维生素E对体外培养的人角膜基质细胞增殖和移行的影响

角膜基质的创伤愈合，尤其是准分子激光屈光性角膜切削术后，引起的角膜上皮下雾状混浊是有害的。用于抑制这一创伤后并发症的药物较多，但由于较严重的毒副作用，从而限制了这些药物的应用。维生素Ｅ作为一种无毒、安全的化合物，已被证明能抑制细胞的增殖。因此，我们观...     

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。     

蛋白激酶C抑制剂对人角膜基质细胞增殖周期的调控作用

目的 探讨蛋白激酶C(PKC)的抑制剂星状孢菌素(ST)对人角膜基质细胞在G1／S时相阶段的调控作用及分子机制。方法 流式细胞仪(FCM)测量PKC抑制剂ST对人角膜基质细胞周期的影响,cyclin／DNA双参数法检测细胞周期蛋白E(cyclin E)蛋白含量。结果 ST使人角膜基质细胞阻滞在G1期,实验组G1期细胞百分比(2．5μg/L ST组为78．4％,5μg/L ST组为90．9％)明显高于对照组(72．4％),并呈现剂量依赖性;cyclin／DNA双参数流式细胞术分析显示ST使G1期细胞cyclin E蛋白水平下降,同时G1期细胞数增加。结论 PKC抑制剂ST能调控人角膜基质细胞的周期,并在G1／S时相发挥其抑制细胞生长的作用。     

增龄对角膜基质载体培养的角膜缘干细胞生物学特性的影响

目的 观察增龄对新西兰兔角膜缘干细胞在同种异体角膜基质上生长、增殖的影响.方法 用酶消化法将不同年龄新西兰兔角膜缘组织制成细胞悬液培养,以相同的细胞密度种植于不同年龄的新西兰兔的角膜基质上,观察角膜缘干细胞在角膜幕质上的生长情况.原位杂交检测ABCG2、ANp63的表达,IPP5.1软件分析图像,流式细胞术测细胞周期,运用SPSS13.0软件对析因设计资料进行方差分析.结果 角膜缘干细胞供体年龄各水平差异有统汁学意义,相同时间内角膜基质培养的青年兔角膜缘干细胞在角膜基质单位面积卜的牛长总而积均较中年和老年高,增殖期细胞所占比例大;而角膜基质供体年龄各水平差异与角膜缘干细胞和角膜基质的交互作用均无统计学意义.结论 在同种异体角膜基质载体上培养的角膜缘干细胞的增殖能力随着年龄的增加而降低.     

MTT法检测转化生长因子-β对人角膜基质细胞增殖的影响

目的探讨转化生长因子-β(TGF-β)对体外培养的人角膜基质细胞增殖的影响.方法体外培养的传2～3代人角膜基质细胞,分别加入0.01,0.1,1,5ng/ml的TGF-β,采用MTT法研究TGF-β对人角膜基质细胞增殖的影响.结果 TGF-β以剂量依赖方式促进人角膜基质细胞增殖,其作用在第2天达到高峰.结论 TGF-β能明显促进人角膜基质细胞增殖,因而可能成为促进角膜溃疡愈合的新方法.     

细胞外基质对角膜创伤修复的作用

细胞外基质在角膜创伤修复过程中起至关重要的作用，其合成代谢受生长因子的调控，两者之间的的关系是是近年来令人感兴趣的新的研究领域，它将有助于阐明角膜创伤修复机制本文概述了细胞外基质的成分、结构、功能和在角膜创伤修复中的作用以及生长因子在务修复过程中对细胞外基质的调控。随着人们对角膜基础研究认识的不断深入，证明角细胞外基质（ＥＣＭ）并非一种被动无活性的结构支架，而是一种活泼的动态物质，它影响着细胞的分     

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。     

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。     

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。     

肝素和干扰素对角膜基质细胞的影响

目的发地求预防或减轻ＰＲＫ术后上皮下浑浊形成的药物。方法 进行兔角膜基质的原代和早期传代培养,并用ＭＴＴ自动比色法检测肝素和干扰素－γ对兔角膜基质细胞增生的影响。结果 肝素浓度≥１０００ｕ／ｍｌ或ＩＦＮ－γ浓度≥５００ｕ／ｍｌ作用４８ｈ和７２ｈ对角膜基质增生均有明显抑制作用。     

Ultrastructure of cultured and cryopreserved human corneal keratocytes.

