抗环血酸抑制胶原酶活性的实验研究

本研究首次在体外观察了抗坏血酸对胶原酶活性的抑制作用。结果表明：抗坏血酸浓度达56．7mmol／L有明显抑制胶原酶活性作用；浓度至85mmoll／L在本反应体系中完全抑制胶原酶活性，加热破坏抗坏血酸则失去抑制作用。这将进一步理解抗坏血酸在治疗角膜碱烧伤及角膜溃疡中的作用。     

胶原酶活性的测定方法介绍——氨基酸分析法

胶原酶活性的测定方法介绍——氨基酸分析法王一徐毅孙伟（第三军医大学西南医院眼科，重庆６３００３８）胶原酶在结缔组织的正常代谢及病理损害中起着重要作用，对于胶原酶的研究国内报道很少。而在国外，对于胶原酶活性的测定已报道众多的方法，如粘度法、同位素法、荧...     

半胱氨酸治疗角膜溃疡的实验研究

文献上曾谈到硷烧伤性角膜溃疡由于胶原溶解酶的释放造成角膜板层溶解和穿孔。任何方式的角膜上皮创伤都将释放出蛋白溶解酶,上皮是胶原溶解酶的来源。钙离子是胶原溶解酶活性的必需因素。半胱氨酸通过两个方面抑制胶原溶解酶活性。第一,直接作用于胶原溶解酶分子,还原酶分子中的二硫键。该作用不可逆;第二,和EDTA一样,通过络合钙离子间接抑制溶解酶的作用。该作用是可逆的。所以半胱氨酸是最好的胶原溶解酶抑制     

人多核白细胞胶原酶潜酶活化的实验研究

对人多核白细胞被激活后释放胶原酶潜酶的活化机制进行了研究。结果表明：激活的多核白细胞可释放明显的胶原酶活性；ＳＯＤ和ＣＡＴ显著抑制释放的潜酶活化，但不影响潜酶的释放。表明活性氧参与胶原酶潜酶的活化，而且最终氧化物为羟自由基（ＯＨ）。     

胶原在眼部的应用

胶原是人体的主要结构蛋白。它具有抵抗原性，良好的组织相容性和可吸收性。胶原具有止血，促伤口愈合功能。角膜胶原膜是良好的药物载体，它不难能起到营养、保护、促进角膜伤口愈合的作用，而且能运载、释放各种药物，在治疗眼科多种疾病上可替代局部频繁点眼和结膜下注射，并在青光眼、白内障术后起到抗纤维增生作用。角膜胶原还是活细胞转运的载体和基因治疗的载体。胶原可作为植入物治疗各种眼病，在整形美容领域也有一定作用。总之，胶原在眼部的应用越来越广泛。     

抗氧化剂治疗角膜溃疡对胶原酶活性影响的观察

目的观察抗氧化剂治疗对角膜组织中胶原酶活性的影响。方法以细菌性角膜溃疡为模型，测定不同抗氧化剂治疗后角膜组织中丙二酰二醛（ｍａｌｏｎｄｉａｌｄｅｈｙｄｅ，ＭＤＡ）含量和胶原酶活性。结果抗氧化剂联合抗菌素治疗较单用抗菌素能更有效地减少组织脂质过氧化和活性胶原酶生成。结论从动物实验角度证明了抗氧化剂治疗对胶原酶活化的影响，并提示抗氧化剂是一种理想的辅助用药，而且应联合用药。     

恶性肿瘤的抑癌基因治疗研究进展

越来越多的证据表明,肿瘤的发生是多因素作用的结果,与癌基因的激活与抑癌基因的失活有关。研究表明几乎一半人类肿瘤都存在抑癌基因的失活,可见抑癌基因失活与肿瘤生长有着密切的关系。因此,将正常的抑癌基因导入肿瘤细胞,去补偿和代替突变或缺失的抑癌基因治疗策略,将成为肿瘤基因治疗中一种重要的治疗模式。就抑癌基因ｐ５３,ＤＣＣ,ｎｍ２３,ＴＩＭＰ,ｐ２１,ｐ１６,ｂｃｌ－Ｘ等在恶性肿瘤基因治疗方面的研究进展作一综述。     

