柠檬精油对Streptococcus mutans疏水、粘附性及srtA基因表达的影响
发布时间：2018-10-22 17:22
【摘要】：目的牙菌斑是龋病的始动因子,变形链球菌(Streptococcus mutans,S.mutans)是牙菌斑中的主要致龋菌。S.mutans粘附在牙齿表面形成牙菌斑,是龋病形成的重要环节。Sortase A酶(编码基因Srt A)介导S.mutans表面蛋白(如表面蛋白Spa P)与细菌细胞壁共价结合的蛋白酶,在S.mutans对牙面的粘附中起着重要作用。本研究在前期工作的基础上进一步分析柠檬精油(lemon essential oil,LEO)对S.mutans疏水性、粘附性、基因srtA的影响,并与LEO的主要成分柠檬烯(limonene,LIM)以及防龋植物产物茶多酚(tea polyphenol,TP)作对比,为天然口腔防龋材料的研发提供实验依据。内容通过体外实验研究经LEO、LIM、TP处理后,S.mutans的表面疏水性、粘附性和srtA、spa P基因表达水平的变化,探讨LEO等减低S.mutans毒性作用从而抑制牙菌斑形成的可能机制。方法1.LEO的提取、抑菌及细胞增殖实验采用水蒸气蒸馏法提取挥发油,测定LEO、LIM、TP对S.mutans的最小抑菌浓度(Minimal inhibitory concentration,MIC),MTT法测定LEO对人脐静脉内皮细胞增殖的影响。2.测定LEO、LIM及TP对S.mutans疏水性、粘附的影响选用微生物黏着碳氢化合物法测定低于MIC五个浓度的LEO、LIM及TP对S.mutans表面疏水性的影响。通过96孔微孔板结晶紫染色法,测定不同浓度植物产物对S.mutans在蛋白包被聚苯乙烯表面粘附的影响,并将结果进行比对。3.测定LEO、LIM及TP对S.mutans的srtA、spa P基因转录水平表达的影响采用实时荧光定量PCR方法,测定不同浓度植物产物处理后S.mutans的srtA、spa P基因的表达水平。4.采用SPSS19.0软件进行数据分析,采用单因素方差分析(ANOVA)进行各实验和对照组总体均数的比较,并计算F值;在F值有意义的前提下,再进行多个实验样本均数之间的两两比较。结果1.LEO、LIM、TP对S.mutans的MIC分别为4.5mg/ml、21mg/ml、4mg/ml。1/20MIC浓度的LEO作用于人脐静脉内皮细胞后,细胞增殖率为92.9%;与空白组相比,1/200MIC浓度的LEO对人脐静脉内皮细胞增殖的影响,差异不具有统计学意义。2.未经药物处理的S.mutans的表面疏水性为76.725%。低于MIC的LEO(1/2MIC~1/20000MIC)、LIM(1/2MIC~1/2000MIC)、TP(1/2MIC~1/20000MIC),对S.mutans表面疏水性具有抑制作用,抑制作用随药物浓度的升高而逐渐增高(FLEO=1013.944,FLIM=482.48,FTP=618.109,P0.05)。比较1/2MIC浓度下三种天然植物成分对S.mutans表面疏水性的抑制作用,LEOLIMTP,差异有统计学意义(F=91.041,P0.05)。相同浓度下,LEO对S.mutans表面疏水性的抑制作用始终高于LIM。1/2MIC、1/20MIC浓度下,LEO对S.mutans表面疏水性的抑制作用高于TP。3.低于MIC的LEO(1/2MIC~1/200MIC)、LIM(1/2MIC~1/20MIC)、TP(1/2MIC~1/200MIC),对S.mutans蛋白包被的聚乙烯表面粘附具抑制作用(FLEO=73.939,FLIM=57.264,FTP=73.410,P0.05)。比较1/2MIC下三种天然植物成分对S.mutans粘附的抑制作用,差异无统计学意义(F=1.43,P0.05)。4.低于MIC的LEO、LIM、TP,对S.mutans的srtA基因表达具有抑制作用;从1/200MIC到1/2MIC的浓度范围内,随着LEO、LIM、TP浓度的逐渐升高,srtA基因的m RNA表达水平呈下降趋势。5.相同浓度下LEO对S.mutans的srtA基因表达的抑制作用始终高于LIM;比较LEO和TP对S.mutans的srtA基因表达影响,1/2MIC和1/20MIC浓度下LEO的抑制作用强于TP。6.低于MIC的LEO、LIM、TP,对S.mutans的spa P基因表达均无抑制作用。结论LEO、LIM、TP在亚抑菌浓度下,可以抑制S.mutans的srtA基因表达,降低S.mutans的表面疏水性,进而影响S.mutans的粘附性,其抑制作用具有浓度依赖性,随药物浓度的升高,抑制作用增强。LEO的抑制作用高于LIM、TP。LEO作为一种天然植物产物,具有较强的防龋应用前景,有望进一步研发安全具有防龋作用的新型植物药物。
[Abstract]:Objective dental plaque is the starting factor of dental caries, Streptococcus mutans (S. mutans) is the primary caries bacteria .S.mutans in dental plaque, which forms plaque on the surface of teeth and is an important link in the formation of dental caries. Sortase A (encoding gene Srt A) mediates the covalent binding of S. mutans surface proteins (e.g., surface protein Spa P) to bacterial cell walls and plays an important role in the adhesion of S. mutans to tooth surfaces. In this study, the effects of lemmon oil (LEO) on hydrophobicity, adhesion and srtA of S. mutans were further analyzed on the basis of the preliminary work, and compared with the main components of LEO, limonene and tea polyphenols and tea polyphenols (TP). and provides an experimental basis for the research and development of natural oral anticaries materials. The surface hydrophobicity, adhesion and srtA, the expression level of S. mutans were studied by means of in vitro experiments, and the possible mechanism of reducing S. mutans toxicity to inhibit the formation of dental plaque was discussed. Methods 1. The extraction, bacteriostasis and cell proliferation of LEO were used to extract volatile oil by steam distillation. The minimal inhibitory concentration (MIC) of LEO, quercetin and TP on the proliferation of human umbilical vein endothelial cells was determined by MTT assay. The effects of LEO, LTP and TP on hydrophobicity and adhesion of S. mutans were determined by microbiological adhesion hydrocarbon method. The effects of LEO, LTP and TP on the hydrophobicity of S. mutans were determined. The effects of different concentration of plant products on the adhesion of S. mutans on the surface adhesion of S. mutans in protein-coated polystyrene were determined through 96-hole microporous plate crystallization violet staining, and the results were compared. The expression levels of srtA and spa P in S. mutans were determined by real-time fluorescence quantitative polymerase chain reaction (PCR). The expression levels of stA and spa P genes in S.mutans were determined by real-time fluorescence quantitative PCR. SPSS19. 