Expressions of matrix metalloproteinases 1 and 3 and their tissue inhibitors in the conjunctival tissue and fibroblasts cultured from conjunctivochalasis

AIM: To investigate the expression of matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3) and their tissue inhibitors of metalloproteinases 1 and 3 (TIMP-1 and TIMP-3) in the conjunctiva of eyes with conjunctivochalasis (CCh). METHODS: The conjunctival tissue was obtained from the CCh patients and controls, the MMPs/TIMPs expression concentration was determined by enzyme-linked immuno-sorbent assay (ELISA) and immunofluorescence staining. The expression levels of MMPs/TIMPs in the CCh fibro-blasts were determined by analyzing its concentration in the cellular supernatant that was abstracted from the in vitro cultured CCh fibroblasts. RESULTS: MMP-1 and MMP-3 levels determined by ELISA were both significantly higher in the CCh group than that in the control group (P=0.042, 0.022, respectively), so was the levels of TIMP-1 (P=0.010). No significant difference in the expression of TIMP-3 in conjunctiva was found between the two groups (P=0.298). The expression of MMP-1 and MMP-3 were both up-regulated significantly in the CCh group (P=0.040, 0.001, respectively) on immuno-fluorescence staining. MMP-1 and MMP-3 expression in the fibroblasts were both significantly higher in the CCh group than that in the control group (P=0.027, 0.001, respectively), while neither the TIMP-1 nor TIMP-3 expression was significantly different between the two groups (P=0.421, 0.237, respectively). CONCLUSION: The overexpression of MMP-1 and MMP-3 in conjunctival tissue and fibroblasts may play an important role in the pathogenesis and development of CCh.     

Role of matrix metalloproteinases in recurrent corneal melting

The aim of this study was to compare the presence and activity of matrix metalloproteinases (MMPs) 1, 2, 3, 7, 8, 9 and 13 in human melted and cadaverous corneas. Twelve melted corneal specimens from three patients with rheumatoid arthritis, one patient with ocular cicatricial pemphigoid and one patient with melting attributed to spastic entropion and ten control corneal buttons were used. The presence of MMPs was detected using indirect enzyme immunohistochemistry. The active forms of MMP-2 and -9 and MMP-3 and -7 were examined by gelatin and casein zymography, respectively. The concentrations of active MMP-1 and -3 were measured using activity assays. Increased immunostaining intensity for MMP-1 and -9 was seen in the corneal epithelium and the anterior stroma of all, and for MMP-2, -3, -7 and -8 of almost all, melted corneas compared to the negative or slightly positive staining of the controls. The posterior stroma showed the presence of MMP-1, -2, -3 and -9 in almost all and of MMP-7 and -8 in half of all melted specimens. A markedly higher level of active MMP-2 was detected in six and active MMP-9 in all of eleven pathologic specimens compared to control specimens, using gelatin zymography. The proenzymes of MMP-3 and -7 and the MMP-7 intermediate cleavage product were detected only in melted corneas using casein zymography. Significantly increased MMP-1 and -3 activity was also found in the melted corneas using activity assays. The markedly increased immunostaining for MMP-1, -2, -3, -7, -8 and -9 as well as the elevated levels of the active forms of MMP-1, -2, -3 and -9 in melted corneal specimens from patients with various diagnoses suggest that although different stimuli may trigger the pathways that lead to the destruction of the extracellular matrix, these enzymes could play a subsequent role in this process.     

基质金属蛋白酶对圆锥角膜的影响

圆锥角膜以进行性角膜前突和变薄,导致不规则散光和视功能损害为特征.大量研究表明,基质金属蛋白酶(matrix metalloproteinases,MMPs)在圆锥角膜基质降解和变薄过程中起关键性作用.紫外光A/核黄素角膜交联术(corneal crosslinking,CXL)是近年来发展起来的唯一能够延缓甚至阻止圆锥角膜病情进展的保守治疗方法.CXL对角膜生物力学和结构的影响研究较多,而对MMPs的影响研究较少.研究表明,CXL后患者泪液中的MMPs出现变化,而对基质中MMPs的影响尚无报道.进一步研究CXL后MMPs的变化有助于理解CXL后圆锥角膜的病理生理进程,以及CXL稳定圆锥角膜病情的作用机制.     

