复方中药金芪滴眼剂对兔角膜上皮及基质细胞增殖的影响

目的：观察复方中药金芪滴眼剂对兔角膜上皮细胞及兔角膜基质层细胞增殖的影响。方法：建立体外培养兔角膜上皮细胞，兔角膜基质层细胞系，用MTT法检测不同浓度金芪滴眼剂对细胞增殖活性的影响。结果：金芪滴眼剂对兔角膜上皮细胞有显著的促进增殖作用。且随浓度的增加。增殖率加大，对兔角膜基质层细胞无促增殖作用。高浓度时有抑制作用。结论：复方中药金芪滴眼剂对兔角膜上皮细胞有较好的保护作用。     

Interferon production and sensitivity of rabbit corneal epithelial and stromal cells

The induction of interferon and the ability of interferon to induce the antiviral state were studied using rabbit corneal epithelial and stromal cells which were cultured for fewer than five passages. Interferon titers in the range of 7000 units/ml were induced in epithelial cell cultures and 76,000 units/ml in stromal cell cultures treated with UV-inactivated bluetongue virus. The interferon induced was stable to pH 2.0 treatment and heating to 56 degrees C for 16 hr. Infection of epithelial and stromal cell cultures with various strains of herpes simplex virus type 1 showed that all strains tested replicated to equivalent titers in the respective cell types, and that no detectable interferon was induced in stromal cells and only trace amounts in epithelial cells. Exogenously supplied rabbit interferon induced the antiviral state in cultures of both cell types restricting the replication of not only encephalomyocarditis virus but also herpes simplex virus. Sixty to ninety units of rabbit interferon reduced HSV-1 virus replication by 50%. Human interferons had less than 27% of the antiviral activity in rabbit cells than they had in a human cell line. The data indicate that exogenously supplied interferon may act to reduce the severity of herpetic keratitis by directly inducing the antiviral state in corneal epithelial and stromal cells. However, interferon endogenously produced by rabbit corneal cells in response to HSV-1 infection probably plays a minor role in the pathogenesis of ocular HSV-1 infections.     

细胞因子对体外牛眼小梁细胞生成SPARC的调节

目的:了解房水中细胞因子对体外培养牛眼小梁细胞合成富含半胱氨酸的酸性分泌蛋白(secretedprotein,acidicandrichincysteine,SPARC)的影响。方法:体外培养牛眼小梁细胞并传3代,应用免疫组化染色法验证SPARC存在于小梁细胞内,并将传3代的小梁细胞无血清培养24h后分别加入房水中的细胞因子bFGF,TGF-β,EGF及IGF-1,72h后提取细胞上清液,并用酶联免疫吸附法(ELISA)测定SPARC的浓度。结果:细胞因子bFGF,TGF-β不影响小梁细胞对SPARC的合成,EGF和IGF-1分别减少和增加小梁细胞对SPARC的合成。结论:细胞因子可通过影响SPARC的生成而调节眼内压。     

Injured corneal epithelial cells promote myodifferentiation of corneal fibroblasts

PURPOSE: To determine whether injured corneal epithelial cells stimulate myodifferentiation in corneal fibroblasts and whether transforming growth factor (TGF)-beta is involved. METHODS: Rabbit corneal fibroblasts were cultured on collagen gel, with or without cocultured corneal epithelial cells or with partially scraped epithelial cells, on a companion plate separated by a permeable membrane. To evaluate fibroblast-induced gel contraction, gel thickness was measured daily relative to the original thickness. Total fibroblasts on the gel were counted. Myofibroblasts were counted by using immunocytochemical identification with anti-alpha-smooth muscle actin (alpha-SMA). TGF-beta was assayed in the media on days 3 and 6. These procedures also were performed in the presence of anti-TGF-beta antibody. RESULTS: Gel contraction, alpha-SMA-positive cells, and total cell number were significantly greater on gels with injured epithelial cells than on gels without epithelial cells or with uninjured epithelial cells, as was TGF-beta concentration in the media. Anti-TGF-beta antibody eliminated these differences. CONCLUSIONS: Injured epithelial cells stimulate myodifferentiation in fibroblasts through one or more soluble factors, including TGF-beta.     