角膜溃疡胶原酶潜酶氧化活化的动物实验研究

目的观察角膜溃疡氧自由基的产生与胶原酶活化的关系。方法以兔角膜溃疡为模型，用ＳＯＤ＋ＣＡＴ联合滴眼治疗和生理盐水滴眼对照１０天后测定角膜组织ＭＤＡ和胶原酶活性。结果治疗组角膜组织中ＭＤＡ及胶原酶活性均显著低于对照组，并在角膜组织匀浆上清中加入胶原酶潜酶活化剂（ＡＰＭＡ）后，对照组胶原酶活性无变化，而治疗组则显著提高，但总酶活力仍低于对照组。结论在体内氧自由基参与胶原酶潜酶活化；也表明氧自由基的清除可减少释放的胶原酶总量。     

胶原在眼部的应用

胶原是人体的主要结构蛋白。它具有低抗原性 ,良好的组织相容性和可吸收性。胶原具有止血 ,促伤口愈合功能。角膜胶原膜是良好的药物载体 ,它不仅能起营养、保护、促进角膜伤口愈合的作用 ,而且能运载、释放各种药物 ,在治疗眼科多种疾病上可替代局部频繁点眼和结膜下注射 ,并在青光眼、白内障术后起到抗纤维增生作用。角膜胶原膜还是活细胞转运的载体和基因治疗的载体。胶原可作为植入物治疗各种眼病 ,在整形美容领域也有一定作用。总之 ,胶原在眼部的应用越来越广泛。     

眼科的抑癌基因

一、抑癌基因的概念抑癌基因的概念最初是由 Kundson于 1985年在对视网膜母细胞瘤的研究中发现并提出的 ,是指当这类基因发生突变、缺失或失活时可引起细胞恶性转化而导致肿瘤的发生。 1986年 Sager将抑癌基因的概念扩大 ,认为所有产生可抑制肿瘤生成和发展的因素都可称为抑癌基因。并将其分为 4类 :1癌基因产物的拮抗物 ,2干扰素 ,3生长抑素 ,4控制染色体稳定的因素。随着研究的深入 ,抑癌基因的概念逐渐被缩窄 ,新的抑癌基因的概念是 :抑癌基因是一大类可抑制细胞生长并能潜在抑制癌变作用的基因群 ,它仅在某一种特定的细胞内起作用。确…     

几种胶原酶抑制剂滴眼液的效果比较

目的 观察比较６种眼科外用药物，为临床筛选较强的抑制胶原酶的眼液。方法 用鼠胶原１、胶原酶及考马斯亮蓝比色法来检测药物抑制胶原酶的能力。结果 ６种药物有不同程度抑制胶原酶作用。其中卡托普利极强、乙酰半胱氨酸和依地酸钠较强，而枸橼酸钠、硫代硫酸钠和四环素较差。结论 ０．５ｇ．Ｌ＾－１卡托普利眼液抑制胶原酶能力极强、副作用小、刺激性极微、药物来源及配制方便。     

Tetracyclines and the treatment of corneal stromal ulceration: a review

PURPOSE: To demonstrate the potential value of tetracyclines in the treatment of corneal ulceration after moderate to severe ocular chemical injuries. METHODS: Review of published materials describing landmarks in the development of tetracyclines as matrix metalloproteinase inhibitors in ophthalmology and related disciplines. RESULTS: Tetracyclines can protect the cornea against proteolytic degradation after moderate to severe ocular chemical injury. They inhibit matrix metalloproteinases by mechanisms independent of their antimicrobial properties, primarily through restriction of the gene expression of neutrophil collagenase and epithelial gelatinase, suppression of alpha1-antitrypsin degradation, and scavenging of reactive oxygen species. CONCLUSION: Oral tetracyclines can be used along with topical tetracycline preparations and other therapeutic agents to inhibit collagenolytic degradation of the cornea after moderate to severe ocular chemical injuries.     

Regulation of collagenase activity in the ulcerating cornea by cyclic-AMP.