全角膜板层带环形结膜移植治疗严重眼表化学烧伤

目的 探讨全角膜板层带环形结膜移植治疗严重眼表化学烧伤的临床效果。方法 对11例(11眼)严重眼表化学烧伤病例，行全角膜板层带环形结膜移植术治疗并进行回顾性研究，对手术方法、时机、效果进行评估。结果 11例中，10例在术后3～7d上皮愈合，1例12d后才愈合。随访1年，全部眼表稳定，角膜透明或半透明，其中6例周边部有少量血管翳侵入，8例层间基质床有新生血管生长，仅1例眼表及角膜血管纤维化愈合。结论 全角膜板层带环形结膜移植治疗严重眼表化学烧伤，术后早期即可获得较为完整的眼表面，由于供体角膜缘干细胞所提供的新上皮，使术眼获得正常眼表型上皮，维持眼表的稳定和生理功能。主要并发症是移植排斥反应。     

基质金属蛋白酶在角膜疾病中的作用

MMP作为降解细胞外基质的酶家族。参与许多生理及病理过程。同样其在角膜表达水平的改变与许多角膜疾病密切相关。随着对其研究的不断深入,使得从一个新的角度来预防和治疗角膜疾病成为可能。所以十分有必要了解MMPs的结构、功能、生物合成的调节及其在角膜疾病中的作用。     

自体结膜瓣覆盖术治疗深层角膜溃疡

目的：评价自体结膜瓣覆盖术对深层角膜溃疡的治疗效果。

方法：分析于2010-05/2011-10在中国医科大学眼科中心行自体结膜瓣覆盖术的14例深层角膜溃疡患者的预后。14例患者中包括6例病毒性角膜炎患者,7例真菌性角膜炎患者和1例细菌性角膜炎患者。进行结膜瓣遮盖术前,真菌性角膜炎患者用氟康唑冲洗角膜溃疡,细菌和病毒性角膜炎患者用庆大霉素液体冲洗角膜溃疡。根据角膜溃疡大小不同采取部分或全部结膜瓣覆盖术。术后根据不同的角膜炎类型使用不同的抗菌素共4wk。所有的患者术后均随访观察结膜瓣存活情况、并发症及视力恢复情况,随访时间达10～18mo。

结果：所有患者经历1次手术后均痊愈(治愈率为100%)。除1例结膜瓣脱落外,其余患者术后2wk自体结膜瓣均存活并与角膜溃疡处贴附紧密(85.71%)。这13例患者的结膜瓣在随访观察期间内逐渐变得透明并发生了不同程度的退缩。1例真菌性角膜炎患者在术后3mo时角膜炎复发,在使用那他霉素眼液点眼3wk后痊愈。1例患者术后3mo时并发浅前房。1例患者术后10mo并发后弹力层膨出。1例患者术后1a并发了青光眼。所有的患者成功地保留了眼球,但视力提高不明显。

结论：自体结膜瓣覆盖术治疗深层角膜溃疡有效,但是术后长期观察存在一定的并发症。     

几丁聚糖对兔结膜上皮细胞和结膜成纤维细胞增殖的影响

目的　研究几丁聚糖对兔结膜上皮细胞和结膜成纤维细胞增殖的影响。方法　采用消化法培养结膜上皮细胞 ,组织块法培养结膜成纤维细胞。通过MTT比色法和细胞计数法测定几丁聚糖对两种细胞增殖的影响。结果　几丁聚糖在浓度 >0 0 3 %时能促进结膜上皮细胞的增殖 ,浓度为 0 12 %时促进作用最明显。结膜成纤维细胞在几丁聚糖浓度 >0 0 3 %时其增殖即受到抑制 ,抑制作用呈浓度依赖性。结论　几丁聚糖有望用于预防睑球粘连     

眼球筋膜囊修补联合结膜瓣覆盖治疗角膜溃疡穿孔

目的:探讨眼球筋膜囊修补联合结膜瓣覆盖治疗角膜溃疡穿孔的治疗效果。方法:对18例18眼不同病因导致的角膜溃疡穿孔,应用自体眼球筋膜囊对角膜穿孔区进行修补,并行结膜瓣覆盖手术治疗。术后观察溃疡愈合情况、外露的角膜情况、术后炎症反应、前房形成情况及并发症如继发青光眼、虹膜粘连等。结果:溃疡愈合17例(94%),成功保住眼球,并且角膜外露部分透明度良好。所有病例术后均未发生严重感染,前房形成。2例(11%)术后继发青光眼,经局部药物治疗眼压控制在正常范围。7例(39%)虹膜粘连,1例(6%)角膜再次穿孔且继发角膜葡萄肿行球内容物剜除术。结论:在供体角膜缺乏时,眼球筋膜囊修补联合结膜瓣覆盖治疗角膜溃疡安全有效,是一种实用的治疗方法。     

