRK术后,角膜存在着脂质过氧化形式介导的自由基损害,降低角膜过氧化损伤可以最大程度地减少PRK术后并发症的发生。     

p38丝裂原活化蛋白激酶抑制剂对兔眼滤过术后肌成纤维细胞分化及细胞外基质合成的作用

背景 各种原因导致滤过手术失败的共同点是滤过通道的成纤维细胞过度增生、分化,导致过度纤维化、瘢痕形成.研究发现,p38丝裂原活化蛋白激酶(MAPK)信号通道在成纤维细胞表型转化过程中发挥重要作用. 目的 探讨p38 MAPK抑制剂SB203580对滤过手术后结膜下纤维化反应的抑制效果及其作用机制.方法 12只清洁级新西兰白兔行常规青光眼滤过手术制作双眼滤过手术模型,模型兔按随机数字表法随机分为单纯滤过手术组、SB203580治疗组和丝裂霉素C(MMC)对照组.SB203580治疗组兔眼滤过术毕立即结膜下注射0.2 g/L SB203580溶液1 ml,MMC对照组兔眼术中用浸泡0.2 g/L MMC的棉片置于术区结膜下及巩膜瓣下3 min.各组兔术后进行裂隙灯显微镜观察,术后1、3、7、10、14d使用Icare回弹式眼压计测量眼压,术后14 d抽取房水0.2ml,并获取手术滤过区结膜下组织标本,用ELISA法检测房水及滤过区结膜组织中的α-平滑肌肌动蛋白( α-SMA)、纤维连接蛋白的表达,用荧光实时定量PGR检测各组兔术眼滤过区结膜组织中ACTA2、结缔组织生长因子(CTGF)和Ⅰ型胶原蛋白α2链(COL1 A2) mRNA的表达. 结果 术后14d,单纯滤过手术组滤过泡血管化、瘢痕形成,SB203580治疗组滤过泡扁平、弥散,MMC对照组滤过泡呈苍白缺血状、扁平弥散.单纯滤过手术组、SB203580治疗组和MMC对照组兔间术前眼压值差异无统计学意义( F=0.065,P=0.937),术后眼压值随着时间的延长逐渐升高,各时间点的总体比较差异有统计学意义(F=32.873,P=0.030).ELISA法检测SB203580治疗组和MMC对照组兔房水及滤过区结膜下组织中的α-SMA质量浓度均明显低于单纯滤过手术组,而MMC组α-SMA质量浓度明显低于SB203580治疗组,差异均有统计学意义(P＜0.05).SB203580治疗组和MMC对照组兔房水及滤过区结膜下组织中的纤维连接蛋白质量浓度均明显低于单纯滤过手术组,而MMC对照组α-SMA质量浓度明显低于SB203580治疗组,差异均有统计学意义(P＜0.05).荧光实时定量PCR检测表明,各组兔术眼滤过区结膜下组织ACTA2、CTGF、COL1A2mRNA表达差异均有统计学意义(P＜0.01),以单纯滤过手术组表达量最高,SB203580治疗组次之,MMC对照组最低,差异均有统计学意义(P＜0.05).结论 p38 MAPK抑制剂SB203580能减少肌成纤维细胞分化及细胞外基质合成,减轻兔眼滤过手术后的组织纤维化反应.     

Nd:YAG激光晶状体前囊膜切除术后房水内丙二醛变化与眼压的关系

探讨Ｎｄ：ＹＡＧ激光晶状丛前囊膜切除术后暂时性眼压升高的发生机制，观察丹参和维生素Ｅ对眼压升高的影响。方法２４只兔随机分为４组，药物且激光手术前后应用丹参和维生素Ｅ。定期观察激光术后眼压和房水内丙二醛含量的变化。结果激光术后发生暂时性眼升高并伴随房水内ＭＤＡ含量明显增加。药物组术后房水内ＭＤＡ含量无明显增加并且术后性眼压升高的幅度明显降低。结论激光前囊膜切除术后暂时性眼压升高与术后房水内ＭＤＡ含量     

