Decellularized human cornea for reconstructing the corneal epithelium and anterior stroma

In this project, we strived to develop a decellularized human cornea to use as a scaffold for reconstructing the corneal epithelium and anterior stroma. Human cadaver corneas were decellularized by five different methods, including detergent- and nondetergent-based approaches. The success of each method on the removal of cells from the cornea was investigated. The structural integrity of decellularized corneas was compared with the native cornea by electron microscopy. The integrity of the basement membrane of the epithelium was analyzed by histology and by the expression of collagen type IV, laminin, and fibronectin. Finally, the ability of the decellularized corneas to support the growth of human corneal epithelial cells and fibroblasts was assessed in vitro. Corneas processed using Triton X-100, liquid nitrogen, and poly(ethylene glycol) resulted in incomplete removal of cellular material. Corneas processed with the use of sodium dodecyl sulfate (SDS) or with sodium chloride (NaCl) plus nucleases successfully removed all cellular material; however, only the NaCl plus nuclease treatment kept the epithelial basement membrane completely intact. Corneas processed with NaCl plus nuclease supported both fibroblast and epithelial cell growth in vitro, while corneas treated with SDS supported the growth of only fibroblasts and not epithelial cells. Decellularized human corneas provide a scaffold that can support the growth of corneal epithelial cells and stromal fibroblasts. This approach may be useful for reconstructing the anterior cornea and limbus using autologous cells.     

Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in treatment of corneal alkali burns in mice

We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in a corneal alkali burn model in mice. An alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. SN50 (10 microg/microl) or vehicle was topically administered daily for up to 12 days. The eyes were processed for histological or immunohistochemical examination after bromodeoxyuridine labeling or for semi-quantification of cytokine mRNA. Topical SN50 suppressed nuclear factor-kappaB activation in local cells and reduced the incidence of epithelial defects/ulceration in healing corneas. Myofibroblast generation, macrophage invasion, activity of matrix metalloproteinases, basement membrane destruction, and expression of cytokines were all decreased in treated corneas compared with controls. To elucidate the role of tumor necrosis factor (TNF)-alpha in epithelial cell proliferation, we performed organ culture of mouse eyes with TNF-alpha, SN50, or an inhibitor of c-Jun N-terminal kinase (JNK) and examined cell proliferation in healing corneal epithelium in TNF-alpha-/- mice treated with SN50. An acceleration of epithelial cell proliferation by SN50 treatment was found to depend on TNF-alpha/JNK signaling. In conclusion, topical application of SN50 is effective in treating corneal alkali burns in mice.     

Structural characterization of the mesangial cell type IV collagenase and enhanced expression in a model of immune complex-mediated glomerulonephritis.

Secretion of glomerular cell-derived matrix metalloproteinases (MMPs) and their specific inhibitors, TIMP-1,2, may play an important role in the turnover of the glomerular extracellular matrix under basal and pathologic conditions. A 66-68 kd MMP secreted by cultured mesangial cells (MC) with activity against Type IV collagen and gelatin was purified and shown by amino-acid sequence analysis to be identical with a Type IV collagenase/gelatinase secreted by certain transformed tumor cell lines. The expression of the mesangial MMP in vivo was limited within the kidney to a small subset of the intrinsic glomerular mesangial cell population. After induction of acute anti-Thy 1.1 glomerulonephritis, there was a large increment in the number of Type IV collagenase-secreting MC, temporally coincident with the development of mesangial hypercellularity. The expression of the MMP inhibitor protein, TIMP-1, was not changed over this period. Ultrastructural studies localized the mesangial MMP to areas of evolving mesangiolysis and at sites of glomerular basement membrane disruption. Enhanced expression of the mesangial cell-derived Type IV collagenase may contribute to the evolution of glomerular injury in this model of immune complex-mediated glomerulonephritis or may be involved in the extensive matrix remodeling process that accompanies this form of glomerular injury.     

Reconstructed human cornea produced in vitro by tissue engineering.

