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

对硫代糖胺聚糖（S-GAG）检测方法进行了改进，适用于阿利新蓝测定。我们使用低pH值和高盐浓度来减少其他多阴离子（如DNA，蛋白质或透明质酸）的干扰。将含4-硫酸软骨素、6-硫酸软骨素、硫酸皮聚糖和硫酸肝素的S-GAGs样品印迹于醋酸纤维素条上，用含有0.05M MgCl^2、0.4M胍- hcl、0.002M硫酸和0.25% Triton X-100的0.2%阿利新蓝溶液在pH 1.5下染色。S-GAG对染料的结合能力存在一定的差异性。硫酸软骨素4对染料的亲和力最大，硫酸肝素对染料的亲和力最弱。DNA、牛血清白蛋白或透明质酸结合的染料量可以忽略不计。这里开发的新方法可以适用于使用扫描密度计的定量分析，线性范围为300ng S-GAG。该方法可直接测定人体生物体液（唾液、血清、滑液）中S-GAG的含量，不受其他大分子聚阴离子的干扰。牙周炎组龈沟液定时取样总S-GAG含量为71.41(]SY.+- . [)20.04ng，显著高于健康组（7.83(]SY.+- . [)2.21ng）和牙龈炎组（10.53(]SY.+- . [)4.28ng） （P<0.001）。

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).