【摘要】：目的牙菌斑是齲病的始動因子,變形鏈球菌(Streptococcus mutans,S.mutans)是牙菌斑中的主要致齲菌。S.mutans粘附在牙齒表面形成牙菌斑,是齲病形成的重要環(huán)節(jié)。Sortase A酶(編碼基因Srt A)介導S.mutans表面蛋白(如表面蛋白Spa P)與細菌細胞壁共價結合的蛋白酶,在S.mutans對牙面的粘附中起著重要作用。本研究在前期工作的基礎上進一步分析檸檬精油(lemon essential oil,LEO)對S.mutans疏水性、粘附性、基因srtA的影響,并與LEO的主要成分檸檬烯(limonene,LIM)以及防齲植物產(chǎn)物茶多酚(tea polyphenol,TP)作對比,為天然口腔防齲材料的研發(fā)提供實驗依據(jù)。內(nèi)容通過體外實驗研究經(jīng)LEO、LIM、TP處理后,S.mutans的表面疏水性、粘附性和srtA、spa P基因表達水平的變化,探討LEO等減低S.mutans毒性作用從而抑制牙菌斑形成的可能機制。方法1.LEO的提取、抑菌及細胞增殖實驗采用水蒸氣蒸餾法提取揮發(fā)油,測定LEO、LIM、TP對S.mutans的最小抑菌濃度(Minimal inhibitory concentration,MIC),MTT法測定LEO對人臍靜脈內(nèi)皮細胞增殖的影響。2.測定LEO、LIM及TP對S.mutans疏水性、粘附的影響選用微生物黏著碳氫化合物法測定低于MIC五個濃度的LEO、LIM及TP對S.mutans表面疏水性的影響。通過96孔微孔板結晶紫染色法,測定不同濃度植物產(chǎn)物對S.mutans在蛋白包被聚苯乙烯表面粘附的影響,并將結果進行比對。3.測定LEO、LIM及TP對S.mutans的srtA、spa P基因轉錄水平表達的影響采用實時熒光定量PCR方法,測定不同濃度植物產(chǎn)物處理后S.mutans的srtA、spa P基因的表達水平。4.采用SPSS19.0軟件進行數(shù)據(jù)分析,采用單因素方差分析(ANOVA)進行各實驗和對照組總體均數(shù)的比較,并計算F值;在F值有意義的前提下,再進行多個實驗樣本均數(shù)之間的兩兩比較。結果1.LEO、LIM、TP對S.mutans的MIC分別為4.5mg/ml、21mg/ml、4mg/ml。1/20MIC濃度的LEO作用于人臍靜脈內(nèi)皮細胞后,細胞增殖率為92.9%;與空白組相比,1/200MIC濃度的LEO對人臍靜脈內(nèi)皮細胞增殖的影響,差異不具有統(tǒng)計學意義。2.未經(jīng)藥物處理的S.mutans的表面疏水性為76.725%。低于MIC的LEO(1/2MIC~1/20000MIC)、LIM(1/2MIC~1/2000MIC)、TP(1/2MIC~1/20000MIC),對S.mutans表面疏水性具有抑制作用,抑制作用隨藥物濃度的升高而逐漸增高(FLEO=1013.944,FLIM=482.48,FTP=618.109,P0.05)。比較1/2MIC濃度下三種天然植物成分對S.mutans表面疏水性的抑制作用,LEOLIMTP,差異有統(tǒng)計學意義(F=91.041,P0.05)。相同濃度下,LEO對S.mutans表面疏水性的抑制作用始終高于LIM。1/2MIC、1/20MIC濃度下,LEO對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),對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)。比較1/2MIC下三種天然植物成分對S.mutans粘附的抑制作用,差異無統(tǒng)計學意義(F=1.43,P0.05)。4.低于MIC的LEO、LIM、TP,對S.mutans的srtA基因表達具有抑制作用;從1/200MIC到1/2MIC的濃度范圍內(nèi),隨著LEO、LIM、TP濃度的逐漸升高,srtA基因的m RNA表達水平呈下降趨勢。5.相同濃度下LEO對S.mutans的srtA基因表達的抑制作用始終高于LIM;比較LEO和TP對S.mutans的srtA基因表達影響,1/2MIC和1/20MIC濃度下LEO的抑制作用強于TP。6.低于MIC的LEO、LIM、TP,對S.mutans的spa P基因表達均無抑制作用。結論LEO、LIM、TP在亞抑菌濃度下,可以抑制S.mutans的srtA基因表達,降低S.mutans的表面疏水性,進而影響S.mutans的粘附性,其抑制作用具有濃度依賴性,隨藥物濃度的升高,抑制作用增強。LEO的抑制作用高于LIM、TP。LEO作為一種天然植物產(chǎn)物,具有較強的防齲應用前景,有望進一步研發(fā)安全具有防齲作用的新型植物藥物。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R780.2

【相似文獻】

相關期刊論文 前1條

1 陸基宗;;檸檬,給健康加分[J];健康;2014年02期

相關會議論文 前2條

1 吳尚;燕鵬華;劉杰;楊志旺;梁麗霞;潘鋒鋒;張輝;雷自強;;溫和條件下Oxone~汶對檸檬精油的氧化[A];全國第十四屆大環(huán)化學暨第六屆超分子化學學術討論會論文專輯[C];2008年

2 李一帆;楊晨;杭天依;李光武;;檸檬精油對DXM肥胖鼠體重的影響及機制[A];中國解剖學會2013年年會論文文摘匯編[C];2013年

相關重要報紙文章 前2條

1 芭芭拉;化妝品配料自己加[N];山西科技報;2001年

2 劉華;家庭異味輕松除[N];保健時報;2007年

相關碩士學位論文 前10條

1 胡君姣;檸檬精油抑制酪氨酸酶機理研究及其在納米乳液中的應用[D];上海應用技術學院;2015年

2 陳思佳;檸檬精油等提取物對變形鏈球菌luxS基因的影響[D];天津醫(yī)科大學;2015年

3 劉雅麗;四種植物提取物抑制Streptococcus mutans LDH活性、清除自由基作用研究[D];天津醫(yī)科大學;2015年

4 趙艷茹;芝麻油開發(fā)為化妝保健油的工藝研究與設計[D];河南工業(yè)大學;2015年

5 張晨;檸檬精油對Streptococcus mutans疏水、粘附性及srtA基因表達的影響[D];天津醫(yī)科大學;2016年

6 孫振江;超臨界CO_2萃取檸檬精油的工藝研究[D];西北大學;2012年

7 趙燕琳;基于嗅吸法的香檸檬精油對人體腦電波的影響[D];上海交通大學;2012年

8 廖玉琴;檸檬果皮精油提取、微膠囊化工藝及抑菌性研究[D];西華大學;2013年

9 田大志;檸檬精油乳液的物理化學穩(wěn)定性研究[D];湖北工業(yè)大學;2014年

10 李一帆;檸檬精油經(jīng)嗅覺通路對DXM肥胖小鼠體重影響研究[D];安徽醫(yī)科大學;2013年

，

本文編號：2287813