PURPOSE: To describe the ultrastructural features of cultured and cryopreserved keratocytes. METHODS: Isolated human keratocytes were cultured with 10% fetal calf serum and 10 ng/ml acidic fibroblast growth factor. The 10% Me2SO and 10% human albumin were used as cryoprotective agents. Cells were cooled at 2 degrees C/min, then thawed at 37 degrees C, and subsequently recultured. They were studied by means of transmission electron microscopy (TEM). RESULTS: TEM of cultured keratocytes before cryopreservation showed a network of intact connecting cells. The average cell thickness was 2.4 microm in cross sections and 5.8 microm in frontal sections. The average nuclear thickness was 1.6 microm in cross sections and 3.7 microm in frontal sections. Nuclei appeared regular and oval in cross sections and indented in frontal sections. Organelles were found in greater amounts in frontal sections than in cross sections. Gap junctions, fenestrations along the cell surface, omega-shaped structures, fibrils, and filamentous networks also were found. Most of the just-thawed, suspended cells were elongated and condensed but had intact plasma membranes. These cells were surrounded by a granular material, corresponding to the albumin-containing thawing medium. Scattered isolated round cells displayed nuclear damage, cell edema, loss of organelles, and cell-membrane disruption. By the end of reculture after cryopreservation, cultured keratocytes displayed the same ultrastructural features as before cryopreservation. CONCLUSION: Cultured human keratocytes display many ultrastructural features of in situ keratocytes. These features are still present after reculture after cryopreservation. Cryopreservation induces necrosis in a small percentage of cells, which seems to be related to a relative lack of cell-membrane protection by the cryoprotectants used.     

Influence on cultured human keratocytes by liposome

AIM: To observe the effects on human keratocytes by cationic liposome Lipofectamine^TM2000 (LF2000), to investigate the efficiency and safe range applied in human keratocytes, and establish basis for gene therapy of human keratocytes. METHODS: Human keratocytes cultured in vivo within 3 to 5 passages were used in experiment after being identified. The effects on proliferation of cultured human keratocytes by LF2000 with different concentrations and time were evaluated By MTT; the effects of LF2000 on the survival rate and its relation with 5,10,20,40,80mg/L concentration and time were detected by trypan blue staining. RESULTS: LF2000's effects on human keratocytes were related with concentration and time. The cellular proliferation and survival rate declined when concentration of LF2000 was above certain level, and this effect increased as time became longer. LF2000 had no effect with concentration under 40mg/L for 24 hours. CONCLUSION：LF2000 did not cause cytotoxicity during a concentration range“tested”, and it is hoped to play an important role in gene therapy of human keratocytes.     

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     

Effects of tranilast on cultured rabbit corneal keratocytes and corneal haze after photorefractive keratectomy

PURPOSE: In vitro and in vivo studies were performed to elucidate the effects of tranilast on cellular proliferation and collagen synthesis. METHODS: Subculturing was carried out using keratocytes from rabbits that underwent photorefractive keratectomy (PRK) and developed corneal haze, and keratocytes from normal rabbit cornea. RESULTS: Tranilast suppressed proliferation in cultured keratocytes from the corneal haze region at doses of 30 and 300 micromol/L and collagen synthesis at doses of 3, 30, and 300 micromol/L. Normal corneal cultures showed suppression of keratocyte proliferation and collagen synthesis only at a high dose of tranilast (300 micromol/L). Betamethasone suppressed proliferation of keratocytes in both haze and normal cornea at a dose of 10 micromol/L, as well as collagen synthesis at respective doses of 1 and 10 micromol/L. Diclofenac sodium suppressed collagen synthesis of keratocytes in haze cornea at a high dose of 100 micromol/L, and in keratocytes in normal cornea, at doses of 10 and 100 micromol/L. In an in vivo study, either 0.5% tranilast, 0.1% betamethasone phosphate eye drops, or a tranilast base solution (control) was instilled four times daily to rabbits that had undergone PRK. Weekly evaluation of the inhibitory effect of these drugs on the development of haze was performed 2 weeks after surgery. Tranilast suppressed haze 6-13 weeks after PRK, but betamethasone phosphate showed no effect. CONCLUSION: These results indicate that tranilast is potentially effective for inhibiting the corneal haze that occurs after PRK.     

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 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