Dibutyryl cyclic-AMP (DBcAMP) and theophylline partially inhibit the appearance of collagenase activity when added to cultures of ulcerating rabbit corneas. Drug treatment with DBcAMP also inhibits degradation of collagen in the explanted corneas as measured by hydroxyproline in the media. The hydrolysis product of DBcAMP, 5′-adenosine monophosphate (5′ AMP) is itself very effective in suppressing collagenase activity and the degradation of explant collagen. The mechanism(s) of the drug effects is not yet known but it is possible that the drugs affect the synthesis and/or the secretion of collagenase. The results suggest that “first messengers” exist which can prevent the secretion of corneal collagenase by raising endogenous cAMP in corneal cells through stimulation of the adenyl cyclase system. Eventual successful treatment of corneal ulceration might well require pharmacologic intervention at multiple levels: the biosynthesis, secretion, activation and activity of collagenase.     

Characterization of human corneal collagenase

Collagenolytic activities have been detected in the culture media from an eye bank cornea and in media from corneal tissues biopsied at the time of surgery. The eye bank cornea was not pathological but was not used for corneal grafting because of the donor age (77). The corneal biopsies were from keratoplasty patients with no history of corneal ulceration. Enzymes from both sources cleave tropocollagen at the same site as do tadpole-tail collagenase and rabbit corneal collagenases, and, because of the restricted cleavage, belong to the class of enzymes known as “tissue collagenases”. The human corneal collagenase activities also degrade reconstituted collagen fibrils. The collagenase activity(ies) from human keratoplasty tissues is inhibited by Calcium-EDTA and N-acetyl-l-cysteine, both of which have been found previously to prevent ulceration in the alkali-burned rabbit cornea and to inhibit collagenases produced by the ulcerating rabbit cornea. The serum antiprotease, α₂-Macroglobulin, has also been found to inhibit the collagenase(s) from keratoplasty tissues, an observation which supports the ophthalmologist's sometime contention that serum inhibits corneal ulceration. It is hoped that the human corneal collagenase(s) produced by the culture of keratoplasty tissues and of eye bank corneas judged unfit for corneal grafting will facilitate the discovery of effective inhibitors of corneal ulceration.     

Inhibition of collagenase activity by extracts of bovine ocular tissues

Bovine eyes were dissected and separate pools of lens, lens capsule, cornea and vitreous were extracted in guanidine, subjected to ultrafiltration, and examined for their effects on collagenolytic activity. Although lens extract was not inhibitory, the cornea and vitreous both contained inhibitors of collagenase. More inhibition was present in the filtrate of the vitreous than in the retentate, whereas the total amount of inhibition in the cornea was distributed almost equally between the two fractions. The inhibition observed was dose dependent. The partially purified inhibitors from cornea and vitreous blocked the activity of human skin and tadpole back skin collagenases, but they failed to inhibit the bacterial (Clostridium histolyticum) collagenase. The inhibitor was stable to heating to 60 degrees for 30 minutes and to trypsinization.     

Corneal ulceration and the serum antiproteases. II. Complexes of corneal collagenases and alpha-macroglobulins.

Previous work has demonstrated that whole rabbit or human serum and the human α₂-macroglobulin (Hα₂-m) inhibit corneal collagenases. In the present study, the ability of rabbit and human serum to cause rabbit corneal collagenase, mol. wt 45 000, to elute in high molecular weight fractions from sieving columns is taken as presumptive evidence for the formation of collagenase-serum protein complexes. The recovery of increased collagenase activity by thiocyanate treatment of serum effluent fractions containing the human α₂-macroglobulin indicates that it is the α₂-m in human serum that complexes rabbit corneal collagenase. This conclusion is supported by the demonstration that purified Hα₂-m transports the rabbit corneal collagenase through a molecular sieve. Moreover, the chromatography of day one culture media from ulcerating rabbit corneas has demonstrated the presence of a significant amount of the total collagenase activity in the high molecular weight (850 000 – 1 000 000) fractions in which rabbit α-macroglobulin (Rα₁-m) was also demonstrated. These observations support the hypothesis that the α-macroglobulins play an important role in the regulation of corneal collagenase activity.Crossed-gel immunoelectrophoretic methods have shown that rabbit and human corneal collagenase preparations perturb the patterns of their respective α-macroglobulins. The perturbed patterns are taken as evidence for the formation of collagenase-α-macroglobulin complexes. The application of crossed-gel methods to the tears of human ulcer patients shows the utility of such methods for examining the status of α₂-m in tears.     

