摘要
菌斑生物膜是粘附于牙齿表面的微生物团体,是龋病发生的始动因子。生物膜中细菌的基因表达和表型明显不同于浮游细菌,对抗生素具有较强的抵抗力。正常情况下,生物膜内部的微生态处于平衡状态。若摄糖频率增加,菌斑更多时间处于低pH状态,利于致龋菌的增殖,平衡向脱矿方向转变,导致龋病发生。因此,去除或控制菌斑生物膜的形成,维持菌斑生物膜的稳态对预防龋病的发生具有重大的意义。中药五倍子富含鞣酸,具有很好的抑菌特性。动物实验及体外研究均表明五倍子可有效防龋,效果与2%氟化物相当,但有关五倍子对菌斑生物膜的影响尚无详细研究。本实验应用人工口腔建立菌斑生物膜模型,观察五倍子水提取物对菌斑生物膜的消除作用以及对菌斑生物膜形成和组成的影响;并通过釉质磨片获取口内菌斑生物膜模型,观察五倍子水提取物对菌斑生物膜活性的影响,旨在进一步探讨五倍子的防龋机理。
实验一 五倍子水提取物对菌斑生物膜的消除作用
本实验对各实验菌的最小抑菌浓度(MIC)进行了测定,并进一步应用人工口腔、菌落计数技术观察了五倍子水提取物对菌斑生物膜的消除作用。结果显示五倍子水提取物对变链菌、粘放菌、血链菌、口腔链球菌的生长均有抑制作用,其中对变链菌、粘放菌和血链菌的MIC为64mg/ml,口腔链球菌为8mg/ml;不同浓度的五倍子水提取物对变形链球菌、粘性放线菌、血链球菌、口腔链球菌形成的单一细菌菌斑生物膜均有一定的消除作用,随
第四军医大学硕士学位论文
着浓度的提高,釉质表面的细菌数逐渐减少,呈浓度依赖性。与
最小抑菌实验相比,发现五倍子水提取物即使采用大于MIC的
浓度,也不能把釉质表面形成的生物膜完全消除,釉质表面仍有
细菌生长。说明与浮游细菌相比,消除生物膜中的细菌需要较大
浓度的五倍子水提取物,这可能与菌斑生物膜的特性有关。
实验二五倍子水提取物对菌斑生物膜形成的影响
本实验应用人工口腔、菌落计数和扫描电镜技术观察五倍子
水提取物对菌斑生物膜形成的影响。结果显示,应用较小浓度的
五倍子水提取物(6mg/m1)处理后的釉质表面其细菌量明显少于
对照组,细菌散在分布于釉质表面,未见细菌团块的堆积;对照
组釉质表面细菌呈块状堆积,可见栅栏状结构和谷穗状结构的形
成。说明五倍子水提取物具有显著的抗生物膜形成作用。
实验三五倍子水提取物对菌斑生物膜组成的影响
本实验应用人工口腔和细菌的选择性培养技术观察五倍子
水提取物对菌斑生物膜组成的影响,从生态学的角度探讨五倍子
的防龋机理。结果显示,应用较小浓度的五倍子水提取物
(6mg/ml)处理后,生物膜中变形链球菌和粘性放线菌的比例明
显降低,血型链球菌和口腔链球菌的比例有所升高。说明五倍子
水提取物可防止变形链球菌在生物膜中的选择性生长,减少菌斑
微生物向有害的方向转变。
实验四五倍子水提取物对口内菌斑生物膜活性的影响
本实验在建立口内菌斑生物膜模型的基础上,应用EB/FDA
染色和CLSM技术观察五倍子水提取物对口内生物膜活性的影
响。结果显示五倍子水提取物处理后,生物膜内细菌的活性明
显低于对照组。提示五倍子水提取物对生物膜中的细菌具有良
好的杀伤效应。
Plaque biofilms, a well-organized microbial community on dental surface, is a major etiological factor of caries. Compared with planktonic bacteria, the bacteria in biofilms have the different gene expression and phenotype, as well as have an increased resistance to antimicrobials. In normal conditions, the microbial ecology of plaque biofilms is in a state of equilibrium, and the bacteria components of biofilms remain relatively stable. If the intake of suger is too frequent, the biofilms will be in a low pH in most of time, which will benefit the proliferation of S.mutans and Lactobacillus, and further lead to caries. So the control on the formation of plaque biofilms and the maintenance of microcosm homeostasis play an important role in preventing caries. Gallnut contains tannin abundantly and can be used as an effective drug in the prevention of caries. The aim of the our research was to investigate the effects of gallnut water extract on the plaque biofilms in vitro, thereby to explore its mechanism of
preventing caries.
