Development of local drug delivery system (LDDS) in periodontal pockets with human lactoferrin and its derivatives

制备了hLF33(GrrrR SVQWC AVSQP EATKC FQWQR NMRKV RGP)、hLF33K(GrrrR SVQWC AVSQP EATKC FQWQR NMKKV RGP)、hLF20-37(C FQWQR NMRKV RGPPV SC)和hLF20-37S(C FQWQR NMRKV RGPPV SC；半胱氨酸残基之间的S-S键)，并测定了它们对脂多糖-LBP结合的抑制作用。这些多肽的作用是HLF的十分之一。在浓度为2.5μm时，各多肽的作用顺序为：hLf33&gt；&gt；hLf33K&gt；&gt；hLf20-37&gt；hLf20-37；hLf20-37S。这些多肽在浓度为2.5μm时无显著差异。这些结果表明，HLFN-末端的第28个氨基酸参与了抑制LBP结合的作用，而半胱氨酸残基之间的S-S键则不参与。这些多肽对肿瘤坏死因子-α的产生也有抑制作用，但它们的抑制作用远低于hFL。THP-1细胞在La，25-二羟基维生素D3存在下预培养18h，细胞转移到ChemoTx96孔一次性小室的过滤器上：下层小室含有每种化学诱导剂，有或没有HLF200μg/m1。平板离心，水中细胞增殖检测试剂盒检测迁移至下腔的细胞数。向内毒素迁移的细胞数明显低于对照组。当内毒素浓度为10 ng/ml时，细胞迁移受到抑制，这种抑制作用可被HLF以剂量依赖的方式部分消除。结果提示，内毒素可抑制THP-1细胞的随机游走，而HLF对细胞游走的抑制具有保护作用。HLF可能在牙周病细菌的炎症反应中调节细胞迁移。

Four peptides, hLF33 (GRRRR SVQWC AVSQP EATKC FQWQR NMRKV RGP), hLF33K (GRRRR SVQWC AVSQP EATKC FQWQR NMKKV RGP), hLF20-37 (C FQWQR NMRKV RGPPV SC) and hLF20-37S (C FQWQR NMRKV RGPPV SC; the S-S bond between cysteine residues) were prepared, and measured for inhibitory effects against the LPS-LBP binding. Effects of these peptides were one-tenth of hLF. No significant differences were observed among the peptides at the concentration of 2.5 μm. However, the rank order of effects for these peptides was as follows at the concentrations of 100 μM: hLf33 >> hLf33K >> hLf20-37 > hLf20-37S. These results suggest that the 28th amino acid from the N-terminal of hLF is involved in inhibitory effects against the LPS-LBP binding, while the S-S bond between cysteine residues does not. These peptides also inhibited TNF-α production, however, inhibitory effects of the peptides were much less than hLF.We further performed chemotaxis assay with 96-well plates. THP-1 cells were preincubated in the presence of la, 25-Dihydroxyvitamin D3 for 18 h. The cells were transferred on the filter of ChemoTx 96-well disposable chamber: the lower chamber contained each chemoattractants in the presence or absence of 200 μg/m1 of hLF. Plates were centrifuged, and the number of cells that migrated to the lower chamber was determined with AQueous cell proliferation assay kit. The number of migrated cells to LPS was significantly lower than the controls. Inhibition of cell migration was observed at concentrations > or = 10 ng/ml of LPS, and the inhibition was partially abrogated by the addition of hLF in a dose-dependent manner. The results suggest that LPS inhibits random migration of THP-1 cells, and that hLF protects against the inhibition of cell migration. hLF may regulate cell migration in an inflammatory response to periodontopathic bacteria.