兔后发性白内障房水中自细胞介素-1β和白细胞介素-6的含量

目的：探讨白内障囊外摘除术后房水中白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)的含量与后发性白内障的关系。方法：28只兔均行白内障晶状体囊外手术，分别于术后1、3、7、14、30d和90d对术眼进行裂隙灯和眼底检查，并抽取房水于-80℃中保存。用酶联免疫吸附试验对术后兔房水中的IL-1β和IL-6含量进行测定。结果：兔房水IL-1β和IL-6含量于术后3、7、14d和30d明显升高，术后7～14d达最高值，且与后囊混浊程度呈正相关。结论：IL-1β和IL-6可能参与后发性白内障的形成。     

兔后发性白内障房水中白细胞介素-1β和白细胞介素-6的含量

目的:探讨白内障囊外摘除术后房水中白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)的含量与后发性白内障的关系.方法:28只兔均行白内障晶状体囊外手术,分别于术后1、3、7、14、30 d和90 d对术眼进行裂隙灯和眼底检查,并抽取房水于-80 ℃中保存.用酶联免疫吸附试验对术后兔房水中的IL-1β和IL-6含量进行测定.结果:兔房水IL-1β和IL-6含量于术后3、7、14 d和30 d明显升高,术后7～14 d达最高值,且与后囊混浊程度呈正相关.结论:IL-1β和IL-6可能参与后发性白内障的形成.     

汉防己甲素抑制自由基眼损伤的实验研究

目的 搪塞汉防己甲素对自由基眼损伤的影响。方法 将２４只实验兔随机分为汉防己甲素组及对照组。术后动态测定房水中丙醛含量。结果 汉防己甲素丙二醛含量明显低于对照组。结论 汉防己甲素能够显著抑制自由基引起的眼损伤。     

地塞米松对电光性眼炎防治作用的实验研究

本文对电光性眼炎家兔角膜上皮的超氧化物歧化酶（ＳＯＤ）活性及脂质过氧化反应主要代谢产物丙二醛（ＭＤＡ）含量进行测量。发现紫外线照射后３小时ＳＯＤ活性降低，伴随上述改变，ＭＤＡ含量增高。用地塞米松在照射前、后滴眼，３小时检测ＳＯＤ和ＭＤＡ，发现其可保护ＳＯＤ，并降低ＭＤＡ含量。透射电镜下显示，角膜上皮细胞的变化主要在其底部细胞，以水肿性改变为主。实验结果提示自由基损伤在急性电光性眼炎的发生、发展过程中起了重要作用，地塞米松可减轻这种变化。     

绿色荧光灯对大鼠视网膜光化学损伤的动态观察

目的观察绿色荧光灯对大鼠视网膜光化学损伤的动态变化过程。方法采用(1900±106.9)Lx绿色荧光灯对SD大鼠进行持续24小时照射，分别于光照前，光照后6小时、6天及14天进行视网膜光镜形态学观察、外核层厚度及FERG检测。结果光照前视网膜形态正常，结构层次清楚，内外节排列整齐规则。光照后6小时视网膜外核层变薄，其厚度减少23.91%，内外节排列紊乱。光照后6天外核层更薄，其厚度减少46.60%。14天外核层较6天增厚，其厚度减少42.40%。FERG的a波和b波振幅于光照后6小时及6天持续降低，至14天仍无显著恢复。结论该模型可能是研究视网膜光化学损伤的较理想的动物模型。(中华眼底病杂志,1998,14:101-103)     

鼠视网膜光化学损伤中的主要组织化学改变

目的：观察大鼠视网膜光化学损伤的主要组织化学改变。方法：对动物模型进行视网膜超微结构观察、丙二醛含量测定、细胞色素氧化酶及镁激活三磷酸腺苷酶活性的组织化学观察。结果：超微结构发现光照后6小时，光感受器细胞核肿胀，内节线粒体肿胀，外节水肿，视网膜色素上皮(retinal pigment epithelitis,RPE)顶端微绒毛消失，溶酶体增多，6天反应加重。14天光感受器细胞及内节已基本正常，外节再生但盘膜排列稀疏，RPE顶端出现微绒毛。观察到光照后6小时及６天，视网膜细胞色素氧化酶及镁激活三磷酸腺苷酶活性下降，丙二醛含量增高，至１４天均有所恢复。结论：脂质过氧化使光感受器细胞膜系统损伤崩解，引起细胞超微结构及酶活性改变，可能与视网膜光化学损伤的发病有关。 (中华眼底病杂志,1998,14:38-40)     

