Dye assay of matrix components in gingival crevicular fluid for destructive activity of alveolar bone

对检测硫酸化糖胺聚糖（S-GAG）的方法进行了修改，以用于阿辛蓝试验。我们使用低pH值和高盐浓度，以尽量减少其他聚阴离子（如DNA、蛋白质或透明质酸盐）的干扰。将S-GAG样品（包括4-硫酸软骨素、6-硫酸软骨素、硫酸皮肤素和硫酸乙酰肝素）印迹到醋酸纤维素条上，并用含有0.05 M MgCl ^2、0.4 M盐酸胍、0.002 M硫酸和0.25% Triton X-100（pH 1.5）的0.2%阿辛蓝溶液染色。S-GAG物质与染料的结合能力存在一些变异性。4-硫酸软骨素对染料的亲和力最强，硫酸乙酰肝素最弱，DNA、牛血清白蛋白或透明质酸与染料的结合量可以忽略不计。本方法可用于S-GAG的定量分析，线性范围可达300 ng，并可直接用于人体体液中S-GAG的测定（唾液，血清，龈沟液中的总S-GAG含量明显高于其他大分子聚阴离子的含量，牙周炎组（71.41 ± 0.001）%（P<0.001），牙周炎组（71.41 ± 0.001）%（P<0.001）。[] 20.04ng）与健康人[7.83（]SY. ±.牙龈炎组（10.53 ± 0.21）ng。[）4.28ng）。

A modification of the method of detecting sulfated glycosaminoglycans (S-GAG) was adapted for use in an Alcian blue assay. We used low pH,and high salt concentration to minimize interference from other polyanions such as DNA,protein, or hyaluronate. The samples of S-GAGs including chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and heparan sulfate were blotted onto cellulose acetate strips and stained with 0.2% Alcian blue solution containing 0.05M MgCl^2,0.4M guanidine-HCl, 0.002M sulfuric acid, and 0.25% Triton X-100 at pH 1.5. There was some variability in the binding ability of S-GAG species to the dye. Chondroitin 4-sulfate showed the greatest affinity for the dye while heparan sulfate had the weakest.The amount of dye bound by DNA,bovine serum albumin, or hyaluronate was negligible. The new method developed here can be adapted as a quantitative assay using a scanning densitometer with a linear range to 300ng of S-GAG.Moreover, this assay method can be used for direct measurement of S-GAG in the human biological fluid (saliva, serum, synovial fluid) without interferences by other macromolecular polyanion.The total S-GAG obtained from the timed samples of gingival crevicular fluid was significantly higher (P<0.001) in the periodontitis subjects (71.41(]SY.+-。[)20.04ng) compared to the healthy (7.83(]SY.+-。[)2.21ng), and gingivitis subjects (10.53(]SY.+-。[)4.28ng).