The collagen shield as a collagenase inhibitor and clinical indicator of collagenase activity on the ocular surface.

Collagen shields applied to the corneas of patients with bacterial keratitis degrade rapidly, often within a few hours. Once treatment brings the infection under control, subsequently applied collagen shields degrade more slowly. In vitro models were established to evaluate the significance of these observations. Twenty-four and 72-hour collagen shields were incubated with collagenase from Clostridium histolyticum. The in vitro rate of digestion of the shields was directly proportional to the concentration of collagenase, with the rate of digestion of the 24-hour shields being greater than that of the 72-hour shields. Therefore, the rate of collagen shield degradation may be a clinically useful index of collagenase activity on the ocular surface. Ultrastructural studies of collagen shields from patients with acute bacterial keratitis revealed irregular degradation of shield matrix with no evidence of adherence of microorganisms or inflammatory cells. Co-incubation of deepithelialized rabbit corneas and collagen shields resulted in inhibition of the digestion of the rabbit corneas when the weight:weight ratio of collagen shield:rabbit cornea was increased to greater than or equal to 2:1. Collagen shields may inhibit corneal collagen degradation in infectious ulceration and melting disorders by effectively competing for collagenase on the ocular surface.     

Collagenase from corneal cell cultures and its modulation by phagocytosis.

The uptake of latex by fibroblasts in confluent primary culture results in the secretion of collagenase at a linear rate for a prolonged period. Phagocytosis might therefore constitute an important level of collagenase regulation in corneal ulceration. The collagenase in cell cultures is present in a latent form (40,000 MW) like that obtained from organ cultures of ulcerating corneas and can be activated proteolytically. Production of the latent collagenase in cell culture depends upon the presence of serum and diminishes greatly when serum is removed from the medium. Collagenase activity can be demonstrated after the latent collagenase has been separated from serum antiproteases in the media. Alternatively, careful titration of the crude media with trypsin to saturate serum antiproteases, to release collagenase from the complex with alpha 2-macroglobulin, and to activate latent collagenase also results in measurable collagenase activity. The collagenase that is secreted cleaves fibrillar type I collagen and cleaves soluble type I collagen into the typical 3/4 and 1/4 length fragments, as demonstrated by SDS-gel electrophoresis and electron microscopy.     

Inhibition of purified collagenase from alkali-burned rabbit corneas

The inhibitory potency of four classes of compounds that inhibit corneal ulceration (thiols, tetracyclines, sodium citrate and sodium ascorbate) was assessed with collagenase purified from culture medium of alkali-burned rabbit corneas. The most potent inhibitor, a beta-mercaptomethyl tripeptide HSCH2(DL)CH[CH2CH(CH3)2]CO-Phe-Ala-NH2, exhibited 50% inhibition (IC50) at approximately 10 nM using the synthetic metalloproteinase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2. The inhibitor was somewhat less potent with type 1 collagen as substrate (IC50 between 1 and 3 microM), possibly because autooxidation of the essential - SH moiety of the inhibitor occurred during the longer time required for assay with the natural substrate. An N-carboxyalkyl tripeptide, CH3(CH2)2(DL)CH-(COOH)-Leu-Phe-Ala-NH2, was less potent (IC50 = 25 microM) than the thiol peptide. N-acetylcysteine, which is used to treat corneal ulceration, gave IC50 values of 2.7 mM and less than 10 mM with the synthetic and natural substrates, respectively. The IC50 values for the tetracyclines using the synthetic substrate were 15, 190 and 350 microM for doxycycline, minocycline and tetracycline, respectively. Inhibition by sodium citrate, but not the tetracyclines, could be reversed by excess Ca2+. Sodium ascorbate did not inhibit collagenase-mediated hydrolysis of either collagen or the synthetic substrate, thus indicating that the mechanism by which this agent inhibits corneal ulceration is not related to inhibition of collagen degradation by collagenase.