Construction of a full-thickness human corneal substitute from anterior acellular porcine corneal matrix and human corneal cells

AIM: To construct functional human full-thickness corneal replacements. METHODS: Acellular porcine corneal matrix (APCM) was developed from porcine cornea by decellulariztion. The biomechanical properties of anterior-APCM (AAPCM) and posterior-APCM (PAPCM) were checked using uniaxial tensile testing. Human corneal cells were obtained by cell culture. Suspending ring was designed by deformation of an acupuncture needle. MTT cytotoxicity assay was used to check the cytotoxicity of suspending ring soaking solutions. A new three-dimensional organ culture system was established by combination of suspending ring, 48-well plate and medium together. A human full-thickness corneal substitute was constructed from human corneal cells with AAPCM in an organ coculture system. Biochemical marker expression of the construct was measured by immunofluorescent staining and morphological structures were observed using scanning electron microscopy. Pump function and biophysical properties were examined by penetrating keratoplasty and follow-up clinical observations. RESULTS: There were no cells in the AAPCM or PAPCM, whereas collagen fibers, Bowman’s membrane, and Descemet’s membrane were retained. The biomechanical property of AAPCM was better than PAPCM. Human corneal cells grew better on the AAPCM than on the PAPCM. There was no cytotoxicity for the suspending ring soaking solutions. For the constructed full-depth human corneal replacements keratocytes scattered uniformly throughout the AAPCM and expressed vimentin. The epithelial layer was located on the surface of Bowman’s membrane and composed of three or four layers of epithelial cells expressing cytokeratin 3. One layer of endothelial cells covered the stromal surface of AAPCM, expressed Na+/K+ATPase and formed the endothelial layer. The construct was similar to normal human corneas, with many microvilli on the epithelial cell surface, stromal cells with a long shuttle shape, and zonula occludens on the interface of endothelial cells. The construct withstood surgical procedures during penetrating keratoplasty. The corneal transparency increased gradually and was almost completely restored 7d after surgery. CONCLUSION: AAPCM is an ideal scaffold for constructing full-thickness corneal replacement, and functional human full-thickness corneal replacements are successfully constructed using AAPCM and human corneal cells.     

游离结膜瓣移植治疗难治性角膜溃疡疗效观察

目的:评价应用游离结膜瓣移植治疗难治性角膜溃疡的临床疗效。方法:收集2011-07/2012-12经药物治疗无效的感染性角膜溃疡患者8例,采用游离结膜瓣移植手术治疗,术中清除角膜表面溶解坏死组织,上穹隆环钻刻切全厚游离结膜瓣,覆盖角膜病变区域,10-0尼龙线间断缝合8～12针,线结埋于角膜基质,配戴角膜接触镜,术后继续药物治疗原发病。结果:全部患者术后感染控制无复发,游离结膜瓣贴敷良好,缝线拆除后结膜瓣无松脱,眼表规则,泪膜涂布良好,术后视力均不同程度提高。结论:游离结膜瓣移植术治疗难治性角膜溃疡安全有效。     

贝伐单抗抑制角膜新生血管的实验研究

目的评价贝伐单抗（avastin）局部应用对小鼠角膜新生血管（CNV）的抑制作用。方法通过碱烧伤建立CNV模型,将30只Balb／c小鼠随机分成5组,A组贝伐单抗1mg／mL每日点眼2次;B组贝伐单抗3mg／mL每日点眼2次;C组贝伐单抗5mg／mL每日点眼2次;D组0．1％地塞米松每日点眼2次;E组生理盐水每日点眼2次。分别于术后3、7、14d观察CNV情况并拍照。术后第14天,处死全部小鼠,行CNV内皮细胞荧光标记,计算CNV所占全角膜面积的比例。结果各组CNV面积为A组（37．11±3．17）％、B组（29．75±3．56）％、C组（18．76±2．55）％、D组（20．91±2．75）％,E组（41．65±2．11）％。各组小鼠CNV面积依次为c组〈D组〈B组〈A组〈E组,C组同A、B、E组比较差异均有统计学意义（P〈0．01）,c组与D组比较差异无统计学意义（P=0．694）。结论局部应用贝伐单抗对小鼠角膜化学烧伤后的CNV有抑制作用。     

