Sulfated proteoglycans synthesized by vascular endothelial cells in culture.
Sulfated proteoglycans synthesized by vascular endothelial cells in culture.
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DOI：
10.1016/s0021-9258(18)33090-4
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发表时间：
1983-02
期刊：
影响因子：
--
通讯作者：
A. Oohira;T. Wight;P. Bornstein
中科院分区：
文献类型：
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作者：
A. Oohira;T. Wight;P. Bornstein
从培养的牛主动脉内皮细胞的培养液和细胞层中分离到代谢标记的蛋白多糖。通过SepharoseCL-4B凝胶过滤、DEAE-纤维素柱层析和CsCl2密度梯度离心法对蛋白多糖进行分级。培养基中含有两种不同的蛋白多糖：一种大的蛋白多糖(Kav=0.07)含有低浮力密度的硫酸乙酰肝素侧链(MR一致性为36,000)，另一种较小的蛋白多糖(Kav=0.45)含有高密度的硫酸软骨素(MR一致性为20,000)。硫酸软骨素蛋白多糖部分含有少量(不到10%)的硫酸皮肤素。在酶抑制剂存在的情况下，用2%十二烷基硫酸钠从细胞层中提取出非常相似的低密度硫酸乙酰肝素蛋白多糖。此外，细胞层中还含有高密度的蛋白多糖，体积小(Kav=0.43)，含有硫酸乙酰肝素侧链(MR=20000)。对培养的人脐静脉内皮细胞合成的蛋白多糖的分析给出了类似的结果，除了这些细胞产生更多的皮肤素硫酸盐和不明原因的过度硫酸软骨素硫酸盐链。形态上不典型的内皮细胞含有大的硫酸乙酰肝素蛋白多糖水平降低。间接免疫荧光和直接免疫电子显微镜均显示牛内皮细胞单层下的基膜样基质与基底膜蛋白多糖抗体发生反应。在电子显微镜下，这种材料由细丝状网络组成，含有直径为10-20 nm的离散的Ru红阳性颗粒，类似于存在于完整动脉基底膜中的颗粒。
Metabolically labeled proteoglycans were isolated both from the culture medium and from the cell layer of cultured bovine aortic endothelial cells. Proteoglycans were fractionated by sequential gel filtration on Sepharose CL-4B, DEAE-cellulose column chromatography, and CsCl density gradient centrifugation. The culture medium contained two distinct proteoglycans: a large proteoglycan (Kav = 0.07) of low buoyant density containing heparan sulfate side chains (Mr congruent to 36,000) and a smaller proteoglycan (Kav = 0.45) of high density containing chondroitin sulfate chains (Mr congruent to 20,000). The chondroitin sulfate proteoglycan fraction contained a small amount (less than 10%) of dermatan sulfate. A very similar low density heparan sulfate proteoglycan was extracted from the cell layer with 2% sodium dodecyl sulfate in the presence of enzyme inhibitors. In addition, there was a high density proteoglycan of small size (Kav = 0.43) containing heparan sulfate side chains (Mr congruent to 20,000) in the cell layer. Analyses of proteoglycans synthesized by cultured human umbilical vein endothelial cells gave similar results, except that these cells produced more dermatan sulfate and unidentified oversulfated chondroitin sulfate chains. Morphologically atypical endothelial cells contained reduced levels of the large heparan sulfate proteoglycan. Both indirect immunofluorescence and direct immunoelectron microscopy revealed that the basement membrane-like matrix under monolayers of bovine endothelial cells reacted with antibodies against the basement membrane proteoglycan isolated from a basement membrane-producing tumor. By electron microscopy, this material was shown to consist of a fine filamentous meshwork containing discrete 10-20-nm diameter ruthenium red positive granules resembling those present in basement membranes of intact arteries.