The aim of the present study was to produce a reconstructed human cornea in vitro by tissue engineering and to characterize the expression of integrins and basement membrane proteins in this reconstructed cornea. Epithelial cells and fibroblasts were isolated from human corneas (limbus or centre) and cultured on plastic substrates in vitro. Reconstructed human corneas were obtained by culturing epithelial cells on collagen gels containing fibroblasts. Histological (Masson's trichrome staining) and immunohistological (laminin, type VII collagen, fibronectin as well as beta1, alpha3, alpha4, alpha5, and alpha6 integrin subunits) studies were performed. Human corneal epithelial cells from the limbus yielded colonies of small fast-growing cells when cultured on plastic substrates. They could be subcultured for several passages in contrast to central corneal cells. In reconstructed cornea, the epithelium had 4-5 cell layers by the third day of culture; basal cells were cuboidal. The basement membrane components were already detected after 3 days of culture. Integrin stainings, except for the alpha4 integrin, were also positive after 3 days. They were mostly detected at the epithelium-stroma junction. Such in vitro tissue-engineered human cornea, which shows appropriate histology and expression of basement membrane components and integrins, provides tools for further physiological, toxicological and pharmacological studies as well as being an attractive model for gene expression studies.     

脱细胞猪角膜基质的基底膜在修复兔角膜损伤中的作用

目的 探讨脱细胞角膜基质支架材料表面保存基底膜样结构对修复角膜损伤的必要性. 方法 将采用反复冻融及酶消化法制备的脱细胞猪角膜基质,移植到兔角膜损伤模型上,通过大体及组织学分别观察材料表面基底膜结构的有无对修复兔角膜基质损伤的影响. 结果 脱细胞猪角膜基质材料表面有基底膜的实验组（n=8）兔角膜损伤全部修复,无穿孔,支架材料与受体角膜整合.材料表面无基底膜的对照组（n=8）中,有6例植入的支架材料溶解,兔角膜穿孔.两组结果比较差异有显著统计学意义. 结论 脱细胞角膜基质支架材料表面具有基底膜样结构,有利于形成正常角膜上皮屏障,从而防止了生物材料的过快降解,有利于材料与宿主基质的整合和改建.     

The Role of Hypoxia in Corneal Extracellular Matrix Deposition and Cell Motility

The cornea is an excellent model tissue to study how cells adapt to periods of hypoxia as it is naturally exposed to diurnal fluxes in oxygen. It is avascular, transparent, and highly innervated. In certain pathologies, such as diabetes, limbal stem cell deficiency, or trauma, the cornea may be exposed to hypoxia for variable lengths of time. Due to its avascularity, the cornea requires atmospheric oxygen, and a reduction in oxygen availability can impair its physiology and function. We hypothesize that hypoxia alters membrane stiffness and the deposition of matrix proteins, leading to changes in cell migration, focal adhesion formation, and wound repair. Two systems—a 3D corneal organ culture model and polyacrylamide substrates of varying stiffness—were used to examine the response of corneal epithelium to normoxic and hypoxic environments. Exposure to hypoxia alters the deposition of the matrix proteins such as laminin and Type IV collagen. In addition, previous studies had shown a change in fibronectin after injury. Studies performed on matrix-coated acrylamide substrates ranging from 0.2 to 50 kPa revealed stiffness-dependent changes in cell morphology. The localization, number, and length of paxillin pY118- and vinculin pY1065-containing focal adhesions were different in wounded corneas and in human corneal epithelial cells incubated in hypoxic environments. Overall, these results demonstrate that low-oxygenated environments modify the composition of the extracellular matrix, basal lamina stiffness, and focal adhesion dynamics, leading to alterations in the function of the cornea. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.     