Hyaluronan Facilitates Corneal Epithelial Wound Healing in Diabetic Rats

We investigated the effect of hyaluronan on corneal epithelial wound healing in rats affected by diabetes. Furthermore, because hyaluronan is thought to affect corneal epithelial wound healing through the mechanism of binding of hyaluronan to provisional fibronectin in the wounded area, we compared the localization of fibronectin immunohistochemically during corneal epithelial wound healing in diabetic and non-diabetic rats. Streptozotocin was used to induce diabetes in half the rats. Two weeks after treatment, the whole corneal epithelium of diabetic and untreated rats was debrided. The rats were divided into groups (seven or eight rats per group), and hyaluronan eye drops at concentrations of 0.03, 0.1, or 0.3%, chondroitin sulfate (3%), or phosphate buffered saline (PBS) was given in eye drops 6 times a day for 4 days, starting immediately after debridement. The area of the corneal epithelial wound was measured immediately after debridement and at 12, 18, 24, 30, 48, 72, and 96 hours afterwards. Although the healing process was similar in non-diabetic and diabetic rats, the healing rate in diabetic rats was slower than that in normal controls. In both diabetic and non-diabetic rats, hyaluronan increased the healing rate in a dose-dependent manner; the difference was significant compared with the PBS-treated group, at hyaluronan doses of 0.1% and 0.3%. However, chondroitin sulfate did not affect corneal epithelial wound closure, regardless of whether the rats were diabetic or not; the healing rates were identical to those of PBS-treated diabetic and non-diabetic controls. In both diabetic and non-diabetic corneas, fibronectin was localized in the corneal subepithelial region, and in streaks between collagen fibers of the stroma. One day after debridement, a layer of fibronectin immunofluorescence was clearly visible on the surface of the denuded stroma. As healing progressed staining of fibronectin diminished at the interface between the new epithelium and the stroma. These changes in localization of fibronectin during corneal epithelial wound healing were similar in both diabetic and non-diabetic rats. Our results demonstrate that hyaluronan facilitates corneal epithelial wound healing in diabetic rats, and suggest that one possible mechanism of its stimulatory effect lies in its binding to a provisional fibronectin matrix, in both diabetic and non-diabetic rats.     

Changes in extracellular matrix components after excimer laser photoablation in rat cornea

PURPOSE: To learn more about corneal wound healing after excimer laser photoablation. METHODS: Observations were made on the chronological changes in type I collagen, fibronectin, laminin, and type IV collagen after excimer ablation of rat cornea. Immunohistochemical techniques were used. RESULTS: There was no obvious change in the localization of type I collagen in the ablated area, but the localization of fibronectin, laminin, and type IV collagen changed remarkably. One day after ablation, immunofluorescent staining for fibronectin increased on the ablated surface. Subsequently, the fluorescence of fibronectin, laminin, and type IV collagen increased remarkably; in particular, the localization of these extracellular matrix proteins was sustained in the shallow layer of the stroma until about 24 weeks after ablation. Hematoxylin-eosin staining indicated that keratocytes temporarily disappeared 1 day after ablation, and activated keratocytes then migrated to the ablated areas. CONCLUSIONS: These results suggest that activated keratocytes might be synthesizing the extracellular matrix components. Therefore sustained responses of keratocytes may be induced by excimer laser photoablation.     

Metabolic changes of the human donor cornea during organ-culture.

