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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 investigate the effect of sialidase gene deletion on the biofilm formation of Porphyromonas gingivalis (P. gingivalis) under different culture conditions. Methods The cells were cultured in a 96-well plate containing 5% sterile defibrinated sheep’s blood, hemin (5μg / mL), and vitamin K (1μg / mL) at different temperatures and concentrations of hydrogen peroxide (H 2 O 2) (W83) and sialidase gene mutant (△ PG0352) were cultured on TSB medium for 4 days. The crystal violet staining and alcohol decolorization were used to detect the formation of biofilm. Results Both P.gingivalis W83 and APG0352 could form biofilms under normal conditions. Compared with P.gingivalis W83, APG0352 formed less biofilms. Biofilm formation of P. gingivalis W83 and △ PG0352 at 34 ℃ and 41 ℃ were less than those under normal culture (37 ℃), both of which were statistically significant (P <0.05). The biofilm formed by W83 and △ PG0352 decreased with the increase of H 2 O 2 concentration. Biofilm formation of △ PG0352 was less than that of W83 at the final concentration of H_2O_2 of 0.1, 0.25 and 0.5 mmol / L (all P <0.05 ): Under alkaline conditions (pH = 9), biofilm formation of both decreased compared with normal conditions (pH = 7.2), and △ PG0352 formed biofilms less than W83; however, under acidic conditions ), Neither strain can form a biofilm. Conclusion The lack of sialidase gene affects biofilm synthesis of P. gingivalis. In non-normal culture conditions, both P. gingivalis W83 and △ PG0352 had weakened biofilm formation ability.