Implication of extracellular matrix metalloproteinases in the course of chronic inflammatory airway diseases

Matrix metalloproteinases (MMPs) are major proteolytic enzymes that are involved in extracellular matrix (ECM) turn over. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) cleave type IV collagen, which is an important constituent of basement membrane. These enzymes play an important role in normal tissue homeostasis, but imbalance between MMPs and their tissue inhibitors (TIMPs) is thought to be a critical factor in regulating tissue remodeling. MMP-2 is produced by fibroblasts, endothelial, and epithelial cells, while MMP-9 is mainly produced by inflammatory cells. The role of MMPs was investigated through biochemical analysis or in situ expression, in the pathogenesis of two chronic inflammatory airway diseases, asthma and nasal polyposis. Both are characterized with the accumulation of active inflammatory cells, matrix remodeling and epithelial changes. Increased levels of MMP-9 and TIMP-1 were found in asthmatic subjects and NP. In NP, MMP-9 expression was detected in epithelial, endothelial and inflammatory cells. In this setting, MMP-9 could play a crucial role in the transmigration of basement membrane components by inflammatory cells leading to inflammatory cell accumulation and maintenance of inflammation in airway. Moreover, MMP-9 may contribute to cell migration, an important mechanism involved in the repair of the respiratory epithelium.     

Matrix metalloproteinase 9 (gelatinase B) is expressed in multinucleated giant cells of human giant cell tumor of bone and is associated with vascular invasion.

Human giant cell tumor (GCT) consists of multinucleated giant cells and mononuclear stromal cells, and is characterized by frequent vascular invasion without distant metastases. To study the role of matrix metalloproteinases (MMPs) in the vascular invasion, we examined production of MMP-1 (tissue collagenase), -2 (gelatinase A), -3 (stromelysin-1), -9 (gelatinase B), and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in GCT. MMP-9 was highly and predominantly expressed in giant cells by both immunohistochemistry and in situ hybridization. Expression of other MMPs was also observed in some cases but was inconstant. Sandwich enzyme immunoassays demonstrated that MMP-9 is the predominant MMP secreted by GCT. There was a definite imbalance between the amounts of MMP-9 and those of TIMPs in the culture media of GCT, leading to detectable gelatinolytic activity in an assay using 14C-gelatin. Gelatin zymography demonstrated the main activity at about 90 kd, which was identified as the zymogen of MMP-9 by immunoblotting. Immunohistochemistry for type IV collagen and laminin, major basement membrane components, showed that disappearance of the proteins is closely associated with MMP-9-positive giant cells. These results indicate the production of MMP-9 by multinucleated giant cells and suggest that the metalloproteinase may contribute to proteolysis associated with vascular invasion and local bone resorption in human GCT.     

Regulation of the biosynthesis of macromolecules of the intercellular corneal matrix]

The influence of a 1M CaCl2 extract and of a collagenase digest of corneal stroma of the biosynthesis of the macromolecules of corneal stroma was investigated. Calf corneas were incubated "in vitro" with radioactive tracers (14C-L-proline; 3H-D-glucosamine or 35SO4) in the presence or absence of the above extracts. After incubation the corneas are submitted to a fractional extraction in order to separate the major macromolecular fractions of the stroma. An increase in incorporation of all tracers is observed in the 1M CaCl2 (CTC), TCA and urea-extracts (containing resp. the diffusible macromolecules, proteoglycans, polymeric collagen and structural glycoproteins) in the presence of the macromolecular extracts and also with the collagenase-hydrolysate of cornea. These results show the existence of a stimulation of the biosynthesis of intracellular matrix macromolecules of the cornea by corneal extracts, probably through positive "feedback" type of mechanism.     

辐照灭菌冷冻干燥羊膜修复碱烧伤

背景：经辐照灭菌的冷冻干燥羊膜便于保存和使用,但羊膜经这种方式处理后其活性是否会降低、其临床疗效是否会受到影响至今仍鲜有报道。 目的：观察辐照灭菌后的冷冻干燥羊膜对兔角膜碱烧伤的修复作用。 方法：取健康成年白兔9只,均用1 mol/L 氢氧化钠烧伤角膜后,左侧眼设为羊膜组植入辐照灭菌后的冷冻干燥的羊膜,右侧眼设为对照组不植入羊膜。植入后30 d内裂隙灯显微镜每日观察角膜透明度、角膜新生血管及角膜上皮修复情况。 结果与结论：羊膜植入后30 d,羊膜组角膜透明度、新生血管及角膜上皮修复情况明显优于对照组(P < 0.05),角膜上皮愈合好,基质纤维排列更为整齐,炎细胞浸润程度轻,新生血管更少。结果证实,兔角膜碱烧伤后植入辐照灭菌后的冷冻干燥羊膜可减轻角膜炎性反应,促进角膜上皮修复,减少角膜新生血管形成。     

