体外培养人角膜基质细胞ICAM-1的表达及调节

目的 探讨细胞间附分子－１（ＩＣＡＭ－１）在角膜基质细胞中表达,以及炎症介质对其表达的调节作用。方法 采用细胞培养、免疫细胞化学和流式细胞技术、观察人角膜基质细胞ＩＣＡＭ－１的表达及脂多糖（ＬＰＳ）和干扰素－γ（ＩＦＮ－γ）对此表达的影响。结果 体外２的基质中基础表达一定量的ＩＣＡＭ－１、ＬＰＳ和ＩＦＮ－γ可上调其表达水平至基础表达的１．４－４．６倍。结论角膜炎症反应中,炎症介质促进基质细胞表达Ｉ     

γ—干扰素对人角膜基质细胞I,Ⅱ型胶原合成的抑制作用

初步探讨γ－ＩＦＮ如何抑制人角膜基质细胞Ｉ，Ⅲ型胶原的合成，用体外培养的２－３代人角膜基质细胞在亚汇合状态下分别加入０．５，５，５０，５００和５０００Ｕ／ｍｌ的γ－ＩＦＮ，共培养４８小时，５００Ｕ／ｍｌ，共培养１２，２４，４８小时，地塞米松作为对照，应用Ｉ，Ⅲ型胶原和纤维连接蛋白（Ｆｉｂｒｏｎｅｃｔｉｎ，ＦＮ）ｃＤＮＡ探针行原位杂交检测ｍＲＮＡ的表达。结果表明，Ｉ，Ⅲ型胶原ｍＲＮＡ的表达与γ－ＩＦ     

脱细胞猪角膜基质体外支持角膜上皮和基质细胞的生长

目的:探讨脱细胞猪角膜基质体外能否支持兔角膜细胞的生长。方法:体外培养兔角膜上皮细胞和基质细胞,并接种到制备的脱细胞猪角膜基质上,倒置相差显微镜和组织学观察细胞生长情况。结果:上皮细胞能在脱细胞猪角膜基质上贴附生长,10d时可形成2~3层的复层结构。基质细胞在脱细胞猪角膜基质上贴附生长后可向材料深层迁徙。结论:制备的脱细胞猪角膜基质体外可支持兔角膜上皮细胞和基质细胞的生长。     

Verapamil对角膜基质细胞影响的研究

目的：观察Ｖｅｒａｐａｍｉｌ（异搏定）对体外培养角膜基质细胞的影响,为其临床应用提供基础。方法：进行兔角膜基质细胞的原代和早期传代培养,并用ＭＴＴ自动比色法检测Ｖｅｒａｐａｍｉｌ对兔角膜基质细胞增殖的影响。结果：Ｖｅｒａｐａｍｉｌ浓度１０～１０００μｇ／ｍｌ作用４８ｈ和７２ｈ对角膜基质细胞增殖有明显抑制作用（Ｐ＜０．０５）。作用７２ｈ抑制率高于作用４８ｈ。结论：Ｖｅｒａｐａｍｉｌ为剂量依赖型和时间依赖型药物,能有效地抑制角膜基质细胞增殖,可望成为调节角膜伤口愈合的新药物。     

房水对培养的角膜基质细胞的影响

目的观察房水对角膜基质细胞生长的作用。方法将新西兰大白兔的角膜去除后弹力层、内皮层和上皮层后,得到基质层,用组织块培养法体外培养角膜基质细胞。在实验组的培养基中加入10％房水,对照组使用常规培养基培养。通过CCK8实验测得角膜基质细胞的吸光度（A）值以分析细胞增生的情况。分别在培养基中加入2．5％、5％、10％、15％、20％的房水,用明胶酶谱法检测基质金属蛋白酶（MMPs）的活性。结果倒置显微镜观察可见培养的角膜基质细胞呈多角形或树枝状,与对照组相比,实验组的细胞生长状态良好,培养的角膜基质细胞数量明显增加。明胶酶谱法显示实验组的条带比对照组清晰。实验第1～5天分别测角膜基质细胞的A值,实验组的检测条带明显强于对照组,〉10％的房水培养组检测的条带明显强于2．5％、5％房水培养组。CCK8检测表明,培养1～5d实验组的角膜基质细胞A值明显高于对照组,差异均有统计学意义（P〈0．05）。结论房水作用于角膜基质细胞后,可促进角膜基质细胞的生长,10％房水对体外培养角膜细胞有促细胞增生的作用。     

