Jester J V, Barry-Lane P A, Cavanagh H D, Petroll W M
Department of Ophthalmology, University of Texas, Southwestern Medical Center at Dallas 75235-9057, USA.
Cornea. 1996 Sep;15(5):505-16.
The effects of serum, transforming growth factor (TGF) beta 1, bFGF, and heparin on in vitro myofibroblast transformation was studied. Primary rabbit corneal keratocytes were grown under serum-free conditions or in media supplemented with serum (10% fetal calf serum), TGF beta 1 (0.1-10 ng/ml), basic fibroblast growth factor (bFGF) (0.1-10 ng/ml), or heparin (10 U/ml). Cells were analyzed for expression of alpha-smooth muscle actin (alpha-SM actin), alpha 5 beta 1 integrin (the high-affinity fibronectin receptor) and fibronectin by immunoprecipitation, Western blotting, and immunofluorescence. Corneal keratocytes grown in the presence of serum showed a typical fibroblast morphology with induction of alpha-SM actin expression in 1 to 10% of cells. Addition of bFGF blocked serum-induced alpha-SM actin expression, whereas addition of TGF beta 1 enhanced alpha-SM actin expression (100%), which in combination with heparin (10 U/ml), led to a pulling apart of the fibroblastic sheet, simulating contraction. Under serum-free conditions, with or without bFGF and heparin, primary corneal fibroblasts appeared morphologically similar to in situ corneal keratocytes, demonstrating a broad, stellate morphology with interconnected processes and no alpha-SM actin expression. Addition of TGF beta 1 to serum-free cultures resulted in a dramatic transformation of corneal keratocytes to spindle-shaped, fibroblast-like cells that expressed alpha-SM actin in 100% of cells and exhibited a 20-fold increase in fibronectin synthesis and a 13-fold increase in alpha 5 beta 1-integrin synthesis. These effects were blocked by the addition of neutralizing antibodies (16 micrograms/ml). Overall these data suggest that TGF beta 1 is a potent modulator of myofibroblast transformation under serum-free conditions. In addition, the growth of keratocytes in serum appears to mimic, in part, in vivo activation and myofibroblast transformation. We conclude that detailed study of TGF beta 1-induced myofibroblast transformation under defined serum-free conditions will provide important insights into the myofibroblast transformation process.
研究了血清、转化生长因子（TGF）β1、碱性成纤维细胞生长因子（bFGF）和肝素对体外肌成纤维细胞转化的影响。原代兔角膜基质细胞在无血清条件下或在补充有血清（10%胎牛血清）、TGFβ1（0.1 - 10 ng/ml）、碱性成纤维细胞生长因子（bFGF）（0.1 - 10 ng/ml）或肝素（10 U/ml）的培养基中培养。通过免疫沉淀、蛋白质印迹和免疫荧光分析细胞中α - 平滑肌肌动蛋白（α - SM肌动蛋白）、α5β1整合素（高亲和力纤连蛋白受体）和纤连蛋白的表达。在有血清存在的情况下培养的角膜基质细胞呈现典型的成纤维细胞形态，1%至10%的细胞中诱导表达α - SM肌动蛋白。添加bFGF可阻断血清诱导的α - SM肌动蛋白表达，而添加TGFβ1则增强α - SM肌动蛋白表达（100%），其与肝素（10 U/ml）联合使用会导致成纤维细胞片层拉开，模拟收缩。在无血清条件下，无论有无bFGF和肝素，原代角膜成纤维细胞在形态上与原位角膜基质细胞相似，呈现宽阔的星状形态，有相互连接的突起，且无α - SM肌动蛋白表达。向无血清培养物中添加TGFβ1会导致角膜基质细胞急剧转化为纺锤形、成纤维细胞样细胞，100%的细胞表达α - SM肌动蛋白，纤连蛋白合成增加20倍，α5β1整合素合成增加13倍。添加中和抗体（16微克/毫升）可阻断这些效应。总体而言，这些数据表明TGFβ1是无血清条件下肌成纤维细胞转化的有效调节因子。此外，血清中角膜基质细胞的生长似乎部分模拟了体内激活和肌成纤维细胞转化。我们得出结论，在明确的无血清条件下对TGFβ1诱导的肌成纤维细胞转化进行详细研究将为肌成纤维细胞转化过程提供重要见解。