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。     

The involvement of matrix metalloproteinases in basement membrane injury in a murine model of acute allergic airway inflammation

BACKGROUND: Airway remodelling in asthma such as subepithelial fibrosis is thought to be the repair process that follows the continuing injury as of chronic airway inflammation. However, how acute allergic inflammation causes tissue injury in the epithelial basement membrane in asthmatic airways remains unclear. Matrix metalloproteinases (MMPs) capable of degrading almost all of the extracellular matrix components have been demonstrated to be involved in cell migration through the basement membrane in vivo and in vitro. OBJECTIVE: We investigated the alterations of matrix construction and the role of MMPs in matrix degradation in the subepithelium during acute allergic airway inflammation. METHODS: Airway inflammation, the ultrastructure of the subepithelium and injury of types III and IV collagen in tracheal tissues from ovalbumin (OVA)-sensitized mice after OVA inhalation with or without the administration of tissue inhibitor of metalloproteinase-2 (TIMP-2) and dexamethasone were evaluated by cell counting in bronchoalveolar lavage (BAL) fluids, electron microscopy and immunohistochemistry, respectively. RESULTS: The disruption of the lamina densa and matrix construction and the decrease of the immunoreactivity for type IV collagen in subepithelium were observed in association with the accumulation of inflammatory cells in airways 3 days after OVA inhalation. This disorganization of the matrix components in the subepithelium, as well the cellular accumulation, was abolished by the administration of TIMP-2 and dexamethasone. The immunoreactivity for type IV collagen in the subepithelium in OVA-inhaled mice returned to the level of that in saline-inhaled mice 10 days after inhalation in association with a decrease of the cell numbers in the BAL fluid. The immunoreactivity for type III collagen was changed neither 3 nor 10 days after OVA inhalation. CONCLUSION: These results suggest that epithelial basement membrane gets injured by, at least in part, MMPs as a consequence of cell transmigration through the membrane during acute allergic airway inflammation.     

Rabbit corneal damage produced by Pseudomonas aeruginosa infection.

Gross, light microscopic, and electron microscopic examination of the rabbit corneal destruction produced by experimental Pseudomonas aeruginosa infections revealed a combination of acute inflammation and liquefaction necrosis of the cornea. Degeneration of the epithelial cells and the start of polymorphonuclear leukocyte infiltration of the cornea occurred initially. These changes were followed by loss of the epithelium, degeneration and loss of the keratocytes and endothelium, loss of the characteristic weblike pattern of the proteoglycan ground substance, dispersal of ultrastructurally normal collagen fibrils, extensive accumulation followed by degeneration of polymorphonuclear leukocytes, and accumulation of plasma proteins and fibrin in the necrotic cornea. Histochemical examination of the cornea suggested a loss of the proteoglycan ground substance but not of collagen. Rabbit corneas injected with Clostridium histolyticum collagenase showed gross and cellular changes similar to those observed during the pseudomonal infections; however, histochemical examination suggested a loss of collagen, and electron microscopy revealed ultrastructurally abnormal collagen fibrils. The results support the idea (i) that a bacterial or host-derived collagenase is not required for extensive corneal damage during a P. aeruginosa corneal infection, and (ii) that a P. aeruginosa corneal infection may severly damage the cornea by producing extensive corneal edema and by causing the loss of the corneal proteoglycan ground substance, thus resulting in dispersal of undamaged collagen fibrils, weakening of the cornea, and subsequent descemetocele formation and corneal perforation by the anterior chamber pressure.     

Amniotic membrane immobilized poly(vinyl alcohol) hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium

Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis.     