角膜基质细胞对淋巴细胞活化的抑制效应

建立人、大鼠和兔角膜基质细胞的原代和传代２。在基质细胞单层上观察了淋巴细胞对丝裂原刺激的增殖反应和单向混合淋巴细胞反应，并进一步分析了基质细胞与细胞培养上清对淋巴细胞活化的影响。结果显示：不同种系来源的角膜基质细胞和细胞上清对小鼠淋巴细胞具有同样的免疫抑制效应，提示：角膜细胞以及产生的可溶怀免疫抑制介质对于角膜移植片免受免疫活性细菌识别和攻击，以及长期存活可能起关一定的作用。     

角膜基质细胞生物学活性与角膜病变

角膜基质细胞是位于角膜基质层中平行排列的胶原纤维板潜在空隙内的扁平细胞。在正常的角膜组织中,角膜基质细胞处于相当稳定的状态。然而,在角膜病变中,角膜基质细胞在发病机制、病理过程及疾病转归中扮演了重要的角色。     

Peters异常：角膜基质中角膜细胞分化及细胞外基质异常

Peters异常以角膜内皮和Descemet膜的缺陷为特征，二与角膜中央的透明性及角膜与晶状体和虹膜的前表面粘连有关。大约50％的病例有前房角发育异常，多为双眼发病。据报道，干扰视神经嵴细胞的迁移和分化有可能导致Peters异常。这种眼部异常可联合全身发育异常，可能是由于在胚胎发育过程中神经嵴细胞机能障碍所致。     

脂质体对人角膜基质细胞影响的实验研究

目的:观察脂质体LipofectamineTM2000对人角膜基质细胞的影响,探索其应用于人角膜基质细胞的可行性及安全范围。方法:体外培养人角膜基质细胞,取其第3-5代细胞鉴定后用于实验。采用MTT法检测不同浓度和时间脂质体对人角膜基质细胞增殖率的影响;采用台盼蓝染色法检测对存活率的影响。结果:脂质体对人角膜基质细胞的影响与浓度和作用时间有关。浓度高于一定水平时可引起细胞增殖率和存活率的下降,浓度相同时作用时间越长下降越明显。浓度低于40mg/L作用24h不会对细胞增殖率和存活率产生明显影响。结论:LipofectamineTM2000在一定范围内不引起细胞毒性,有望在角膜基质细胞的基因治疗中发挥重要作用。     

角膜基质细胞研究进展

角膜基质细胞,存在于角膜基质层中,在正常情况下,处于静止状态。但当角膜损伤时,不同上皮来源的因子及环境信号,将影响角膜基质细胞的应答反应,决定着角膜能否完全被修复或形成角膜瘢痕。本文就角膜基质细胞的分布、形态、受激后的应答反应情况及与角膜疾病的关系作一综述。     

In vitro effects of antiviral agents on human keratocytes

PURPOSE: To study the effects of antiviral agents on human keratocytes in vitro. METHODS: Cultured human keratocytes were incubated with either ganciclovir, idoxuridine, trifluridine, or cidofovir at concentrations from 0.0001 to 10 mg/mL. Phase-contrast microscopy and XTT (sodium [2,3-bis [2-methoxy-4-nitro-5-sulphophenyl]-2h-tetrazolium-5-carboxanilide, inner salt) colorimetric assay were performed after 24, 48, and 72 hours of incubation. RESULTS: When adjustments were made for time of incubation and concentration, trifluridine reduced cell viability significantly more than ganciclovir, idoxuridine, and cidofovir (p<0.001, three-way analysis of variance). There was significant time-and dose-dependent reduction of cell viability (p<0.001) with trifluridine and cidofovir. After a 72-hour incubation with ganciclovir or idoxuridine, cell viability was reduced as compared with 24- and 48-hour incubation (p<0.001); only the effects of the highest concentration tested (1.0 mg/mL) were significantly different from those of the lower concentrations (p<0.002). At a concentration of 1.0 mg/mL, trifluridine and cidofovir produced moderate to severe signs of cytotoxicity, whereas ganciclovir and idoxuridine displayed much less severe morphologic signs. CONCLUSIONS: Our results indicate that antiviral agents may have both time- and concentration-related toxic effects on stromal keratocytes. These findings may impact the selection of the most appropriate antiviral drug when it is needed to treat infections involving the corneal stroma.     