PURPOSE: To study metabolic changes of the human cornea during organ-culture. Morphological changes have been extensively studied, whereas changes in human corneal metabolism have not been investigated yet. MATERIAL AND METHODS: 106 human corneas were stored for 1, 7, 15, 18, 21 and 28 days in a closed-system under standard eyebank conditions. After storage, glucose, lactate, ATP, ADP and AMP concentrations were determined in each cornea. RESULTS: Glucose concentration decreased during the first two weeks with a minimum on day 15. ATP and ADP concentrations increased during the same period of time, but had their minimum later, on day 18. Lactate increased during the culture period up to day 21 and decreased thereafter. CONCLUSION: From these data we conclude that the human cornea recovers during organ-culture, especially during the first two weeks. The changes occurring after a fortnight might be related to the artificial culture conditions. Nevertheless, the metabolic status is better than in post-mortem corneas. The changes may be partly avoided by changing the medium after at least two weeks of organ-culture.     

TIMP-2基因转染后豚鼠形觉剥夺眼后极部巩膜Ⅰ型胶原和纤维连接蛋白的表达

背景细胞外基质（ECM）蛋白和酶参与眼球后极部巩膜主动的重塑过程,主要通过影响I型胶原蛋白（Col-Ⅰ）纤维连接蛋白（FN）发挥作用。目的通过对形觉剥夺性近视（FDM）豚鼠经脉络膜上腔注入转染有TIMP-2基因的脂质体,观察基质金属蛋白酶抑制剂-2（TIMP-2）对细胞外基质中Ⅰ型胶原蛋白（Col-Ⅰ）和FNmRNA表达的影响。方法半透明眼罩遮盖36只豚鼠的右眼14d建立FDM动物模型,用随机数字表法分组后分别于右眼脉络膜上腔注入5p,1TIMP-2脂质体质粒、空质粒和生理盐水,每组12只眼,左眼为自身对照。另12只豚鼠持续遮盖右眼为模型对照组。分别于脉络膜上腔注药后的2、7、14d过量麻醉处死豚鼠,获取眼球后极部巩膜组织,用RT-PCR法分别检测各组豚鼠眼巩膜组织中Col-Ⅰ和FNmRNA的表达。结果TIMP-2组豚鼠后极部巩膜Col-Ⅰ mRNA表达降低,FNmRNA表达升高,与自身对照相比差异均有统计学意义（P〈0．05）。注入TIMP-2后,Col-I mRNA表达水平从第2天开始升高,第7天达到高峰,第14天时有所回落;FNmRNA表达水平则呈相反变化。二者在第7天、第14天的表达与其他3组间比较差异均有统计学意义（P〈0．05）。C01．ImRNA和FNmRNA表达水平在注射后第7天与第2天或第14天比较差异均有统计学意义（P〈0．01）。结论TIMP-2注入FDM豚鼠脉络膜上腔可上调Col-I mRNA表达,下调FNmRNA表达,早期可有减缓豚鼠FDM巩膜重塑的作用。     

t-PA抑制白内障术后眼内纤维膜形成的实验研究

目的：研究白内障手术注射t-PA抑制眼内纤维蛋白渗出及眼内纤维膜的形成。方法：成年青紫兰兔行晶体囊外摘除，部分后房型人工晶体植入，术后不同时间、不同浓度结膜下注射t-PA，分别于1、3、7、14、30天摘除眼球，病理常规切片，光镜观察。结果：注射t-PA后，眼内发生显著的病理形态变化，主要有四方面改变：减轻前房纤维蛋白及炎症细胞的渗出；减少虹膜、睫状体细胞的增生抑制眼内纤维膜的形成；减弱晶体囊膜增厚及皱褶的程度。结论：t-PA具有显著的抑制纤维蛋白渗出及眼内纤维膜形成的作用，不同浓度t-PA均有相同作用，以术后第7天效果明显。t-PA是治疗后发性白内障的安全、有效药物。     

Immunocytochemical study of extracellular matrix components during lens and neural retina regeneration in the adult newt.

