中药复方提取液对牙周可疑致病菌和人牙周膜细胞作用的临床和实验研究
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摘要
目的
牙周病是口腔常见病和多发病，是一种发生在牙周支持组织上以细菌为主的感染性疾病，是成年人口腔中病理性失牙最主要的原因。牙周可疑致病菌是牙周病的主要致病因素，是引起牙周组织慢性破坏性疾病的始动因子。消除致病因子和阻断牙周病的发生发展是预防和治疗牙周病的重要手段，如采用龈上下洁刮治术或药物治疗，控制引起牙周病的牙菌斑。长期以来，许多学者对中医药在牙周病的病因、治疗和预防方面都进行了大量的研究，特别是二十世纪八十年代以来，天然药物防治口腔疾病逐渐受到国内外学者的关注。本研究采用名中医干祖望教授推荐的经验方，制成中药复方提取液进行了相关的临床和实验研究，并且按照世界卫生组织《口腔健康调查基本方法》进行牙周健康状况的调查。
了解人群牙周健康流行状况，既为开展口腔预防保健提供了基线资料，又进一步说明研究开发防治牙周病药物具有十分重要的意义；探讨中药复方提取液减少牙茵斑的作用和寻找一个合适的用药方法；观察中药提取物对牙周病可疑致病菌的杀菌作用；观察中药复方提取液在不同浓度下对人牙周膜成纤维细胞(HPLF)增殖和总蛋白含量的变化以及观察中药复方提取液对HPLF形态和超微结构的影响。为研究该中药防治牙周病的机制提供实验依据。
方法
本课题研究分实验研究、临床研究和流行病学研究三个部分
1、实验研究
中药复方提取液：选用江苏省中医院名中医干祖望教授推荐的防治牙周病的方药(以金银花、紫花地丁、蚤休、芦根等组成)，中药材来源于江苏省中医院药剂科。将中药材加5倍量水提取3次，过滤合并滤液，90％乙醇沉淀，离心后去除沉淀，取上清液，浓缩至所需浓度1g／ml作为原液。原液于沸水中煮沸10min，备用。
实菌验株及培养基：选用国际标准菌株(由上海交通大学附属第九人民医院口腔医学研究所提供)：牙龈卟啉单胞菌(Porphyromonas gingivalis，Pg)ATCC 33277、具核梭杆菌(Fusobacterium rlucleatum，Fn)ATCC 25586、伴放线放线杆菌(Actinobacillus actinomycetemcomitans，Aa)ATCC Y4。用专性厌氧培养基(杭州天和微生物试剂有限公司提供)传代培养。变形链球菌(st reptOCOCCUS mutans，sm)NCTC Ingbritt。用(Ms)轻唾培养基(杭州天和微生物试剂有限公司提供)传代培养。
细胞培养：取正畸需要拔除的无龋、无牙周病的10～16岁青少年前磨牙，随即放置于含有100u／ml青霉素、100μg／ml链霉素预冷的DMEM培养基中，在超净工作台内，无菌条件下，刮取牙根中1／3的牙周膜组织，剪成1mm×1mm×1mm小块，平铺于25ml培养瓶中，加入含有20％血清、100u／ml青霉素、100μg／ml链霉素的DMEM培养基2ml，在CO_2培养箱中，37℃、5％CO_2饱和条件下培养4 h后，翻转培养瓶，继续培养。有细胞游出后，每隔3天换液，细胞长满瓶底壁后，以0.25％胰蛋白酶消化，传代。凡免疫组化角蛋白阴性、波形蛋白阳性的4～10代细胞用于实验。
配制系列浓度中药提取液：取1g／ml中药贮存液，用5％血清的DMEM培养基将其稀释成0g／ml(对照组)、1×10~(-7)g／ml、1×10~(-6)g／ml、1×10~(-5)g／ml、1×10~(-4)g／ml、1×10~(-3)g／ml、1×10~(12)g／ml不同浓度的条件培养基。
体外敏感试验操作程序(微量液体稀释法)：采用TSA血平板培养基。厌氧培养18小时(90％N_2 5％CO_2 37℃)。保存之细菌复苏，经生化鉴定后接种于相应琼脂平板。挑取琼脂表面的单个菌落混悬于相应液体培养基中(1个1mm菌落／ml)培养，培养相应时间后使菌液浓度呈10~(-6)CFU／ml备用。
分别吸取两种样品原液。药品液为原液对倍稀释(1：1，1：2，1：4……)。体外敏感试验采用微量液体稀释法检测MBC(最小杀菌浓度)。
MBC测定：用无菌接种环挑取肉眼观察无细菌生长的微孔板中培养物，划线接种于相应平板，放置适宜培养条件下孵育。48h培养后观察，无细菌生长的最低药物浓度为MBC。
MTT法测定细胞增殖：取第5代细胞，用0.25％胰蛋白酶消化，以1×10~5／ml的细胞浓度接种到96孔板，每孔100μl，培养24h后，吸弃原有培养液，每孔加入条件培养液：200μl，每个浓度8孔。分别于培养1d、3d、5d后，每孔加入MTT(5mg／ml)20μl，37℃，5％CO_2饱和条件下培养4h，吸弃培养液，每孔加入DMSO150μl，振荡10min，于酶标仪测定OD值，波长为490nm。仅含有5％血清的培养基作为对照组。
考马斯亮蓝染色法测定细胞总蛋白：取第5代细胞，用0.25％胰蛋白酶消化，以1×10~5／ml的细胞浓度接种到96孔板，每孔100μl，培养24h后，吸弃原有培养液，每孔加入条件培养液200μl(不包括1×10~(-2)g／ml)，每个浓度5孔。分别于培养1d、3d、5d后，每孔加入100μl0.1％TritonX-100，室温下振荡30min，吸取20μl，转移到另一块96孔板，每孔加入考马斯亮蓝染液200μl，室温下振荡10min，用分光光度计在595nm波长处测定溶液的OD值。仅含有5％血清的培养基作为对照组。
超微结构：取第5代细胞，用0.25％胰蛋白酶消化接种到新的培养瓶中，培养24h后，吸弃原培养液，加入1×10~(-3)g／ml浓度条件培养液培养5d，以不含中药提取液的5％DMEM培养基作为对照组。第5天用0.25％胰蛋白酶消化，分别收集在离心管中，1000r／min，10min离心后，弃上清液。2％戊二醛固定细胞团块2h，室温下系列丙酮脱水，EPON812包埋，超薄切片后透射电镜观察。
2、临床研究
中药复方提取液同实验研究，并将其配制成中药低浓度组(0.2g／ml，用蒸馏水稀释)、中药高浓度组(0.4g／ml，用蒸馏水稀释)、阳性对照组(1／2浓度的口泰)、阴性对照组(蒸馏水)4个组分别编为A、B、C、D 4个组。采用双盲法进行实验观察，提供漱口液者不参加实验检查，进行牙周指数检查的人员是经过培训的口腔医生，直接给幼儿提供漱口液并进行监督的是幼儿园老师。实验开始前由口腔医生对幼儿进行牙周指数的检查，再由幼儿园老师安排幼儿漱口，要求每天含漱2次，每次用量10ml，含漱2分钟后吐干净，连续二周，最后一次漱口完后1hr由口腔医生检查牙周指数。整个实验过程中幼儿不改变平时的生活习惯。测量牙周指数，包括菌斑指数(plaqueindex，PLI)和牙龈指数(gingival index，GI)。
