Development of endotoxin-detection reagents for periodontal treatments with human lactoferrin

1.与LFS的结合能力：以多粘菌素B为基础，测定乳铁蛋白衍生多肽与LFS结合的抑制作用。人乳铁蛋白(HLf)、hLf33K和hLf33R的抑制作用分别为0.2 nM、0.08 nM和0.05 nM。2.对THP-1细胞释放炎性细胞因子的影响：HLF、hLf33K或hLf33R分别作用于THP-1细胞，检测细胞培养上清液中肿瘤坏死因子-α(TN-x)水平和乳酸脱氢酶(LDH)活性，HLF、hLf33K和hLf33R对THP-1细胞释放炎性细胞因子的影响。3.对THP-1细胞趋化能力的影响：用E-cob中提取的内毒素刺激THP-1细胞，用Boyden小室法测定细胞的趋化能力。加入HLF、hLf33K或hLf33R后，趋化性显著降低，但抑制效果无显著差异。4.检测染料的选择及其与HLF的结合我们咨询了Kiya Chemical Ca关于食用染料与HLF的结合方法。他们说，一旦批准的食用染料一旦与HLF结合，将不再被允许用于食品。此外，我们咨询了Sigma Genosys Co.，关于在合成过程中将该染料与HLF衍生的多肽结合的可能性，似乎很难用该染料合成多肽。我们将在未来探索直接与内毒素结合的染料。

1.Binding capability to LFS: Inhibitory effects of LPS-IBP binding were measured for lactoferrin-derived peptides on the basis of polymyxin B. Inhibitory effects of human lactofenin, (hLf), hLf33K and hLf33R were 0.2 nM, 0.08 nM and 0.05 nM, respectively. There were significant differences among the effects.2.Effects on the inflammatory cytokine release from THP-1 cells : LPS from E cob was added to THP-1 cell culture with hLf, hIf33K or hLf33R, and TNF-a levels and LDH activity in culture supernatant were measured TN-(x levels and LDH activity were significantly decreased upon addition of hLf, hLf33K or hLf33R, however, there was no significant difference among the inhibitory effects. Cell growth of THP-1 measured by cellular intake of dyes was not affected with any reagents.3.Effects on chemotaxis of THP-1 cells : THP-1 cells were stimulated with LPS from E cob and chemotaxis was determined by Boyden chamber method. Chemotaxis were significantly decreased upon addition of hLf, hLf33K or hLf33R, however, there was no significant difference among the inhibitory effects.4.Selection of detection dye and binding of the dye to hLf We consulted Kiriya Chemical Ca about the binding method of the food dye to hLf. They told that approved food dyes once bound to hLf would be no longer allowed to use for food In addition, we consulted Sigma Genosys Co. about the possibility of binding the dye to hLf-derived peptides during synthesis It seemed to be difficult to synthesize the peptide with the dye. We would explore the dye that binds directly to LPS in future.