We have conducted an immunocytochemical study of fibronectin, laminin, heparan sulfate proteoglycans and nidogen-entactin during lens and neural retina regeneration in the adult newt from 0 to 60 days. In the normal eye, fibronectin was detected in the corneal stroma and Descemet's membrane, in dorsal and ventral irises and lens capsule but not in Bowman's membrane of the cornea. In normal neural retina, fibronectin was found in Bruch's and inner limiting membranes. Heparan sulfate proteoglycans gave a slight signal in both irises and the lens capsule. Nidogen-entactin distribution in the cornea was similar to that of fibronectin; it was absent from the stroma of both irises, and the signal was weak in the pigmented iris epithelium. Nidogen-entactin was not detected in the lens capsule and inner limiting membrane of the neural retina but was present in Bruch's membrane. During the first 15 days of lens regeneration, fibronectin and nidogen-entactin decreased but did not disappear from the pupillary margin of both irises, and no signal was obtained for laminin and heparan sulfate proteoglycans. From day 15 to day 60 fibronectin and nidogen-entactin increased in both irises and lens capsule. The signal for laminin was restricted to the lens capsule. Heparan sulfate proteoglycans gave a slight signal in both irises and in the lens capsule. During the first 25 days of neural retina regeneration, fibronectin was the first to appear in Bruch's membrane and the cell border of the new neuroepithelium and remained during the entire process. Laminin appeared after 41 days in the inner limiting and Bruch's membranes, but by day 50 it appeared as a weak signal only in the inner limiting membrane. Heparan sulfate proteoglycans were not detected at any of the regeneration stages studied. Nidogen-entactin was only detected in Bruch's membrane and around the cells and blood vessels of the new neural retina. Later it was detected in the inner limiting membrane but not in Bruch's membrane. Thus, the results obtained showed that extracellular matrix components do change during both lens and neural retina regeneration. These changes may play an important role during both regenerating processes.     

Oral flavonoids, chromocarb diethylamine salt and cyaninosides chloride, to eliminate lipoperoxidation postvitrectomy

This study was undertaken to determine the concentration of malondialdehyde, an end product of lipoperoxidation, in lens and retinal tissue postvitrectomy associated with oral administration of antioxidant flavonoids cyaninosides chloride and chromocarb diethylamine salt or N -acetylcysteine. Fifty adult pigmented rabbits were divided into five groups: (1) controls (normal eyes, malondialdehyde concentration in lens and retina); (2) vitrectomy with BSS Plus (malondialdehyde level measured 2hr after vitrectomy); (3) vitrectomy with BSS Plus and pretreatment with oral cyaninosides chloride 100mg kg day(-1)for 3 weeks (malondialdehyde level measured 2hr after surgery); (4) vitrectomy with BSS Plus and pretreatment with oral chromocarb diethylamine salt 100 mg kg day(-1)for 3 weeks (malondialdehyde level measured 2hr after surgery); and (5) vitrectomy with BSS Plus and pretreatment with oral N -acetylcysteine 200 mg kg day(-1)for 3 weeks (malondialdehyde level measured 2hr after surgery). Lens and retina samples were used to determine malondialdehyde levels using ion-pairing high performance liquid chromatography. Statistical analysis was done using analysis of variance (P<0.05). The content of malondialdehyde in the normal lens was 0.036 +/- 0.017 microg g(-1); in the vitrectomized groups the malondialdehyde concentrations were as follows: (2) 0.027 +/- 0.013 microg g(-1); (3) under detection limit (detection limit=1.75x e-3 microg g(-1)); (4) under detection limit; and (5) 0.020 +/- 0.006 microg g(-1). The results showed that the malondialdehyde concentration in the normal retina was 1.160 +/- 0.361 microg g(-1), while in the vitrectomized groups with or without pretreatment (cyaninosides chloride, chromocarb diethylamine salt, and N -acetylcysteine) the malondialdehyde levels were 2.091 +/- 0.982 microg g(-1), 0.069 +/- 0.024 microg g(-1), 0.082 +/- 0.027 microg g(-1), and 0.215 +/- 0.134 microg/g(-1), respectively, all significantly different from the normal eyes (P<0.05). Vitrectomy induced increased malondialdehyde levels in the retina. Oral flavonoids are an effective protective therapy for surgically induced lipoperoxidation, especially in the retina.