1. The role of of gallnut water extract in eradicating plaque biofilms
In this part, the minimal inhibitory concentrations(MIC) of all experimental strains were determined, and the role of gallnut water extract in eradicating plaque was further investigated by using the artificial mouth and the technology of colony counting. The results
showed that gallnut water extract could inhibit the growth of S.mutans, A.viscosus, S.sanguis and S.oralis. The MIC of S.mutans was 64mg/ml, which was same as A.viscosus and S.sanguis. The MIC of S.oralis was 8mg/ml; The plaque biofilms formed by S.mutans, A.viscosus, S.sanguis and S.oralis could be eradicated effectively by different concentrations of gallnut water extract respectively. With the increasing concentration, the number of bacteria was reduced gradually, and the effect was dose-dependent. Compared with the experiment in determining the MIC, it was found that even though higher concentration than MIC of gallnut water extract was adopted, the plaque biofilms couldn't be eradicated completely. It suggested that the eradication of plaque biofilms need a higher concentration of gallnut water extract comparing with the planktonics, which might correlate with the propert of plaque biofilms.
2. The effects of gallnut water extract on the formation of plaque biofilms
The artificial mouth, the technology of colony counting and scanning electron microscope(SEM) were used to investigate the effects of gallnut water extract on the formation of plaque biofilms. The results of colony counting showed that the amount of bacteria on enamel fragments treated with gallnut water extract was less than that of control team, and the difference between two groups was significant. The observation with SEM revealed that enamel fragments treated with gallnut water extract had no biofilm formed, the bacteria were distributed scatteredly, while the mature biofilm occurred on the control enamel fragments . So we can draw a conclusion that Chinese nutgall can inhibit the formation of oral
biofilm significantly.
3. The effects of gallnut water extract on the composition of plaque biofilms
The artificial mouth and the technology of selective culture of
bacteria were used to observe the effects of gallnut water extract on the composition of plaque biofilms. The results showed that gallnut water extract could reduce the proportion of S.mutans and A.vicosus, and increase the proportion of S.sanguis and S.oralis, It suggested that gallnut water extract can prevent the selective growth of S.mutans in biofilms, and can reduce the harmful conversion of microbial.