3、流行病学研究
参考世界卫生组织(WHO)《口腔健康调查基本方法》(第四版)调查方法，采用多阶段、分层、等容量、随机抽样的方法，调查江苏省35-44岁和65-74岁两个年龄组的城乡居民1584人，男女各半，城乡人数各半。
结果
1、中药复方提取液对各实验细菌均有杀菌作用，对牙周常见可疑致病菌牙龈卟啉单胞菌、伴放线放线杆菌、具核梭杆菌和常见致龋菌变形链球菌的MBC值分别为7.8125、3.90625、7.8125和7.8125 mg／ml。
2、中药复方提取液浓度在1×10~(-7)×10~(-3)g／ml，均能明显促进人牙周膜成纤维细胞增殖，与对照组比较，有显著性差异，且1×10~(-3)g／ml作用效应最大。中药复方提取液浓度在1×10~(-4)～1×10~(-3)g／ml，能够明显增加人牙周膜成纤维细胞总蛋白的合成，与对照组比较，有显著性差异。在此范围内，中药复方提取液的作用显示出一个明显的浓度依赖效应和时间依赖效应。
3、与对照组比较，用药组细胞的内质网、线粒体等明显增多。
4、应用含金银花、紫地丁等组成的含漱液漱口可降低牙周指数，与对照组之间有高度显著差异。
5、流行病学调查表明：(1)中老年城乡居民牙周健康的检出率和平均区段数仅为0.70％和0.50；牙龈出血检出率和平均区段数为67.74％和1.78；牙结石检出率和平均区段数为93.93％和4.59；浅牙周袋检出率和平均区段数为32.07％和0.56；深牙周袋检出率和平均区段数为7.46％和0.10。(2)65-74岁年龄组牙周袋检出率和平均区段数高于35-44岁年龄组(P〈0.01)。(3)牙周附着丧失记分为2、3、4的检出率和平均区段数均为65-74岁组高于35-44岁组(P〈0.01)。
结论
1、浓度为7.8125mg／ml的中药复方提取液在不破坏牙周局部生态平衡的情况下可有效杀灭牙周细菌。
2、中药复方提取液可促进人牙周膜成纤维细胞增殖及蛋白质合成。
3、中药复方提取液能促进细胞的代谢和蛋白质的合成，与细胞增殖实验和总蛋白测定结果一致。
4、金银花、紫地丁等组成的中药复方漱口液可有效去除牙菌斑，能够发挥药物的抗茵、消炎作用。
5、牙周病是口腔常见病、多发病，牙结石是人们口腔卫生面临的一个重要问题。因此，应加强牙周病的预防工作，开展社区口腔卫生服务，提高居民口腔健康水平；并且说明研究开发防治牙周病的药物具有广阔的应用前景。
Objective
Periodontal disease is a common ailment and frequently occurring illness in oral cavity.It is a bacterial infection disease occurring in periodontally supporting tissue, and it is themost important reason that causes pathological teeth-losing in adults. Suspiciousperiodontally pathogenic bacteria is the main causative agent of periodontal disease, and theinitiation factor to the chronically destructive disease in dental capsule. The chief way toprevent and cure periodontal disease is to eliminate the causative agent and obstruct itsformation and development. For instance, Curettage around gum or drug treatment cancontrol the bacterial plaque. For many years, many scholars have done considerable studyon the cause, treatment and precaution from the perspective of Chinese Medicine. Andsince 1980s, the use of naturally occurring drugs in preventing and treating oral disease hasincreasingly drawn the attention of the scholars both abroad and at home.
This study shows the clinical and experimental research with respect to theprescriptional extract from Chinese crude drug, which is based on the empirical formularecommended by Professor Gan Zuwang, a famous TCM doctor. And a survey onperiodontal health-status has been made on the basis of Basic Approaches to the Survey ofPeriodontal Health by the WHO.
To gain a profound understanding of the situation of periodontal health, so as to collectbasic information,and further demonstrate that searching for and improving the medicinethat can prevent and cure periodontal disease is very meaningful. To discover the functionof prescription extract on reducing bacterial plaque and find a proper medication method. To examine the bactericidal effect of the exact on relative pathogenic bacteria. To observethe influence of the exact with different density on the multiplication of HPLF and on thechange of total protein level. And to observe its effect on the form and theultramicrostructure of the HPLF. All these will help provide experimental data for the studyof preventing and treating mechanism for periodontal disease from the aspect of ChineseMedicine.
