伊犁黑蜂蜂胶对变形链球菌属及其生物膜代谢影响研究  

In vitrostudy of effect in Yili dark propolis on Mutans Streptococci metabolism in both planktonic and biofilm state

作　　者：祖力卡尔江.阿合买提 李艳[2] 孟凡琦[1] 赵今[1] 

机构地区：[1]新疆医科大学第一附属医院牙体牙髓科.新疆维吾尔自治区口腔医学研究所,乌鲁木齐830054 [2]新疆医科大学第五附属医院口腔科,乌鲁木齐830011

出　　处：《新疆医科大学学报》2015年第7期843-849,共7页Journal of Xinjiang Medical University

基　　金：新疆地方病分子生物学实验室开放课题基金(XJDX0208-2011-06)

摘　　要：目的探讨伊犁黑蜂蜂胶乙醇提取物(Ethanol extract of Propolis,EEP)与氟化钠(NaF)对致龋变形链球菌属产酸、产糖代谢关键酶乳酸脱氢酶(Lactate dehydrogenase,LDH)与葡萄糖基转移酶(Glycosyltransferase,GTF)及基因表达的影响及其防龋机制。方法分别培养游离状态与生物膜状态下生长的变形链球菌、远缘链球菌,以含梯度浓度EEP的脑心浸液培养基(Brian Heart Infusion,BHI)作为实验组,50mg/L NaF的BHI培养基作为阳性对照组,以不添加任何药物的BHI培养基作为阴性对照组。各组与不同生长状态的细菌作用18h。采用还原性辅酶I氧化法测定乳酸脱氢酶活性。硫酸铵盐析结合超滤离心法提取GTFs粗酶,蒽酮法测定GTFs活性。RT-qPCR法测定各组LDH编码基因ldh表达和各组GTFs编码基因gtfb、gtfc、gtfd表达。结果在游离状态与生物膜状态下,变形链球菌、远缘链球菌各实验组和NaF组LDH活性均明显受到抑制(P<0.05)。在游离状态和生物膜状态下,EEP组、NaF组变形链球菌GTFs活性没有变化(P>0.05)。游离状态时,变形链球菌、远缘链球菌实验组和NaF组ldh表达明显受到抑制(P<0.05);生物膜状态下,变形链球菌实验组在1、1/2、1/4MBEC浓度时ldh表达受到抑制(P<0.05),远缘链球菌实验组在1、1/2、1/4、1/8MBEC浓度时ldh表达受到抑制(P<0.05),NaF组细菌在ldh表达差异没有统计学意义(P>0.05)。游离状态与生物膜状态下,变形链球菌实验组gtfb、gtfc、gtfd基因表达与阴性对照组差异无统计学意义(P>0.05)。游离与生物膜生长状态下,变形链球菌NaF对gtfb基因表达有促进作用(P<0.05)。结论伊犁黑蜂蜂胶是通过抑制产变形链球菌属糖代谢途径LDH活性及其转录来抑制变形链球菌产酸,从而达到防龋的效果。Objective To expolore effect of ethanol extract of Propolis （EEP）and NaF on bacteria′s lactate dehydrogenase（LDH）and glycosyltransferase（GTFs）activity and transcription of related coding gene on mechanism of Caries-preventive.Methods Streptococcus mutans and Streptococcus sobrinus in both planktonic and biofilm state were incubated,which were interacting with BHI culture medium which con-taining various concentration of EEP;BHI culture medium which containing 50mg/L NaF,and BHI cul-ture medium were interacted for 18 hours.Coenzyme I reduced method was used to assay LDH activity of each group.GTFs crude enzyme was extracted from supernatant ;anthrone method was used to calculate GTF activity .Total RNA was extracted by Trizol,and levels of ldh gtfb,gtfc,gtfd were tested by means of RT-qPCR.Results The significant inhibition of LDH activity found between all study groups and NaF group compared to negative control in both planktonic and biofilm of Streptococcus mutans and Streptococ-cus sobrinus after interaction （P〈0.05）.As for planktonic state of Streptococcus mutans and Streptococ-cus sobrinus,after interaction with all groups,significant down-regulation of ldh was detected in both study group and NaF group （P〈0.05）.ldh down-regulation ratio in study group was greater than NaF group （P〈0.05）;to the state of biofilm,Streptococcus mutans after interaction with all groups,study groups contain MBEC,1/2,1/4MBEC EEP significantly down-regulated ldh expression compared to the negative control （P〈0.05）;for Streptococcus sobrinusbiofilm,all concentrations of EEP in study group significantly down-regulate ldh expression compared to the negative control （P〈0.05）.While NaF group in neither Streptococcus mutans and Streptococcus sobrinus didn＇t show difference to negative control （P 〉0.05）.In both planktonic and biofilm state of Streptococcus mutans,expression of gtfb,gtfc,gtfd weren＇t affected by EEP in study group,as for positive control group,NaF upregulated the gtfb ex

关 键 词：蜂胶 变形链球菌属 乳酸脱氢酶 葡萄糖基转移酶 荧光定量PCR 

分 类 号：R780.1[医药卫生—口腔医学]