In vitro effects of fluoroquinolone and aminoglycoside antibiotics on human keratocytes

PURPOSE: The purpose of this study was to assess the cytotoxic effects of the fluoquinolone ofloxacin and the aminoglycoside netilmicin on stromal human keratocytes in vitro. METHODS: Cultured human keratocytes were exposed to various concentrations of ofloxacin or netilmicin (0.16-5.0 mg/mL). Both cell proliferation (MTT assay) and cell morphology (phase-contrast microscopy) were evaluated after 1, 4, 12, and 24 hours of incubation. Measurement of annexin V binding performed in association with the dye exclusion test using propidium iodide (PI) was also performed by FACS analysis after 4 hours of exposure. RESULTS: Both antimicrobials induced dose- and time-dependent morphologic changes in keratocytes, yet the effects of netilmicin were minimal. After 24 hours of exposure, both drugs induced a dose-dependent inhibition of cell proliferation; however, ofloxacin demonstrated significantly more toxic effects than netilmicin (t test for ED50 values, P < 0.0001). Statistical differences between 2 antibiotics start at concentrations above 1.25 mg/mL (ANOVA with post-hoc test, P < 0.01). Expression of the apoptotic marker annexin V was unaffected by antibiotic exposure, whereas the uptake of the necrotic marker PI was increased by ofloxacin (5 mg/mL) but not by netilmicin (ofloxacin versus netilmicin, ANOVA, P < 0.05). CONCLUSIONS: Relative effects of aminoglycosides and fluoroquinolones on stromal keratocytes appear to be different: netilmicin was shown to be significantly less toxic than ofloxacin. This finding is particularly relevant in deciding the optimal antibiotic to be applied in clinical situations in which the epithelium is absent or compromised, as after photorefractive keratectomy, alkali burns, or ulcerative keratitis.     

The effects of organ-culture on the density of keratocytes and collagen fibers in human corneas

PURPOSE: Keratocytes are important in regaining corneal transparency during wound healing after surgery or trauma. Hitherto, there are still controversies concerning the effects of organ culture on the density and integrity of keratocytes and collagen fibers. The current study aimed at a systematic analysis of the effects of organ-culture on the morphology and density of keratocytes and collagen fibers. METHODS: Human corneas were organ-cultured in MEM for 7 (n = 17, 3 pairs), 14 (n = 18, 9 pairs) and 21 days (n = 18, 9 pairs). Of the pairs one cornea was processed in swollen condition and the fellow cornea after reversal of swelling in MEM plus Dextran. Eleven post-mortem corneas (PM) and 11 fresh corneas obtained from melanoma patients were used as controls. Stromal thickness, number of keratocyte profiles (corrected for swelling), number and diameter of collagen fibers were measured in light microscopical sections and electron micrographs. RESULTS: Stromal swelling due to organ-culture resulted in large keratocyte profiles with many vacuoles and large distances between collagen fibers in the posterior stroma. In contrast both keratocytes and distances between collagen fibers were not affected in the anterior stroma. After reversed-swelling the posterior corneal stroma was similar to that in fresh controls, indicating that the swelling process is largely reversible. The initial decrease in keratocyte density (18%) in the early post-mortem period did not progress during 21 days of organ culture. CONCLUSION: With respect to the morphology and density of keratocytes and collagen fibers it can be concluded that donor corneas remain suitable for transplantation up to at least 21 days after organ-culture.     

Toxic effects of mitomycin-C on cultured corneal keratocytes and endothelial cells.

Improper use of mitomycin-C in ocular medication may result in damage to corneal cells. In this study, the toxic effects of mitomycin-C on cultured porcine keratocytes and endothelial cells were estimated by MTT, 3H-thymidine uptake and cellular counting assay methods. It was found that mitomycin-C caused a dose-dependent toxic effect to keratocytes and endothelial cells. Both cells were treated with mitomycin-C at the concentration ranging from 100, 10, 1, 0.1 to 0.01 microg/ml for 3 min, 5 min or 100 min. The 50% inhibitory dose (ID50) of mitomycin-C to keratocytes and endothelial cells as measured by MTT assay was 0.40, 0.18, 0.16 mg/ml and 0.27, 0.15, 0.14 mg/ml, respectively, after 3, 5 and 100 minutes drug treatment. The ID50 for keratocytes and endothelial cells as measured by 3H-thymidine uptake immediately, 1 day and 7 days after 100 minutes mitomycin-C treatment was 0.3, 0.0002, 143.2 microg/ml and 45.1, 101.1, 450.2 microg/ml, respectively. The ID50 for keratocytes and endothelial cells as measured by cellular counting 1 day and 7 days after mitomycin-C treatment was 232.5, 109.7 microg/ml and 239.9, 367.5 microg/ml, respectively. It is concluded that mitomycin-C is more toxic to cellular proliferation in cultured corneal keratocytes than in endothelial cells.     