4. The effects of gallnut water extract on the vitality of plaque biofilms model in the oral cavity
The technology of EB/FDA staining and CLSM were used to observe the effects of gallnut water extract on the vitality of of plaque biofilms on the basis of establishing a plaque biofilms model in the oral cavity. The results showed that gallnut water extract could decrease the vatility of plaque b
引文
1 Marsh PD. Host defense and microbial homeostasis: role of microbial interactions.J Dent Res.1989, 68:1567-1575
2 周学东,胡涛,李继遥.牙菌斑生态防治新概念.国外医学口腔医学分册,1997,24(6):323
3 Van der Hoeven JS, Camp PJM. Synergistic degradation of muscin by Streptococcus oralis and Streptococcus sanguis in mixed chemostat cultures. J Dent Res.1991, 70:1041-1044
4 Marsh PD. Host defenses and microbial homeostasis: role of microbial interactions. J Dent Res.68:1567-1575
5 Marsh PD.Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res. 1994 Jul,8(2):263-71
6 Marsh PD. The role of chemostats in the evaluation of antimicrobial agents for use in dental products. Microb Ecol Hlth Dis.1993,6:147-149
7 Bradshaw D J, Mckee AS, MarshPD. Effects of pH on the stability of a microbial community. J Dent Res. 1989b, 68:930
8 Svanberg M. Streptococcus mutans in plaque after mouthrinsing with buffers of varying pH value. J Dent Res. 1980, 54:872-880
9 Bradshaw DJ, Mckee AS, MarshPD. Prevention of population shifts in oral microbial communities in vitro by low fluoride concentrations. J Dent Res.1990, 69:436-441
10 Hamilton IR, Bowden GH. Response of freshly isolated strains of Streptococcus mutans and Streptococcus mitior to change in pH in the presence and absence of fluoride during growth in continuous culture. Infect Immun. 1982 Apr,36(1):255-62
11 Marsh PD. Microbiological aspects of the chemical control of
plaque and gingivitis. J Dent Res. 1992, 71:1431-143812 Donoghue HD. Can the colonization resistance of the oral microflora be enhanced? Microb Ecol Hlth Dis. 1990,3:ⅰ-ⅳ
13 Tanzer JM. On changing the cariogenic chemistry of coronal plaque. J Dent Res1989, 68:1576-1587
14 Hillman JD, Socransky SS. The theory and application of bacterial interference to oral diseases. In : Myers HM, editor. New biotechnology in oral research. Basel: Karger, 1-17
15 Heasman PA, Seymour RA. Pharmacological control of periodontal disease. I. Antiplaque agents. J Dent. 1994 Dec,22(6):323-35
16 Williams MJ, Adams D, Hillam DG, Ashley KC. The effect of hexetidine 0.1% in the control of dental plaque. Br Dent J. 1987 Nov 7,163(9):300-2
17 Ashley FP, Skinner A, Jackson P, Woods A, Wilson RF. The effect of a 0.1% cetylpyridinium chloride mouthrinse on plaque and gingivitis in adult subjects. Br Dent J. 1984,157(6):191-6
18 Ciardi JE, Bowen WH, Rolla G. The effect of antibacterial compounds on glucosyltransferase activity from Streptococcus mutans. Arch Oral Biol. 1978,23(4):301-5
19 Mandel ID. Chemotherapeutic agents for controlling plaque and gingivitis. J Clin Periodontol. 1988 Sep,15(8):488-98
20 Maruniak J, Clark WB, Walker CB, Magnusson I, Marks RG, Taylor M, Clouser B. The effect of 3 mouthrinses on plaque and gingivitis development. J Clin Periodontol. 1992,19(1): 19-23
21 Weiss EI, Lev-Dor R, Kashamn Y, Goldhar J, Sharon N, Ofek I. Inhibiting interspecies coaggregation of plaque bacteria with a cranberry juice constituent [published erratam appear in J Am
Dent Assoc 1999 Ja n;130(1):36 and 1999,130(3):332 J Am Dent Assoc. 1998,129(12):1719-23
