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。     

体外培养人角膜基质细胞β1整合素的表达

目的探讨β1整合素在角膜创伤愈合中的作用。方法采用免疫荧光细胞计数仪检测法。结果体外培养的人角膜基质细胞β1整合素表达的阳性率平均为95．7％，阴性表达率为4．3％。结论在角膜创伤愈合中，角膜基质细胞β1整合素的表达可能极高，从而促进角膜基质细胞与细胞外基质的粘附。     

核因子кB在人角膜基质细胞中的表达

目的 探讨核因子кＢ（ＮＦ－кＢ）在人角膜基质成纤维细胞中的基础表达及激活情况。方法 取体外培养的第２或３代人角膜基质细胞,同步化后分为２组：无血清的ＤＭＥＭ液培养的不加药组;含质量浓度为１０μｇ／ｍｌ脂多糖的ＤＭＥＭ液培养６小时的加药组。均采用免疫荧光抗体染色、流式细胞仪计数法和核蛋白提取物凝胶迁移率法检测。结果 流式细胞计数法示,角膜基质细胞内ＮＦ－кＢ的基础表达率为９．４％,脂多糖作用后升高     

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。     

原生质体诱导酶对体外培养的人角膜基质细胞活性的影响

目的:检测用以制备烟曲霉菌原生质体的诱导酶对体外培养的人角膜基质细胞活性的影响.方法:将浓度为1g/dL蜗牛酶、1g/dL纤维素酶及0.1g/dL溶壁酶的复合诱导酶液与人角膜基质细胞共同培养15min,4h和8h,采用四氮唑盐代谢法(MTT法)检测不同作用时间的诱导酶对人角膜基质细胞的影响,台盼兰染色法检测诱导酶对人角膜基质细胞存活率的影响与其作用时间的关系.结果:共培养15min及4h后细胞形态未见明显变化,8h后细胞间隙略变小,偶见脱壁漂浮细胞,MTT实验显示诱导酶培养到8h时MTT值仍无明显下降,台盼兰染色显示8h内诱导酶对细胞存活率影响较小.结论:用以制备烟曲霉菌原生质体的诱导酶短时间内对体外培养的人角膜基质细胞活性影响较小,这一浓度的复合诱导酶用于动物模型及人细胞学实验较为安全.     

脂质体介导 Bcl-xL 基因体外转染人角膜基质细胞的动态表达

目的:观察脂质体LipofectamineTM(LF)2000介导Bcl-xL基因在人角膜基质细胞中表达的时间规律。方法:Bcl-xL/LF2000载体复合物转染人角膜基质细胞,转染后1～15d,采用定量RT-PCR及流式细胞计数法检测目的基因Bcl-xL表达阳性细胞率,观察目的基因在角膜基质细胞中表达的时间变化规律。结果:RT-PCR及流式细胞计数法均显示:在转染后1d开始检测到Bcl-xL表达阳性细胞,阳性率在转染后3d最高(48.3%),此后逐渐降低,转染后15d有少量细胞仍表达Bcl-xL基因。结论:LF2000能有效介导外源基因Bcl-xL转染人角膜基质细胞。     

蛋白激酶Cα在人角膜基质细胞中的表达

目的 观察蛋白激酶Ca（PKCα）在原位及体外培养的人角膜基质细胞中的表达。方法标本来源于北京大学第一医院眼库。人角膜基质细胞体外原代及传代培养，应用免疫组织化学法检测PKCα在培养的人角膜基质细胞中的表达；制作人正常角膜组织冰冻切片，观察PKCα在角膜基质细胞中的表达。结果免疫组织化学法检测显示：正常人原位角膜基质细胞对PKCα无明显表达，而体外培养的人角膜基质细胞有明显阳性表达。结论特殊的角膜创伤模型——体外培养人角膜基质细胞中PKCα有明显阳性表达，提示PKC在角膜基质细胞增生过程中扮演了重要角色，抑制PKC的活性有可能成为治疗角膜瘢痕愈合的一个新途径。     