The effects of various levels of intraocular pressure on the rabbit's outflow system

The effects of various levels of intraocular pressure on the morphology of the rabbit outflow system were investigated. In a total of 20 rabbits, intraocular pressure was maintained at various levels between 0–50 mmHg in the experimental eye and at 20 mmHg in the control eye for 1 hr. Flow rates into the eyes were measured by following the movement of a miniscus through a calibrated capillary tube.Progressive increase in pressure lead to distension and enlargement of the ciliary cleft. The meshwork tissues stretched and distended, and the vessels of the angular aqueous plexus had pressure-sensitive giant vacuoles in their endothelium. These vessels were prone to closure at pressures of 30 mmHg and greater. Difficulties associated with quantitation of giant vacuoles in this species were discussed.     

The effects of growth factors and conditioned media on the proliferation of human corneal epithelial cells and keratocytes

• Background: As growth factors play an important role in epithelial wound repair, we evaluated the effect of exogenous growth factors in the presence and absence of corneal epithelial and keratocyte conditioned medium on human corneal epithelial cell and keratocyte proliferation. • Methods: Preconfluent cultures of human corneal epithelial cells or stromal keratocytes were exposed to varying concentrations of EGF, TGF-β or bFGF in the presence or absence of human corneal epithelial or stromal keratocyte conditioned medium. Cell numbers were determined after 48 h incubation. RIA and ELISA were used to quantify the levels of EGF, TGF-β and bFGF in conditioned media. • Results: EGF and bFGF increased, while TGF-β decreased, the proliferation of both cell types in a dosedependent manner. Epithelial cell conditioned medium inhibited, and keratocyte conditioned medium stimulated, the proliferation of both cell types. The proliferative effects of EGF, TGF-β and bFGF in the presence of keratocyte conditioned medium were additive for both cell types. By contrast, the addition of exogenous growth factors was unable to overcome the inhibitory potential of epithelial conditioned medium. Both conditioned media contained significant levels of bFGF, but TGF-β levels in epithelial conditioned medium were up to 5 times greater than that in keratocyte conditioned medium. • Conclusions: The results indicate that corneal cells maintain tissue homeostasis and modulate the wound healing response via paracrine/autocrine pathways.     

The effects of growth factors and conditioned media on the proliferation of human corneal epithelial cells and keratocytes

Background: As growth factors play an important role in epithelial wound repair, we evaluated the effect of exogenous growth factors in the presence and absence of corneal epithelial and keratocyte conditioned medium on human corneal epithelial cell and keratocyte proliferation. • Methods: Preconfluent cultures of human corneal epithelial cells or stromal keratocytes were exposed to varying concentrations of EGF, TGF-β or bFGF in the presence or absence of human corneal epithelial or stromal keratocyte conditioned medium. Cell numbers were determined after 48 h incubation. RIA and ELISA were used to quantify the levels of EGF, TGF-β and bFGF in conditioned media. • Results: EGF and bFGF increased, while TGF-β decreased, the proliferation of both cell types in a dose- dependent manner. Epithelial cell conditioned medium inhibited, and keratocyte conditioned medium stimulated, the proliferation of both cell types. The proliferative effects of EGF, TGF-β and bFGF in the presence of keratocyte conditioned medium were additive for both cell types. By contrast, the addition of exogenous growth factors was unable to overcome the inhibitory potential of epithelial conditioned medium. Both conditioned media contained significant levels of bFGF, but TGF-β levels in epithelial conditioned medium were up to 5 times greater than that in keratocyte conditioned medium. • Conclusions: The results indicate that corneal cells maintain tissue homeostasis and modulate the wound healing response via paracrine/autocrine pathways. Received: 6 February 1997 Revised version received: 2 April 1997 Accepted: 10 April 1997