Nakazawa K, Morita A, Nakano H, Mano C, Tozawa N
Section of Radiochemistry, Faculty of Pharmacy, Meijo University, Nagoya.
J Biochem. 1996 Jul;120(1):117-25. doi: 10.1093/oxfordjournals.jbchem.a021372.
The corneal stromal cells from 2-day-old chicks were cultured on plastic dishes or within three-dimensional collagen gel in the presence or absence of growth factor (EGF, bFGF, PDGF, TGF-beta 1, or their combinations). The cells were labeled with [35S] sulfate and [3H]-glucosamine, and the radio-labeled proteoglycans were examined. Keratan sulfate was synthesized to some extent (15.4-16.9% of total synthesis for medium fraction; 8.0% for cell layer fraction) in a primary culture even when the cells were cultured on plastic dishes, although the values were very much lower than that (42.7%) in the stromal fraction of organ culture of corneal explants. The primary culture in collagen gel showed some increase in the proportion of keratan sulfate synthesis as compared with the culture on plastic. Among growth factors, addition of EGF to the culture in gel caused a further increase in the proportion of keratan sulfate synthesis. bFGF and TGF-beta 1 increased proteoglycan synthesis as a whole to some extent, but chondroitin sulfate/dermatan sulfate synthesis was increased preferentially and, consequently, the proportion of keratan sulfate synthesis to total synthesis was decreased. PDGF also caused some decrease in the proportion. In the culture after one passage (secondary culture), the keratan sulfate synthesis decreased markedly (8.6-8.3% of total synthesis for medium fraction; 2.7% for cell layer or gel fraction) and a large chondroitin sulfate/dermatan sulfate proteoglycan appeared whether the cells were cultured on plastic or in collagen gel. But, when the medium was changed to CG medium (serum-free medium) in the middle of either primary or secondary cultures, the keratan sulfate synthesis (27.8% for medium fraction; 15.6% for gel fraction) was maintained at the level of that of the primary culture in gel. EGF and bFGF were not additive to the effect of CG medium on the keratan sulfate synthesis in the secondary culture. Instead, EGF and bFGF stimulated hyaluronic acid synthesis in the culture. The mechanism of these changes in the expression type of proteoglycan and their significance remain to be clarified.
将2日龄雏鸡的角膜基质细胞培养于塑料培养皿中或三维胶原凝胶内，分别添加或不添加生长因子（表皮生长因子、碱性成纤维细胞生长因子、血小板衍生生长因子、转化生长因子-β1或它们的组合）。用[35S]硫酸盐和[3H] - 葡糖胺对细胞进行标记，并检测放射性标记的蛋白聚糖。即使细胞在塑料培养皿中培养，在原代培养中也会合成一定量的硫酸角质素（培养基部分占总合成量的15.4 - 16.9%；细胞层部分占8.0%），尽管这些值远低于角膜外植体器官培养基质部分的合成量（42.7%）。与在塑料上培养相比，在胶原凝胶中的原代培养显示硫酸角质素合成比例有所增加。在生长因子中，向凝胶培养物中添加表皮生长因子会导致硫酸角质素合成比例进一步增加。碱性成纤维细胞生长因子和转化生长因子-β1在一定程度上整体增加了蛋白聚糖的合成，但硫酸软骨素/硫酸皮肤素的合成优先增加，因此硫酸角质素合成占总合成的比例降低。血小板衍生生长因子也导致该比例有所下降。在传代一次后的培养（传代培养）中，无论细胞是在塑料上还是在胶原凝胶中培养，硫酸角质素的合成均显著下降（培养基部分占总合成量的8.6 - 8.3%；细胞层或凝胶部分占2.7%），并且出现了大量的硫酸软骨素/硫酸皮肤素蛋白聚糖。但是，在原代或传代培养中期将培养基更换为CG培养基（无血清培养基）时，硫酸角质素的合成（培养基部分占27.8%；凝胶部分占15.6%）维持在凝胶中原代培养的水平。表皮生长因子和碱性成纤维细胞生长因子对传代培养中CG培养基促进硫酸角质素合成的作用无相加效应。相反，表皮生长因子和碱性成纤维细胞生长因子刺激了培养物中透明质酸的合成。蛋白聚糖表达类型的这些变化机制及其意义仍有待阐明。