0 software was used for data analysis. A single factor analysis of variance (ANOVA) was used to compare the overall results of each experiment and the control group, and F value was calculated. Results 1. The MIC of LEO, CA125 and TP to S. mutans were 4.5mg/ ml, 21mg/ ml and 4mg/ ml respectively. The ratio of LEO of 1/ 200MIC to human umbilical vein endothelial cells was 92.9%, and the difference was not statistically significant compared with blank group. The surface hydrophobicity of S.mutans without drug treatment was 76. 725%. LEO (1/ 2MIC ~ 1/ 20000MIC), (1/ 2MIC ~ 1/ 2000MIC), TP (1/ 2MIC ~ 1/ 20000MIC), which were lower than MIC, inhibited the hydrophobicity of S. mutans surface. The inhibitory effect was increased with the increase of drug concentration (FLEO = 1013. 944, FLIM = 482.48, FTP = 618,109, P0.05). The inhibitory effect of three natural plant components on the surface hydrophobicity of S. mutans was compared at 1/ 2MIC. The difference was statistically significant (F = 91. 0, P0.05). In the same concentration, the inhibitory effect of LEO on the surface hydrophobicity of S. mutans was always higher than that of the surface hydrophobicity of S.mutans, and the inhibitory effect of LEO on the surface hydrophobicity of S. mutans was higher than that of TP. LEO (1/ 2MIC ~ 1/ 200MIC) was lower than MIC (1/ 2MIC ~ 1/ 20MIC), TP (1/ 2MIC ~ 1/ 200MIC), and the surface adhesion of polyethylene coated with S. mutans protein was inhibited (FLEO = 73. 939, FLIM = 57. 264, FTP = 73. 410, P0.05). The inhibitory effect of three natural plant components on the adhesion of S. mutans in 1/ 2MIC was not statistically significant (F = 1.43, P0.05). The expression of srtA gene was inhibited from 1/ 200MIC to 1/ 2MIC, and the mRNA expression level of srtA gene decreased in the concentration range from 1/ 200MIC to 1/ 2MIC. The inhibitory effect of LEO on the expression of srtA gene of S. mutans was always higher than that of LEO at the same concentration. The inhibitory effect of LEO and TP on the expression of srtA gene in S. mutans was stronger than that of TP. 6. There was no inhibitory effect on the expression of P gene in the spa of S.mutans, which was lower than that of the MIC. Conclusion The expression of the srtA gene of S. mutans can be inhibited by LEO, CA125 and TP in subinhibitory concentration, so that the surface hydrophobicity of S. mutans can be reduced, and the adhesion of S. mutans can be reduced, and the inhibition effect of the S. mutans has a concentration dependence, and the inhibition effect is enhanced with the increase of drug concentration. The inhibitory effect of LEO is higher than that of TP, TP. LEO, which is a natural plant product, has strong anticaries application prospect, and is expected to further develop new plant drugs with anti-caries function.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R780.2
本文编号：2287813
[Abstract]:Objective dental plaque is the starting factor of dental caries, Streptococcus mutans (S. mutans) is the primary caries bacteria .S.mutans in dental plaque, which forms plaque on the surface of teeth and is an important link in the formation of dental caries. Sortase A (encoding gene Srt A) mediates the covalent binding of S. mutans surface proteins (e.g., surface protein Spa P) to bacterial cell walls and plays an important role in the adhesion of S. mutans to tooth surfaces. In this study, the effects of lemmon oil (LEO) on hydrophobicity, adhesion and srtA of S. mutans were further analyzed on the basis of the preliminary work, and compared with the main components of LEO, limonene and tea polyphenols and tea polyphenols (TP). and provides an experimental basis for the research and development of natural oral anticaries materials. The surface hydrophobicity, adhesion and srtA, the expression level of S. mutans were studied by means of in vitro experiments, and the possible mechanism of reducing