Gelatinase A expression and localization in human breast cancers. An in situ hybridization study and immunohistochemical detection using confocal microscopy

The gelatinase A (72 kDa type IV collagenase) is a matrix metallo-proteinase which degrades basement membrane collagens. Various studies emphasize its role in stromal invasion of cancers, but there is some controversy about its origin. Gelatinase A was localized by immunohistochemistry using confocal microscopy in 15 human mammary carcinomas. In addition, the cells responsible for the synthesis of this enzyme were detected by in situ hybridization. Most invasive and non-invasive tumour cells were labelled by immunohistochemistry. Of particular interest was the pattern observed in some pre-invasive areas. Gelatinase A was found in fibroblasts in close contact with pre-invasive tumour clusters. Confocal observation allowed a more precise localization of gelatinase A to the periphery of tumour clusters along the basement membranes and in peritumour fibroblasts. The malignant epithelial cells were negative by immunohistochemistry in these areas. By in situ hybridization, mRNAs encoding gelatinase A were detected only in fibroblasts in close contact with pre-invasive and well differentiated tumour clusters. These findings support the hypothesis that peritumour fibroblasts produce gelatinase A and that breast cancer cells may bind this enzyme to their cell surface and/or internalize it.     

Tissue inhibitor of metalloproteinase-1 moderates airway re-epithelialization by regulating matrilysin activity

Obliterative bronchiolitis (OB) is the histopathological finding in chronic lung allograft rejection. Mounting evidence suggests that epithelial damage drives the development of airway fibrosis in OB. Tissue inhibitor of metalloproteinase (TIMP)-1 expression increases in lung allografts and is associated with the onset of allograft rejection. Furthermore, in a mouse model of OB, airway obliteration is reduced in TIMP-1-deficient mice. Matrilysin (matrix metallproteinase-7) is essential for airway epithelial repair and is required for the re-epithelialization of airway wounds by facilitating cell migration; therefore, the goal of this study was to determine whether TIMP-1 inhibits re-epithelialization through matrilysin. We found that TIMP-1 and matrilysin co-localized in the epithelium of human lungs with OB and both co-localized and co-immunoprecipitated in wounded primary airway epithelial cultures. TIMP-1-deficient cultures migrated faster, and epithelial cells spread to a greater extent compared with wild-type cultures. TIMP-1 also inhibited matrilysin-mediated cell migration and spreading in vitro. In vivo, TIMP-1 deficiency enhanced airway re-epithelialization after naphthalene injury. Furthermore, TIMP-1 and matrilysin co-localized in airway epithelial cells adjacent to the wound edge. Our data demonstrate that TIMP-1 interacts with matrix metalloproteinases and regulates matrilysin activity during airway epithelial repair. Furthermore, we speculate that TIMP-1 overexpression restricts airway re-epithelialization by inhibiting matrilysin activity, contributing to a stereotypic injury response that promotes airway fibrosis via bronchiole airway epithelial damage and obliteration.     

Action at a distance: systemically administered adult stem/progenitor cells (MSCs) reduce inflammatory damage to the cornea without engraftment and primarily by secretion of TNF-α stimulated gene/protein 6

Previous reports demonstrated that the deleterious effects of chemical injury to the cornea were ameliorated by local or systemic administration of adult stem/progenitor cells from bone marrow referred to as mesenchymal stem or stromal cells (MSCs). However, the mechanisms for the beneficial effects of MSCs on the injured cornea were not clarified. Herein, we demonstrated that human MSCs (hMSCs) were effective in reducing corneal opacity and inflammation without engraftment after either intraperitoneal (i.p.) or intravenous (i.v.) administration following chemical injury to the rat cornea. A quantitative assay for human mRNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) demonstrated that less than 10 hMSCs were present in the corneas of rats 1-day and 3 days after i.v. or i.p. administration of 1 × 10(7) hMSCs. In vitro experiments using a transwell coculture system demonstrated that chemical injury to corneal epithelial cells activated hMSCs to secrete the multipotent anti-inflammatory protein TNF-α stimulated gene/protein 6 (TSG-6). In vivo, the effects of i.v. injection of hMSCs were largely abrogated by knockdown of TSG-6. Also, the effects of hMSCs were essentially duplicated by either i.v. or topical administration of TSG-6. Therefore, the results demonstrated that systemically administered hMSCs reduce inflammatory damage to the cornea without engraftment and primarily by secretion of the anti-inflammatory protein TSG-6 in response to injury signals from the cornea.     