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

AIM: To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix (APCM) in vitro. METHODS: The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin (HE) staining and 4’, 6-diamidino-2-phenylindole (DAPI) staining. Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, and then cell proliferative ability was evaluated by MTT assay. To construct a human corneal anterior lamellar replacement, corneal fibroblasts were injected into the APCM and cultured for 3d, followed by culturing corneal epithelial cells on the stroma construction surface for another 10d. The corneal replacement was analyzed by HE staining, and immunofluorescence staining. RESULTS: Histological examination indicated that there were no cells in the APCM by HE staining, and DAPI staining did not detect any residual DNA. The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells. At 10d, a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed, and the injected corneal fibroblasts distributed within the scaffold. The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of vimentin in the stroma. CONCLUSION: Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo.     

前房冲洗及结膜瓣掩盖治疗化脓性角膜炎

目的探讨伴有前房积脓的化脓性角膜溃疡行前房冲洗联合结膜瓣掩盖术的效果。方法12例药物治疗无效的化脓性角膜溃疡，在处理溃疡面后，以角膜穿刺刀在颞下及鼻下方角膜缘处做两个相对的穿刺1：3，以8U／mL庆大霉素液冲洗前房。前房积脓冲洗干净后，制作桥状结膜瓣，覆盖于溃疡面上，以10—0尼龙缝线固定。前房充气，加压包扎。术后继续用药，10天拆线，14天剪除结膜瓣两端。结果覆盖的结膜瓣萎缩变白形成角膜白斑，周边角膜透明，前房积脓消失。术后视力增加，视力最好者达0．3。结论前房冲洗联合结膜瓣掩盖术治疗药物不能控制的化脓性角膜溃疡，效果良好，并为二期角膜移植创造了条件。此法操作简便，效果确切，不需特殊材料。     

改良式角膜缘干细胞移植治疗原发性翼状胬肉的手术体会

目的:探讨原发性翼状胬肉的手术治疗术式及疗效。方法:回顾2006-08/2009-01,对收住我院的原发性翼状胬肉患者40例43眼,均行改良式角膜缘干细胞移植术,术后观察角膜上皮恢复情况、结膜伤口愈合情况和胬肉复发情况等。结果:本组病例术后随访1～30mo,绝大多数患者角膜上皮和结膜伤口愈合良好,有4例患者术后复发。结论:改良式角膜缘干细胞移植术治疗原发性翼状胬肉复发率低,可以广泛开展,但需要手术者娴熟的技术和足够的耐心。     

结膜瓣遮盖术治疗难治性角膜溃疡

目的探讨应用结膜瓣遮盖术治疗难治性角膜溃疡的临床疗效。方法选取22例(22眼)难治性角膜溃疡患者,其中单纯疱疹病毒性角膜溃疡4眼,真菌性角膜溃疡7眼(发生角膜穿孔2眼),细菌性角膜溃疡8眼,严重的化学烧伤1眼,穿透性角膜移植术后植片溃疡2眼。所有患者均进行局部病灶清除+结膜瓣遮盖术,并针对原发病进一步药物治疗。术后随访3～6个月,观察结膜瓣血供、前房及视力等情况。结果 20眼(包括2眼角膜穿孔眼)角膜溃疡1次手术后治愈;2眼术后结膜瓣溶解脱落,其中1眼细菌性角膜溃疡眼行再次结膜瓣遮盖术后治愈;另1眼真菌性角膜溃疡眼反复2次行结膜瓣遮盖术,感染未控制,最终发生结膜瓣自溶。21眼视力不变或提高,其中光感5眼,数指～0.1者12眼,0.2～0.5者5眼。21眼前房形成良好,眼压基本正常。结论只要手术适应证选择适当、手术操作细致,结膜瓣遮盖术是治疗难治性角膜溃疡的有效方法之一。