The expression of matrix metalloproteinases and their inhibitors in corneal fibroblasts by alarmins from necrotic corneal epithelial cells

Purpose

Sterile ulceration is frequently observed in the cornea following persistent corneal epithelial damage. We examined the effect of alarmins released by necrotic corneal epithelial cells (HCE) on the production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by corneal fibroblasts.

Methods

IL-1α and high-mobility group box 1 protein (HMGB1) released into the supernatant derived from necrotic HCE cells were measured with enzyme-linked immunosorbent assay (ELISA). MMPs and TIMPs produced by corneal fibroblasts, stimulated with the supernatant from necrotic HCE cells, were analyzed and measured with protein array and ELISA. To investigate dynamic expression of alarmins in the corneal epithelium, we used immunohistochemistry to observe the expression of human IL-1α in the corneal epithelium of human IL-1α Tg mice with or without cryopexy. We also investigated the expression of MMPs in corneal stroma of the mice treated with cryopexy, using RT-PCR.

Results

We detected IL-1α and HMGB-1 in the supernatant of necrotic HCE cells. These supernatants increased the expression of MMP-3 and MMP-1, and decreased that of TIMP-1 and TIMP-2 in human corneal fibroblasts. Almost always these were inhibited by IL-1 receptor antagonist. Recombinant IL-1α increased the production MMP-3 and MMP-1 in corneal fibroblasts. After cryopexy of the epithelium of human IL-1α Tg mice, the expression of human IL-1α was recognized in the cytoplasm but not nucleus of epithelial cells. The level of MMP-3 and MMP-1 mRNAs was elevated in the corneal stroma in mice treated with cryopexy.

Conclusion

Alarmins, especially IL-1α, released from necrotic HCE cells may play an important role in the expression of MMPs and TIMPs by corneal fibroblast, resulting in sterile ulceration.

     

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.     

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     

人角膜基质细胞诱导人骨髓间充质干细胞分化为角膜上皮细胞的初步研究

目的 探讨人骨髓间充质干细胞（MSC）体外分化为角膜上皮细胞的可行性.方法 实验研究.分别取第3代人MSC和自行培养的第3代人角膜基质细胞共同培养1周,实验组在Transwell共培养体系中培养,对照组不放置Transwell小室培养.1周后,观察实验组和对照组中人MSC光镜特征、间接免疫细胞化学染色和电镜结构,对被诱导分化的细胞进行综合鉴别.结果 第3代人MSC和人角膜基质细胞在体外培养条件下均能够较快贴壁生长.两种细胞共同培养1周后,可见部分细胞形态上呈上皮细胞特征,单克隆抗体AE1染色呈阳性,电镜下可见微绒毛、桥粒和张力丝等典型上皮细胞结构特征.结论 体外培养的人MSC在人角膜基质细胞的诱导下可能会分化为人角膜上皮样细胞.     

The mechanisms of cell membrane resealing in rabbit corneal epithelial cells

PURPOSE: To examine membrane repair mechanisms in rabbit corneal epithelial (RCE) cells. METHODS: Microneedle puncture and fluorescent dye loss were used to wound membranes and assay resealing, respectively. Different repair mechanisms were detected pharmacologically and with antisense oligonucleotides. RESULTS: The RCE cells rapidly reseal plasma membranes by calcium-dependent exocytotic mechanisms that exhibit both facilitated and potentiated responses to multiple wounding. The facilitated response was inhibited by specific inhibitors of protein kinase C (PKC) and brefeldin A, and the potentiated response was blocked by inhibitors of cAMP-dependent protein kinase (PKA). Reduction of myosin IIA inhibited the facilitated response, and reduction of IIB inhibited the initial resealing. CONCLUSIONS: RCE cells rapidly repair plasma membrane disruptions. At a second wound at the same site, PKC stimulated vesicle formation from the Golgi apparatus, resulting in more rapid membrane resealing for a facilitated response. The RCE cell also contains a PKA-dependent global potentiation of membrane resealing.     

