Montgomery R I, Lidholt K, Flay N W, Liang J, Vertel B, Lindahl U, Esko J D
Department of Biochemistry, School of Medicine, University of Alabama, Birmingham 35294.
Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11327-31. doi: 10.1073/pnas.89.23.11327.
Stable cell lines that synthesize heparin have been established from the Furth murine mastocytoma. The parental line (MST) divides in suspension every 14-18 h in growth medium supplemented with fetal bovine serum or defined growth factors. Adherent subclones were selected by adhesion to plastic culture vessels. Both adherent and nonadherent cells contain about 0.4 micrograms of glycosaminoglycan hexuronic acid per 10(6) cells, composed of 80% heparin and 20% chondroitin sulfate E. Deaminative cleavage of MST heparin by HNO2 at pH 1.5 released disaccharides that were similar in composition to those obtained from commercial heparin, except that disaccharides containing 3,6-O-desulfated GlcN units were not found. Greater than 90% of the glycosaminoglycans were stored in cytoplasmic granules, and challenge of the cells with dinitrophenylated bovine serum albumin and anti-dinitrophenyl IgE released a portion of the stored material. Growth studies of subclones showed that MST cells tolerate a 10-fold variation in glycosaminoglycan content. Incubation of cells with sodium chlorate reduced glycosaminoglycan sulfation by > 95% without affecting cell growth. Thus, granule glycosaminoglycans appear to be nonessential for growth of MST cells.
已从弗思小鼠肥大细胞瘤建立了合成肝素的稳定细胞系。亲代细胞系(MST)在补充有胎牛血清或特定生长因子的生长培养基中每14 - 18小时在悬浮液中分裂一次。通过贴附于塑料培养容器选择贴壁亚克隆。贴壁细胞和非贴壁细胞每10⁶个细胞均含有约0.4微克糖胺聚糖己糖醛酸,其中80%为肝素,20%为硫酸软骨素E。在pH 1.5条件下,亚硝酸对MST肝素进行脱氨基裂解,释放出的二糖组成与从商业肝素获得的二糖相似,只是未发现含有3,6 - O - 去硫酸化葡糖胺单元的二糖。超过90%的糖胺聚糖储存在细胞质颗粒中,用二硝基苯基化牛血清白蛋白和抗二硝基苯基IgE刺激细胞会释放出一部分储存物质。亚克隆的生长研究表明,MST细胞能耐受糖胺聚糖含量10倍的变化。用氯酸钠孵育细胞可使糖胺聚糖硫酸化减少> 95%,而不影响细胞生长。因此,颗粒糖胺聚糖似乎对MST细胞的生长并非必不可少。