S. mutans toxicity to inhibit the formation of dental plaque was discussed. Methods 1. The extraction, bacteriostasis and cell proliferation of LEO were used to extract volatile oil by steam distillation. The minimal inhibitory concentration (MIC) of LEO, quercetin and TP on the proliferation of human umbilical vein endothelial cells was determined by MTT assay. The effects of LEO, LTP and TP on hydrophobicity and adhesion of S. mutans were determined by microbiological adhesion hydrocarbon method. The effects of LEO, LTP and TP on the hydrophobicity of S. mutans were determined. The effects of different concentration of plant products on the adhesion of S. mutans on the surface adhesion of S. mutans in protein-coated polystyrene were determined through 96-hole microporous plate crystallization violet staining, and the results were compared. The expression levels of srtA and spa P in S. mutans were determined by real-time fluorescence quantitative polymerase chain reaction (PCR). The expression levels of stA and spa P genes in S.mutans were determined by real-time fluorescence quantitative PCR. SPSS19. 0 software was used for data analysis. A single factor analysis of variance (ANOVA) was used to compare the overall results of each experiment and the control group, and F value was calculated. Results 1. The MIC of LEO, CA125 and TP to S. mutans were 4.5mg/ ml, 21mg/ ml and 4mg/ ml respectively. The ratio of LEO of 1/ 200MIC to human umbilical vein endothelial cells was 92.9%, and the difference was not statistically significant compared with blank group. The surface hydrophobicity of S.mutans without drug treatment was 76. 725%. LEO (1/ 2MIC ~ 1/ 20000MIC), (1/ 2MIC ~ 1/ 2000MIC), TP (1/ 2MIC ~ 1/ 20000MIC), which were lower than MIC, inhibited the hydrophobicity of S. mutans surface. The inhibitory effect was increased with the increase of drug concentration (FLEO = 1013. 944, FLIM = 482.48, FTP = 618,109, P0.05). The inhibitory effect of three natural plant components on the surface hydrophobicity of S. mutans was compared at 1/ 2MIC. The difference was statistically significant (F = 91. 0, P0.05). In the same concentration, the inhibitory effect of LEO on the surface hydrophobicity of S. mutans was always higher than that of the surface hydrophobicity of S.mutans, and the inhibitory effect of LEO on the surface hydrophobicity of S. mutans was higher than that of TP. LEO (1/ 2MIC ~ 1/ 200MIC) was lower than MIC (1/ 2MIC ~ 1/ 20MIC), TP (1/ 2MIC ~ 1/ 200MIC), and the surface adhesion of polyethylene coated with S. mutans protein was inhibited (FLEO = 73. 939, FLIM = 57. 264, FTP = 73. 410, P0.05). The inhibitory effect of three natural plant components on the adhesion of S. mutans in 1/ 2MIC was not statistically significant (F = 1.43, P0.05). The expression of srtA gene was inhibited from 1/ 200MIC to 1/ 2MIC, and the mRNA expression level of srtA gene decreased in the concentration range from 1/ 200MIC to 1/ 2MIC. The inhibitory effect of LEO on the expression of srtA gene of S. mutans was always higher than that of LEO at the same concentration. The inhibitory effect of LEO and TP on the expression of srtA gene in S. mutans was stronger than that of TP. 6. There was no inhibitory effect on the expression of P gene in the spa of S.mutans, which was lower than that of the MIC. Conclusion The expression of the srtA gene of S. mutans can be inhibited by LEO, CA125 and TP in subinhibitory concentration, so that the surface hydrophobicity of S. mutans can be reduced, and the adhesion of S. mutans can be reduced, and the inhibition effect of the S. mutans has a concentration dependence, and the inhibition effect is enhanced with the increase of drug concentration. The inhibitory effect of LEO is higher than that of TP, TP. LEO, which is a natural plant product, has strong anticaries application prospect, and is expected to further develop new plant drugs with anti-caries function.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R780.2
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