β1整合素过表达促进体外兔角膜上皮细胞黏附和迁移

目的:探讨β1整合素过表达对角膜上皮细胞黏附和迁移的影响机制。方法:将β1整合素-GFP融合蛋白真核细胞重组表达质粒转染兔角膜上皮细胞,观察转染细胞的融合基因表达以及细胞的黏附和迁移能力。检测β1整合素转染对角膜上皮细胞粘着斑激酶(FAK)磷酸化的影响。结果:成功将β1整合素-GFP融合蛋白转染至兔角膜上皮细胞并使其过表达;β1整合素过表达能够明显增加角膜上皮细胞的黏附和迁移能力(P<0.05)并促进FAK磷酸化(P<0.05)。结论:β1整合素过表达能够明显促进角膜上皮细胞的黏附和迁移。     

Serum-free culture of porcine and rabbit corneal epithelial cells

To better define the growth requirements for corneal epithelial cells, methods for serum-free culture were established. Sheets of corneal epithelium from eyes of swine and rabbits were obtained by dispase treatment of corneal buttons, and single cells and small clumps were obtained by further dissociation with trypsin/EDTA. Growth of cells with epithelial-like morphology was readily achieved in low calcium MCDB 153 medium containing epidermal growth factor, insulin, hydrocortisone, and bovine pituitary extract. Primary cultures could be subcultivated at least four times at 1:6 split ratios. Examination of cultured corneal epithelial cells by transmission electron microscopy demonstrated a relatively undifferentiated phenotype. Elevation of calcium in the medium caused the reappearance of desmosomal junctions and a more typical corneal epithelial morphology.     

Inhibitory effect of diclofenac sodium on the proliferation of rabbit corneal epithelial cells in vitro

Purpose:To investigate the inhibitory effect of diclofenac sodium on rabbit corneal epithelial cells(RCECs)in vitro and explore its pharmacological mechanism.Methods: The fresh rabbit cornea was cultured to get the primary RCECs,and RCECs of passage 2 were used for the research.The cells were divided into experimental groups,the cells in which were incubated with different concentrations(18.18,27.27,36.36,45.45,54.55 μg/ml)of dielofenac sodium,and control group.The effect of diclofenac sodium on the proliferation of cells was measured by methyl thiazolyl thiazolium(MTT)assay 24,48 and 72 h after incubation.While the RCECs were divided into experimental groups,the cells in which were incubated with 9 and 12.5μg/ml diclofenac sodium,and control group.The cell cycle and apoptotic rate were observed by flow cytometer.Results:MIT assay showed that diclofenac sodium had obvious inhibitory effect on RCECs,and the inhibition rate was increasing along with the increase of the concentration of diclofenac sodium and the incubation time(P<0.05).Flow cytometer showed that after incubation with dielofenac sodium,the cells in G0/G1 phase were obviously increased,the apoptosis cusp and apoptotic rate were increased.Conclusion: Diclofenac sodium has obvious inhibitory effect on RCECs,which was dosage-dependent,and it may function by inducing cell apoptosis and ceasing cells cycles.     

角膜缘上皮细胞体外培养、冻存和复苏的实验研究

目的 建立兔角膜缘上皮细胞体外培养、冻存和复苏的方法。方法 兔角膜缘上皮细胞组织块接种培养。取第三代细胞进行冻存。于冻存后第2周，3、6个月复苏细胞。用MTT法测细胞生长曲线。结果 兔角膜缘上皮细胞体外生长良好。培养细胞AE1单克隆抗体染色阳性，PAS染色阴性。冻存细胞复苏成功。冻存细胞复苏后生长曲线良好。结论 兔角膜缘上皮细胞可以在体外培养、冻存和复苏。     