22 Sato S, Yoshinuma N, Ito K, Tokumoto T, Takiguchi T, Suzuki Y, Mural S. The inhibitory effect of funoran and eucalyptus extract-containing chewing gum on plaque formation. J Oral Sci. 1998,40(3):115-7
23 Saito N. Anti-caries effects of polyphenol compound from Camellia sinensis. Nichidai Koko Kagaku. 1990,16(2): 154-63
24 Ooshima T, Minami T, Aono W, Tamura Y, Hamada S. Reduction of dental plaque deposition in humans by oolong tea extract. Caries Res. 1994,28(3):146-9
25 王少虎.中草药防龋研究进展.国外医学口腔医学分册.2001,28 (1):49-51
26 Kozai K, Miyake Y, Kohda H, Kametaka S, Yamasaki K, Suginaka H, Nagasaka N. Inhibition of glucosyltransferase from Streptococcus mutans by oleanolic acid and ursolic acid. Caries Res. 1987,21(2):104-8
27 Wu-Yuan CD, Chen CY, Wu RT. Gallotannins inhibit growth, water-insoluble glucan synthesis, and aggregation of mutans streptococci. J Dent Res. 1988 Jan,67(1):51-5
28 王艳,唐荣银,陈强,等.中药五倍子防龋的动物试验研究[J].牙体牙髓牙周病学杂志.2001,11(3):172
29 黄正蔚,周学东等.中药五倍子对口腔致龋菌影响的体外研究.华西药学杂志.2002,17(2):104-106
30 李伟丽,唐荣银,陈强,等.五倍子抑龋作用的实验研究.牙体牙髓病学杂志.2002,12(5):258
31 李艳君,唐荣银,吕昕,刘斌.五倍子提取液对牙本质龋损时胶原蛋白分解的影响.牙体牙髓病学杂志.2002,12(5):252
32 朱秀丽,陈强,唐荣银等.五倍子对5种常见牙周细菌抑制作用的体外研究.牙体牙髓病学杂志.2002,12(5):255
33 王志良 五倍子水提取物对牙周炎症抗炎机理的实验研究[学位论文].第四军医大学口腔医学院 2003
34 王静,唐荣银,王志良等.五倍子水提取物对人牙周膜成纤维细胞保护作用的实验研究.牙体牙髓牙周病学杂志.2003.13(5):260—263
35 McKee AS, McDermid AS, Ellwood DC, Marsh PD。The stablishment of reproducible, complex communities of oral bacteria in the chemostat using defined inocula. J Appl Bacteriol. 1985,59(3):263-75
36 Herles S, Olsen S, Afflitto J, Gaffar A. Chemostat flow cell system: an in vitro model for the evaluation of antiplaque agents. J Dent Res. 1994,73(11):1748-55
37 Bradshaw DJ, Marsh PD, Schilling KM, Cummins D. A modified chemostat system to study the ecology of oral biofilms. J Appl Bacteriol. 1996,80(2):124-30
38 乔伟民,樊明文.口腔恒化器的研制.口腔医学纵横 1997,13:148
39 乔伟民,樊明文等.应用口腔恒化器形成人工牙菌斑的扫描电镜研究.湖北医科大学学报 1998,19(4):368—370
40 李德懿,李宗林等.可控模拟口腔环境的改良MD-300恒化器的建立及应用.临床口腔医学杂志.2002,18(4)
41 Kinniment SL, Wimpenny JWT, Adams D. Microbiology. 1996, 142(pt3): 631-638
42 Kinniment SL, Wimpenny JW, Adams D, Marsh PD. The effect of chlorhexidine on defined, mixed culture oral biofilms grown in a novel model system. J Appl Bacterial. 1996,81(2):120-5
43 Pratten J, Barnett P, Wilson M. Composition and susceptibility to chlorhexidine of multispecies biofilms of oral bacteria. Appl Environ Microbiol. 1998, 64(9):3515-9
44 Pratten, R Bedi, and M Wilson. An in vitro study of the effect of fluoridated milk on oral bacterial biofilms. Applied and Environmental Microbiology. 2000, 66(4): 1720-1723
45 D.E. Hudson, H.D. Donoghue and C.J. Perrons, A laboratory microcosm (artificial mouth) for the culture and continuous pH measurement of oral bacteria on surfaces. Journal of Applied Bacteriology. 1986,60:301-310
46 C.J. Perrons and H.D. Donoghue, Colonization resistance of defined bacterial plaques to Streptococcus mutans implantation on teeth in a model mouth. Journal of Dental Research. 1990, 69:483-488
47 Sissons CH, Wong L, Cutress TW. Patterns and rates of growth of microcosm dental plaque biofilms. Oral Microbiol Immunol. 1995,10(3):160-7
48 Shu, L. Wong, J.H. Miller and C.H. Sissons, Development of multi-species consortia biofilms of oral bacteria as an enamel and root caries model system. Archives of Oral Biology. 2000,45:27-40
49 李鸣宇,刘正,朱彩莲.人工口腔牙菌斑生物膜药敏实验模型的建立.实用口腔医学杂志.2003,19(4):318—320
50 周泽渊,倪龙兴,吴补领,黄晓峰,王胜朝.用激光共聚焦显微镜研究菌斑生物膜的结构.牙体牙髓牙周病学杂志.2001,11(3):152-153
51 Zaura-Arite E, van Marie J,et al. Conofocal microscopy study of undisturbed and chlorhexidine-treated dental biofilm. Dent Res.
