不同培养条件下唾液酸酶基因缺失对牙龈卟啉单胞菌生物膜形成的影响研究  

Effects of sialidase gene deletion on Porphyromonas gingivalis biofilm formation under stressful environmental conditions

作　　者：佟彤[1,2] 李琛[1] 徐晓宇[1] 杨雪[1] 郭艳[3] 

机构地区：[1]中国医科大学附属口腔医院牙周科辽宁省口腔医学研究所牙周病学研究室辽宁省口腔疾病转化医学研究中心,辽宁沈阳110002 [2]鞍山市双山医院口腔科,辽宁鞍山114000 [3]中国医科大学附属口腔医院中心实验室辽宁省口腔疾病重点实验室,辽宁沈阳110002

出　　处：《中国实用口腔科杂志》2017年第7期436-440,共5页Chinese Journal of Practical Stomatology

基　　金：国家自然科学基金(81200785);辽宁省自然科学基金(2015010496-301)

摘　　要：目的探讨在不同培养条件下唾液酸酶基因缺失对牙龈啉单胞菌(Porphyromonas gingivalis,P.gingivalis)生物膜形成的影响。方法不同温度、过氧化氢(H_2O_2)浓度及pH值条件下,在96孔板中用含有5%无菌脱纤维羊血、氯化血红素(5μg/mL)和维生素K(1μg/mL)的TSB培养基培养P.gingivalis野生株(W83)和唾液酸酶基因突变株(△PG0352)4d,结晶紫染色、酒精脱色后,采用酶标仪检测生物膜的形成情况。结果P.gingivalis W83和APG0352在正常条件下均能形成生物膜,与P.gingivalis W83相比,APG0352形成的生物膜较少。P.gingivalisW83和△PG0352在34℃和41℃条件下生物膜形成均较正常培养条件下(37℃)形成的生物膜少,差异均有统计学意义(均P<0.05)。随着H_2O_2浓度的增加,W83和△PG0352形成的生物膜均逐渐减少;在H_2O_2终浓度分别为0.1、0.25和0.5 mmol/L时,△PG0352形成的生物膜均比W83少(均P<0.05):在碱性条件下(pH=9),两者的生物膜形成均较正常条件下(pH=7.2)减少,并且△PG0352形成生物膜比W83少;但在酸性条件下(pH=5),两种菌株均不能形成生物膜。结论唾液酸酶基因缺失影响P.gingivalis的生物膜合成。在非正常培养条件下,无论是P.gingivalis W83还是△PG0352,其生物膜的形成能力均有所减弱。Objective To compare the biofilm formation of P.gingivalis W83 and its sialidase-deficient mutant strain （△PG0352） under stressful conditions.Methods P.gingivalis W83 and △PG0352 were cultured in trypticase soy broth （TSB）and supplemented with 5 μghnL hemin and 1 μg/mL menadione under stressful conditions,including temperature,oxidatie stress and pH values.Biofilms were formed on the 96-well plates for four days,and stained with crystal violet.Detect the biofilm formation with spectrophotometer.Results Both P.gingivalis W83 and △PG0352 strains could form biofilms in normal culture conditions.P.gingivalis W83 formed more biofilm,compared to △PG0352 （P 〈 0.05）.The biofilm formations of P.gingivalis W83 and △PG0352 decreased in 34℃ and 41 ℃ （P 〈 0.05）.As the concentration of H2O2 increased,biofilm formations of both strains decreased more and more seriously.The biofilm formations of △PG0352 were less than those of P.gingivalis W83,when the ultimate concentration of hydrogen peroxide was 0.1,0.25 and 0.5 mmol/L.Both P.gingivalis W83 and △PG0352 formed less biofilm in alkaline condition （pH =9）,△PG0352 even less,and were unable to form biofilm in acidic condition （pH =5）.Conclusion Sialidase gene deletion can affect P.gingivalis biofilm formation.Biofilms formations of both P.gingivalis W83 and △PG0352 will decrease under stressful conditions.

关 键 词：牙龈卟啉单胞菌 唾液酸酶 生物膜 

分 类 号：R78[医药卫生—口腔医学]