富组蛋白中和牙周可疑致病菌内毒素作用的实验研究

The Neutralizing Effect of Histidine-rich-polypeptides on LPS from Suspected Periodontal Pathogens

目的 :研究人腮腺唾液富组蛋白对牙周可疑致病菌内毒素的中和作用。方法 :采用改良Westphal热酚水法提取并纯化牙龈卟啉菌 (4 7 A株 )和具核梭杆菌 (临床分离株 )的内毒素 ,用鲎试验试管法观察HRP 5对这两株细菌的内毒素的中和作用。结果 :在本实验条件下 ,HRP 5 (10 μg/ml)能轻度抑制大肠杆菌、牙龈卟啉菌及具核梭杆菌的内毒素 (1ng/ml)的鲎活性。结论 :相同浓度的HRP 5对需氧的大肠杆菌和专性厌氧的牙龈卟啉菌及具核梭杆菌的内毒素 ,具有相同的中和作用 ,提示富组蛋白可能在维护牙周健康中发挥一定作用。

Objective:Human parotid histidine rich polypeptides (HRPs) are a family of lowmolecular weight, cationic polypeptides. HRP 1, HRP 3 and HRP 5 comprise 85%～90% of the total HRPs and are called major HRPs. There were many researches indicating the antimicrobial activities of HRPs. Recently, HRPs were reported to have an inhibitory action on the lipopolysaccharide (LPS) of E.coli, with HRP 5 being the most effective one among the three major HRPs. Since the LPS of oral gram negative bacteria is thought to be one of the important etiological factors during the development of periodontal diseases, our experiment was aimed to investigate the neutralizing effect of human parotid HRP 5 on the LPS of anaerobic suspected periodontal pathogens, which have different chemical structures and biological activities compared with LPS of aerobic E.coli.Methods: By using of preparative acid urea polyacrylamide gel electrophoresis (AU PAGE), HRP 5 was purified from parotid saliva collected from healthy adults. Two stains of suspected periodontal pathogens, Porphyromonas gingivalis (P.g, 47 A) and Fusobacterium nuceatum (F.n, separated from subgingival plaque of a patient with adult periodontitis) were mass cultured. After harvested in the midlogarithmic phase, they were washed and lyophilized. The LPS of dried bacteria were extracted by the modified Westphal hot phenol water procedures and purified by the enzyme digestion plus ultracentrifugation. Limulus test was applied to test the neutralizing effect of HRP 5 on the LPS induced gelation of Limulus amoebocyte lysate. In brief, the standard LPS of E.coli. or extracted LPS of P.g or F.n, was preincubated with HRP 5 in a tube separately in room temperature for 10 minutes. Then the reagent of Limulus amoebocyte lysate was added in all the tubes, continued the incubation in 37℃ for one hour. After that, the gelation level of every tube was observed. Results: LPS extracted from P.g and F.n both showed good purity and strong activities to induce gelation of Limulus amoebocyte lysate. The gelation induced by LPS (1ng/ml) of these two anaerobic suspected periodontal pathogens were weakly inhibited by HRP 5 (10μg/ml),\ similar to that observed with standard LPS of E.coli.\ To get a complete neutralizing effect on LPS, it may be important to increase the concentration of HRP 5.Conclusion: It appeared that HRPs could neutralize the endotoxic properties of LPS of suspected periodontal pathogens, therefore may contribute to periodontal health.\ The pre sent investigation further confirmed that HRPs are important components of the host non immune defense system.\;