兔角膜内皮、上皮及基质细胞体外培养扩增的研究

目的 建立角膜上皮、基质及内皮细胞体外培养扩增的简单稳定的方法，为组织工程化角膜的构建提供种子细胞。方法 内皮细胞与后弹力层在培养基中孵育后消化法获原代细胞，胰酶消化去除表层上皮后取角膜缘，组织块法培养角膜缘上皮细胞，基质细胞应用胶原酶消化法获原代培养，各细胞融合后胰酶消化依次传代培养。结果 原代内皮细胞4—5d融合成单层细胞，可连续传6—7代。上皮细胞1周左右生长融合，连续传3—4代后细胞形态改变。基质细胞接种6—7d后近融合，传代后增殖明显，可连续传10代。结论依据角膜组织特征选择合适的方法体外分离、培养角膜3种细胞成分，可获连续传代扩增的角膜细胞。     

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.     

Morphological characteristics and intercellular connections of corneal keratocytes

PURPOSE: To investigate the morphological characteristics of keratocytes and the interconnection of keratocytes with adjacent keratocytes using the flat preparation method and scanning electron microscopy with a frontal section of the human corneal stroma. METHODS: The thin, corneal collagen lamellae were carefully dissected from the cornea (n=7), which had been stained by the flat preparation method. The remaining tissue was fixed in 3% glutaraldehyde and observed by transmission electron microscopy following the frontal section. RESULTS: The flat preparation revealed the corneal fibroblasts between the lamellae of the collagen fibers and showed that the ramifying cellular processes of the keratocytes were in contact with the cytoplasmic processes or cell bodies of neighboring fibroblasts. Two types of discrete subpopulations of keratocytes were identified: a smaller, cellular type of keratocyte with spindle-shaped nucleus with heterochromatin, and a larger, cellular type with a large indented nucleus with relatively scanty cytoplasm. Collagen fibers ran parallel to each other toward the fenestration of the cytoplasmic wall of the keratocyte. CONCLUSIONS: These flat preparation method results showed that the keratocytes within the corneal stroma are interconnected with the adjacent keratocytes, which indicates the presence of a functional communicating network through the keratocyte circuits within the stroma. A smaller, cellular type of keratocyte with spindle-shaped nucleus was morphologically differentiated from a larger, cellular type with a large, indented nucleus by flat preparation and transmission electron microscopy.     

Expression of CD34 and L-selectin on human corneal keratocytes

PURPOSE: To investigate the expression of CD34, a hematopoietic stem cell marker and an adhesion molecule, and its ligand L-selectin in the human cornea. METHODS: Seventeen normal adult human corneal specimens were studied by immunohistochemistry using a panel of monoclonal antibodies against all three classes of the hematopoietic stem cell marker CD34 and its ligand L-selectin. An additional six corneal specimens were used for protein extraction and analysis by Western blotting, using the CD34 and L-selectin antibodies. PCR was used to determine expression of mRNA for CD34 and L-selectin in the corneal specimens. RESULTS: Only corneal keratocytes showed positive immunostaining for all three classes of CD34. Western blotting confirmed the expression of CD34 by these cells and mRNA expression for CD34 in the corneal stroma was demonstrated by PCR. For L-selectin, positive staining around keratocytes was noted on immunohistochemistry but L-selectin could not be detected either by Western blotting or PCR. CONCLUSIONS: Normal human corneal keratocytes express all three classes of CD34. The expression of this adhesion molecule on corneal keratocytes suggests that it may have a role in keeping the keratocytes anchored in their microniche, between the collagen lamellae. The positive staining for L-selectin found by immunohistochemistry but not by Western blotting or PCR would indicate the presence of either another ligand from the selectin family or a cross-reactive epitope on corneal keratocytes.     

维甲酸对人角膜基质细胞增殖和移行的影响

目的 探讨维甲酸对角膜基质创伤愈合的可能的作用。方法 利用细胞计数法、ＭＴＴ法观察维甲酸对培养的人角膜基质细胞增殖的影响;利用缺损闭合实验观察维甲酸对培养的人角膜基质细胞移行的影响。结果 ＭＴＴ法和细胞计数法显示,维甲酸抑制体外培养的人角膜基质细胞增殖,抑制作用呈剂量、时间依赖性（Ｆ检验,Ｐ〈０．０５）,抑制率在１１．４％～３９．２％。细胞计数法还显示,０．５％苔芬兰染色示各组活细胞均在９０％以上     

Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

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.