CAJ | 학술논문

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，50 mg／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18 h。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。目的：探討伊犛黑蜂蜂膠乙醇提取物（Ethanol extract of Propolis，EEP）與氟化鈉（NaF）對緻齲變形鏈毬菌屬產痠、產糖代謝關鍵酶乳痠脫氫酶（Lactate dehydrogenase， LDH ）與葡萄糖基轉移酶（Glycosyltransferase，GTF）及基因錶達的影響及其防齲機製。方法分彆培養遊離狀態與生物膜狀態下生長的變形鏈毬菌、遠緣鏈毬菌，以含梯度濃度 EEP 的腦心浸液培養基（Brian Heart Infusion，BHI）作為實驗組，50 mg／L NaF 的 BHI 培養基作為暘性對照組，以不添加任何藥物的 BHI 培養基作為陰性對照組。各組與不同生長狀態的細菌作用18 h。採用還原性輔酶 I 氧化法測定乳痠脫氫酶活性。硫痠銨鹽析結閤超濾離心法提取 GTFs 粗酶，蒽酮法測定 GTFs 活性。RT-qPCR 法測定各組 LDH 編碼基因 ldh 錶達和各組 GTFs 編碼基因 gtfb 、gtfc 、gtfd 錶達。結果在遊離狀態與生物膜狀態下，變形鏈毬菌、遠緣鏈毬菌各實驗組和 NaF 組 LDH 活性均明顯受到抑製（P ＜0．05）。在遊離狀態和生物膜狀態下，EEP 組、NaF 組變形鏈毬菌 GTFs 活性沒有變化（P ＞0．05）。遊離狀態時，變形鏈毬菌、遠緣鏈毬菌實驗組和 NaF 組 ldh 錶達明顯受到抑製（P ＜0．05）；生物膜狀態下，變形鏈毬菌實驗組在1、1／2、1／4MBEC 濃度時 ldh 錶達受到抑製（P ＜0．05），遠緣鏈毬菌實驗組在1、1／2、1／4、1／8MBEC 濃度時ldh 錶達受到抑製（P ＜0．05），NaF 組細菌在 ldh 錶達差異沒有統計學意義（P ＞0．05）。遊離狀態與生物膜狀態下，變形鏈毬菌實驗組 gtfb 、gtfc 、gtfd 基因錶達與陰性對照組差異無統計學意義（P ＞0．05）。遊離與生物膜生長狀態下，變形鏈毬菌 NaF 對 gtfb 基因錶達有促進作用（P ＜0．05）。結論伊犛黑蜂蜂膠是通過抑製產變形鏈毬菌屬糖代謝途徑 LDH 活性及其轉錄來抑製變形鏈毬菌產痠，從而達到防齲的效果。
목적：탐토이리흑봉봉효을순제취물（Ethanol extract of Propolis，EEP）여불화납（NaF）대치우변형련구균속산산、산당대사관건매유산탈경매（Lactate dehydrogenase， LDH ）여포도당기전이매（Glycosyltransferase，GTF）급기인표체적영향급기방우궤제。방법분별배양유리상태여생물막상태하생장적변형련구균、원연련구균，이함제도농도 EEP 적뇌심침액배양기（Brian Heart Infusion，BHI）작위실험조，50 mg／L NaF 적 BHI 배양기작위양성대조조，이불첨가임하약물적 BHI 배양기작위음성대조조。각조여불동생장상태적세균작용18 h。채용환원성보매 I 양화법측정유산탈경매활성。류산안염석결합초려리심법제취 GTFs 조매，은동법측정 GTFs 활성。RT-qPCR 법측정각조 LDH 편마기인 ldh 표체화각조 GTFs 편마기인 gtfb 、gtfc 、gtfd 표체。결과재유리상태여생물막상태하，변형련구균、원연련구균각실험조화 NaF 조 LDH 활성균명현수도억제（P ＜0．05）。재유리상태화생물막상태하，EEP 조、NaF 조변형련구균 GTFs 활성몰유변화（P ＞0．05）。유리상태시，변형련구균、원연련구균실험조화 NaF 조 ldh 표체명현수도억제（P ＜0．05）；생물막상태하，변형련구균실험조재1、1／2、1／4MBEC 농도시 ldh 표체수도억제（P ＜0．05），원연련구균실험조재1、1／2、1／4、1／8MBEC 농도시ldh 표체수도억제（P ＜0．05），NaF 조세균재 ldh 표체차이몰유통계학의의（P ＞0．05）。유리상태여생물막상태하，변형련구균실험조 gtfb 、gtfc 、gtfd 기인표체여음성대조조차이무통계학의의（P ＞0．05）。유리여생물막생장상태하，변형련구균 NaF 대 gtfb 기인표체유촉진작용（P ＜0．05）。결론이리흑봉봉효시통과억제산변형련구균속당대사도경 LDH 활성급기전록래억제변형련구균산산，종이체도방우적효과。
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 expression.Con-clusion Possible mechanism of Yili dark Propolis inhibiting acid production of Mutans Streptococci is in-hibiting LDH activity as well as down regulating ldh gene expression in both planktonic and biofilm state. Propolis has little affect on the GTF activity and gtfs gene expression.