Ribault D, Khatib A M, Panasyuk A, Barbara A, Bouizar Z, Mitrovic R D
INSERM U.349, Paris, France.
Arch Biochem Biophys. 1997 Jan 15;337(2):149-58. doi: 10.1006/abbi.1996.9767.
The effects of human recombinant epidermal growth factor (EGF) on rat articular chondrocytes from humeral and femoral head cartilage of 21-day-old Wistar rats were analyzed. The cells were cultured under standard conditions as monolayers. Cell proliferation was studied by [3H]thymidine incorporation and determination of DNA content, proteoglycan synthesis by [35S]sulfate incorporation, and collagen synthesis by [3H]proline incorporation. The presence of specific receptors was confirmed by [125I]-EGF binding and that of EGF and EGF-receptor (EGF-R) mRNA by reverse transcription and the polymerase chain reaction. EGF (0.5-2.5 ng/ml) stimulated [3H]thymidine incorporation and increased DNA content of cultures. The effect was strongest when serum concentration was low (< or =1%) and was lost at high (> or =7.5%) serum concentrations. The EGF-induced effect on deoxynucleic acid synthesis was inhibited by transforming growth factor-beta and tyrphostin, a tyrosine kinase inhibitor that blocks the phosphorylation of tyrosine residues on EGF-R. Cultured rat articular chondrocytes possess a single class of high-affinity binding sites (Kd 0.18 nM). There were about 4.5 x 10(9) binding sites per microgram of DNA or about 37,800 binding sites per cell with 8.3 pg DNA per cell. Cultured cells contained EGF mRNA and EGF-R mRNA. Incubation of cells with EGF for 24 h decreased the EGF mRNA transcripts and increased the EGF-R mRNA levels. These findings suggest that EGF probably takes part in the regulation of chondrocyte activity under normal and presumably pathological conditions.
分析了人重组表皮生长因子（EGF）对21日龄Wistar大鼠肱骨头和股骨头软骨来源的大鼠关节软骨细胞的作用。细胞在标准条件下作为单层进行培养。通过[³H]胸苷掺入法和DNA含量测定研究细胞增殖，通过[³⁵S]硫酸盐掺入法研究蛋白聚糖合成，通过[³H]脯氨酸掺入法研究胶原蛋白合成。通过[¹²⁵I]-EGF结合证实特异性受体的存在，通过逆转录和聚合酶链反应证实EGF和EGF受体（EGF-R）mRNA的存在。EGF（0.5 - 2.5 ng/ml）刺激[³H]胸苷掺入并增加培养物的DNA含量。当血清浓度低（≤1%）时，该作用最强，而在高血清浓度（≥7.5%）时该作用消失。转化生长因子-β和酪氨酸激酶抑制剂 tyrphostin可抑制EGF诱导的脱氧核糖核酸合成，tyrphostin可阻断EGF-R上酪氨酸残基的磷酸化。培养的大鼠关节软骨细胞具有一类高亲和力结合位点（Kd 0.18 nM）。每微克DNA约有4.5×10⁹个结合位点，或每个细胞约有37,800个结合位点，每个细胞含8.3 pg DNA。培养的细胞含有EGF mRNA和EGF-R mRNA。用EGF孵育细胞24小时可降低EGF mRNA转录本水平并增加EGF-R mRNA水平。这些发现表明，EGF可能在正常及可能的病理条件下参与软骨细胞活性的调节。