Growth and characterization of rabbit corneal cells in vitro

Primary organ cultures of rabbit corneal epithelium, stroma, and endothelium were established by microdissection. Secondary cultures of epithelial cells, keratocytes, and endothelial cells were established by serial passage. The doubling time for epithelial cells and keratocytes was 18 h, and endothelial cells doubled their number in 5 days. Ultrastructural studies demonstrated characteristic morphological, nuclear, and cytoplasmic features of corneal epithelial cells, keratocytes, and endothelial cells and confirmed the identity of the cell lines. The purity of the three distinct cell types was ascertained by indirect immunofluorescence techniques, using antibodies against keratin, which identified epithelial cells, and fibronectin, which identified keratocytes and endothelial cells. The indirect immunofluorescence technique represents a simple method to screen an aliquot of a cell suspension and determine the purity of corneal cells grown in vitro.     

Cytokine regulation of C3 and C5 production by human corneal fibroblasts.

Recent investigations have suggested that cytokines play important roles during inflammation and host defense, primarily by regulating the diverse functions of immunologic cells (e.g. lymphocytes and monocytes). However, much less is known about the capacity of cytokines to also regulate the functions of resident tissue cells. We hypothesize that during inflammation, cytokines (e.g. monokines and lymphokines) directly regulate the expression of inflammatory precursors and mediators, such as the third and fifth complement components, by resident ocular cells and are therefore important in the local regulation of ocular inflammation. To test this hypothesis we developed an in vitro culture system utilizing isolated human corneal fibroblasts and examined the effects of specific cytokines, i.e. interleukins and interferons, on the production of the third and fifth components of the complement system. Human corneal fibroblasts were cultured in the presence of varying concentrations (1-500 U ml-1) of interleukin 1 alpha, interleukin 1 beta, interleukin 2, interferon alpha and interferon gamma for 48 hr at 37 degrees C, 5% CO2. The supernatants were then evaluated for antigen levels for the third and fifth components of complement using specific enzyme-linked immunospecific assays. These studies revealed that both interleukin 1 alpha and interleukin 1 beta induced seven to tenfold increases in the levels of the third component. Similarly interferon alpha and interferon gamma stimulated an approximate four and ninefold dose-dependent increase, respectively, in the production of the third component. Analysis of the effect of interleukin 2 on third component production demonstrated that higher concentrations (100 U ml-1) were required to induce a fivefold increase in the production of the third component.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-adrenergic and serotonergic responsiveness of rabbit corneal epithelial cells in culture

Rabbit corneal epithelial cell cultures were established from Dispase-treated anterior corneas. In culture medium containing cholera toxin, insulin and epidermal growth factor, these cells proliferated in vitro in the absence of any contaminating cells. Following subculture, cells retained epithelial morphology and the ability to synthesize cAMP in response to beta-adrenergic stimulation, but lacked the ability to respond to serotonergic stimulation. Retention of the beta-adrenergic system in culture serves as a functional epithelial cell marker; whereas expression of serotonergic responsiveness may be regulated by developmental or extrapithelial systems that are absent in these cell cultures.     

Movements of cultured corneal epithelial cells and stromal fibroblasts in electric fields

The effects of an externally applied direct-current electric field on the movement of cultured rabbit corneal epithelial cells and stromal fibroblasts were studied. After a latency of approximately 20 minutes in an electric field, both epithelial cells and stromal fibroblasts became spindle shaped and underwent galvanotropism by aligning their long axes perpendicular to the applied electric field. The electric field stimulus thresholds for galvanotropic movements in epithelial cells and stromal fibroblasts were 4V/cm and 6 V/cm, respectively. After an additional latency of 30 minutes, both cell types manifested galvanotaxic movements: epithelial cells commenced migration in the cathodal (downfield) direction and stromal fibroblasts in the anodal (upfield) direction. For both types of cells, ruffled membranes and lamellipodia were abundant at the leading edges of migrating cells and cell processes underwent retraction at the trailing edges. At field strengths of above 10 V/cm, evidence of cellular damage (manifested by cellular rounding and detachment), attributable to the electric field treatment, was observed after 4 hours. These preliminary results suggest that galvanotaxic responses could be exploited clinically in the enhancement of corneal wound healing.