Human platelet-derived mitogens. I. Identification of insulinlike growth factors I and II by purification and N alpha amino acid sequence analysis
Human platelet-derived mitogens. I. Identification of insulinlike growth factors I and II by purification and N alpha amino acid sequence analysis
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人血小板衍生的有丝分裂原。
DOI：
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发表时间：
1989
期刊：
影响因子：
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通讯作者：
D. Sirbasku
中科院分区：
文献类型：
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作者：
Kenneth P. Karey;H. Marquardt;D. Sirbasku
人血小板裂解物在无血清成分确定培养基中对MCF-7人乳腺癌细胞具有有效的促有丝分裂活性。因为这些活性不能被已知的血小板有丝分裂原（如血小板衍生生长因子或转化生长因子β）所取代，我们试图通过纯化和N α氨基酸测序来鉴定乳腺癌细胞有丝分裂原。用硫酸铵沉淀法浓缩过期血小板的乙酸提取物，然后用SephadexG-50和Bio-GelP-10柱在0.5mol/L乙酸中进行分离。两个主要的活动，通过分子筛的方法解决和分馏进一步通过反相高效液相色谱法（HPLC）。完成纯化（70，000至870，000倍），得到摩尔重量为7，400的产物，其通过碘化、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和放射自显影测定为均匀的。通过N α氨基酸微测序鉴定这些因子为胰岛素样生长因子I（IGF-I）和II（IGF-II）以及截短的IGF-I。在剂量反应实验中，血小板衍生的IGF-I和IGF-II促进MCF-7细胞的多次分裂，ED 50值分别为12和100 pg/mL。血小板衍生的IGF-I和IGF-II的比活性和其他生物测定特征与重组产生的人生长因子相似。这是首次报道从人血小板裂解液中纯化胰岛素样生长因子。
Human platelet lysates contained potent mitogenic activities for MCF-7 human breast-cancer cells in serum-free-defined media. Because these activities were not replaced by known platelet mitogens, such as platelet-derived growth factor or transforming growth factor beta, we sought to identify the breast cancer cell mitogens by purification and N alpha amino-acid sequencing. Acetic acid extracts of outdated human platelets were concentrated by ammonium sulfate precipitation and fractionated on Sephadex G-50 and Bio-Gel P-10 columns in 0.5 mol/L acetic acid. Two major activities were resolved by molecular sieve methods and fractionated further by reverse-phase high-performance liquid chromatography (HPLC). Purifications (70,000 to 870,000-fold) were accomplished yielding mol wt 7,400 products that were homogeneous as determined by iodination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography. The factors were identified as insulinlike growth factor I (IGF-I) and II (IGF-II) and truncated IGF-I by N alpha amino acid microsequencing. In dose-response experiments, platelet-derived IGF-I and IGF-II promoted multiple divisions of the MCF-7 cells with ED50 values of 12 and 100 pg/mL, respectively. The specific activities and other bioassay characteristics of platelet-derived IGF-I and IGF-II were similar to those of recombinant-produced human growth factors. This is the first report of the purification of insulinlike growth factors from human platelet lysates.