92-kd gelatinase is actively expressed by eosinophils and stored by neutrophils in squamous cell carcinoma.

Tumor invasion and metastasis are assisted by multiple proteinases that degrade basement membrane and stromal matrix components. We used in situ hybridization with 35S-labeled RNA probes and immunohistochemistry to localize cellular sites of 92-kd gelatinase production in sections of invasive squamous cell carcinoma. Signal for enzyme messenger RNA was detected only in numerous eosinophils that surrounded the tumor nodules, and immunohistochemical staining verified the presence of enzyme protein in these granulocytes and also revealed strong reactivity in neutrophils. No resident or other migratory cell type was positive for gelatinase messenger RNA or protein, and no signal was detected by either assay in samples of healthy skin. These data indicate that eosinophils have the capacity to synthesize actively 92-kd gelatinase, whereas neutrophils store and probably release the enzyme on demand. Because of the capacity of 92-kd gelatinase to degrade both basement membrane and interstitial extracellular matrix molecules, the expression, delivery, and secretion of this metalloproteinase by granulocytes may be critical for tumor invasion.     

Differences in proliferation and invasion by normal,transformed and NF1 Schwann cell cultures are influenced by matrix metalloproteinase expression

Loss of negative growth regulation and high invasive potential are neoplastic traits often associated with abnormal expression of matrix metalloproteinases (MMPs). We previously found MMP-3 (stromelysin/transin) was secreted by quiescent rat Schwann cell cultures and expressed potent antiproliferative activity. In the present study we observed that human Schwann cells and cutaneous neurofibroma Schwann cell cultures secreted abundant MMP-3 and their proliferation was inhibited by autologous and rat Schwann cell conditioned media. Antiproliferative activities were depleted by immunoadsorption with anti-stromelysin antibodies. In contrast, plexiform neurofibroma cultures did not secrete MMP-3 and failed to respond to Schwann cell antiproliferative activities associated with MMP-3. Quiescent Schwann cells constitutively secreted low levels of MMP-2 (gelatinase A) and showed a low invasion potential in filter-based assays of basement membrane invasion. Cyclic AMP elevation, which profoundly influences cell differentiation, increased the invasion potential of rat Schwann cells and caused a corresponding increase in secretion of MMP-2. Schwann cells immortalized by protracted elevation of CAMP, as well as a schwannoma cell line (D6P2T), also rapidly invaded a reconstituted basement membrane and over-expressed MMP-2. Similarly, neurofibroma Schwann cells were highly invasive and secreted up to 10-fold more MMP-2 than normal human Schwann cells. Additionally, only cutaneous neurofibroma Schwann cell cultures secreted MMP-9 (gelatinase B) and MMP-1 (interstitial collagenase) and also invaded native type I collagen barriers. Cultures of normal Schwann cells and plexiform neurofibroma tumor expressed little or no MMP-1 and did not invade type I collagen barriers. These results suggest a role for MMPs in the control of proliferation and invasion by Schwann cells and in the formation of peripheral nerve sheath tumors.     

脱细胞猪角膜表面基底膜成分的检测

背景：前期实验显示脱细胞猪角膜具有良好的组织相容性,可以支持角膜细胞和皮肤上皮细胞的生长。 目的：检测脱细胞猪角膜是否保存了利于角膜上皮细胞生长的重要组织结构—基底膜。 方法：利用荧光抗体对脱细胞猪角膜表面的基底膜成分(层粘蛋白和Ⅳ型胶原)进行免疫组织化学检测,荧光显微镜下观察脱细胞猪角膜表面是否保存了基底膜成分。 结果与结论：免疫荧光染色显示脱细胞猪角膜前基质表面层粘蛋白和Ⅳ型胶原呈阳性表达,与新鲜猪角膜表面基底膜的荧光表达相同,表明脱细胞猪角膜保存了利于角膜上皮细胞生长的基底膜。