2001, 80(5):1436-40
52 Suci PA, Mittelman MW, Yu FP, Geesey GG. Investigation of ciprofloxacin penetration into Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother. 1994,38(9):2125-33
53 Stewart PS, Roe F, Rayner J, Elkins JG, Lewandowski Z, Ochsner UA, Hassett DJ. Effect of catalase on hydrogen peroxide penetration into Pseudomonas aeruginosa biofilms. Appl Environ Microbiol. 2000,66(2):836
54 Anderl JN, Franklin MJ, Stewart PS. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother. 2000,44:1818-1824
55 B Hoyle. Pseudomonas aeruginosa biofilm as a diffusion ba rrier to piperacillin. Antimicrob Agents Chemother. 1992,36: 2054-2056
56 Dunne WM Jr, Mason EO Jr, Kaplan SL. Diffusion of rifampin and vancomycin through a Staphylococcus epidermidis biofilm. Antimicrob Agents Chemother. 1993, 37:2522-2526
57 Tuomanen E, Cozens R, Tosch W, Zak O, Tomasz A. The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth. J Gen Microbiol. 1986,32 ( Pt 5):1297-304
58 Tuomanen E, Durack DT, Tomasz A. Antibiotic tolerance among clinical isolates of bacteria. Antimicrob Agents Chemother. 1986,30(4):521-7
59 Brown MR, Allison DG, Gilbert P. Resistance of bacterial biofilms to antibiotics: a growth-rate related effect? J Antimicrob Chemother. 1988,22(6):777-80
60 Wentland EJ, Stewart PS, Huang CT, McFeters GA. Spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm. Biotechnol Prog. 1996 May-Jun,12(3):316-21
61 Evans DJ, Allison DG, Brown MR, Gilbert P. Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate. J Antimicrob Chemother. 1991 Feb,27(2):177-84
62 Duguid IG, Evans E, Brown MR, Gilbert P. Growth-rate-in dependent killing by ciprofloxacin of biofilm-derived Staph ylococcus epidermidis; evidence for cell-cycle dependency. J Antimicrob Chemother. 1992 Dec, 30(6):791-802
63 Duguid IG, Evans E, Brown MR, Gilbert P. Effect of biofilm culture upon the susceptibility of Staphylococcus epidermidis to tobramycin. J Antimicrob Chemother. 1992 Dec,30(6): 803-10
64 Desai M, Buhler T, Weller PH, Brown MR. Increasing resistance of planktonic and biofilm cultures of Burkholderia cepacia to ciprofloxacin and ceftazidime during exponential growth. J Antimicrob Chemother. 1998 Aug,42(2): 153-60
65 Brooun A, Liu S, Lewis K. A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother. 2000,44(3):640-6
66 Jaffe A, Chabbert YA, Semonin O. Role of porin proteins OmpF and OmpC in the permeation of beta-lactams. Antimicrob Agents Chemother. 1982,22(6):942-8
67 A.P. Pugsley, C.A. Schnaitman, Outer membrane proteins of Escherichia coli.Ⅶ. Evidence that bacteriophage-directed protein 2 functions as a pore. J. Bacteriol. 1978, 133:1181-1189
68 Prigent-Combaret C, Vidal O, Dorel C, Lejeune P. Abiotic
surface sensing and biofilm-dependent regulation of gene expression in Eschericia coli. J Bacteriol. 1999, 181:5993-6002