目的 筛选能与集落细胞刺激因子(granulocyte-colony stimulatingfactor,G-CSF)结合,并能调节血细胞生长的活性物质,为研究药理作用提供实验依据.方法 采用毛细管电泳(capillary ehetrophoresis,CE)方法研究15种不同链长和不同硫酸基含量的硫酸寡聚糖(Dextran sulfate,DexS)和G-CSF的相互作用.采用细胞生物学实验观察被筛选出的寡聚糖与G-CSF结合后对小鼠白血病细胞(bipotential murine hemopoietic cellline,NFS-60)增生、细胞周期、细胞表面抗原的表达和细胞形态的影响,以证明毛细管电泳所筛选出的DexS具有促进细胞分化等生物学活性.结果 除肝素寡聚糖、降解的卡拉胶、低分子量右旋Dexs、非硫酸化的寡聚糖以及碱性壳寡糖外,其余10种寡聚糖与G-CSF均有相互作用.DexS500、λ卡拉胶、ι卡拉胶、κ卡拉胶和具有抗凝活性的低分子量肝素(LMWH)-1能抑制NFS-60细胞生长,其作用NFS-60细胞后相对吸光度值分别为31.33±2.08、37.67±2.08、45.00±1.20、43.00±2.65、74.67±1.52,与对照组100.00±3.00比较,差异有统计学意义(t值分别为27.94、26.71、26.42、26.61、10.54,P均<0.01).形态学结果表明,LMWH、λ卡拉胶和相对分子质量为500 000的硫酸葡聚糖能有效地诱导早幼粒细胞过渡到中性晚幼粒细胞.流式细胞术分析结果表明,用LMWH和λ卡拉胶分别处理细胞后,其表面的CD11b和CD18表达量增加.在硫酸葡聚糖(DexSS00)和λ或ι卡拉胶作用后,细胞表面CD34含量增加.用不同的寡聚糖作用后,其CD34表达量降低.用壳寡糖处理后,CDs含量几乎保持不变.细胞周期分析结果表明,在LMWH、DexS500和λ或ι卡拉胶作用后,S期NFS-60细胞减少,而壳寡糖处理后其细胞增加.结论 毛细管电泳技术能有效地筛选出与G-CSF有相互作用的酸性寡聚糖,相互作用的强弱与寡聚糖的碳链长度和酸性基团的含量及其基团位置有关.%Objective To screen out the bioactive sulfate saccharides interacted with granulocyte colony-stimulating factor (G-CSF) and provide some scientific data on their pharmacology. Methods The interactions between G-CSF and 15 sulfate saccharides with various chain lengths and extent of sulfation were studied by capillary electrophoresis. Furthermore, the effects of these G-CSF binding saccharides on the proliferation and differentiation of leukemia cell line and NFS-60 were also investigated. Results Except for the hepatosaccharide, degradable carrageenan, Dextran sulfate (DexS) with a low molecular mass the nonsulfated sancharide and alkaline chitosan oligosaccharide, the rest of saccharides had interact with G-CSF.DexS500, λ carrageenan, ι carrageenan, κ carrageenan and low molecular weight heparin-1(LMWH-1)with anticoagulant activity could inhibit the NFS-60 growth. Compared with the absorbency of the control group (100.00±3.00) after these saccharides interacted with the NPS-60 cells, their relative absorbencies were 31.33±2.08,37.67±2.08,45.00±1.20,43.00±2.65 and 74.67±1.52, respectively. Their differences had a statistical significance(t=27.94,26.71,26.42,26.61,10.54,P<0.01). The morphological analytic results showed that the LMWH and lambda carrageenan as well as DexS with a molecular mass of 500 000 induce effectively promyclocyte to transit metamyelocyte. Flow cytometry results showed enhanced expressions of CD11b, CD18 and CD45 after treatment with the LMWH, lambda carrageenan and DexS500,respectively. Cell cycle analysis indicated that the S phase population decreased after treatment with these saccharides. Conclusions Capillary electrophoresis is an effective medicine-screening technique that is used to screen some of these saccharides which could bind to G-CSF. And it is found that the interactions are correlated with the chain length, the extent and the position of sulfation of these saccharides.
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