Methods
This study includes the following three parts: experimental research, clinical researchand epidemiologic research.
1.Experimental research
The prescriptional Exact: It is based on the empirical formula recommended byProfessor Gan Zuwang, which includes honeysuckble, herba, rhizome and phragmitis. Thecrude drug is from the pharmaceutical division of the Jiangsu Provincial Hospital of TCM.The crude drug in 5 times amount of time is extracted three times. All the colature isfiltered, then the 90% ethanol is added, and the precipitation is removed. And the clearsupernatant liquid is chosen, and is condensed to 1 g/ml, which is the stock solution that isneeded in the study. And the stock solution is prepared for use after being in boiling waterfor 10 minutes.
Experimental strain and the nutrient medium: They are of international standard,which include Porphyromonas gingivalis (ATCC 33277), Fusobacterium nucleatum (ATCC25586), and Actinobacillus actinomycetemcomitans (ATCC Y4). They are cultivated byserial subcultivation with profession anaerobian medium. Streptococcus mutans(NCTCIngbritt) is from serial subcultivation with saliva mudium.
Cell Culture: Take a bicuspid tooth for a youngster between 10 to 16 who has amisshapen problem but has no periodontal disease or odontosphacelism, and put it in thecooled DMEM mudium which has in it penicillin of 100U/ml and phytomycin of 100μg/ml.In the germ free condition, on the clean working table, one third of the tissue ofalveolodental ligament in the dental root is scraped and cut into small pieces of 1 mm×1 mm×1mm, then displayed in a culture flask of 25ml volume. Put into the flask 2 ml DMEMnutrient medium including 20% serum, 100U/ml penicillin and phytomycin 100μg/ml, andplace it into CO_2 incubato, with a fine condition of 37℃and 5%CO_2, for development. After 4 hours, reverse the flask for development. When cell emigration appears, the culturefluid is changed every 3 other days. When the flask bottom is full of cells, trypsinization,the density of 0.25%, helps its serial subcultivation. Those cells from the 4th generation tothe 10th generation, which are negative in ceratin and positive in vimentin, are used in theexperiment.
The preparation of the drug extract with different density: Dilute a lg/ml stocksolution with DMEM medium of 5% serum, into 0g/ml(control group), 1×10~(-7)g/ml,1×10~(-6)g/ml、1×104g/ml、1×10~(-4)g/ml、1×10~(-3)g/ml、1×10~(-2)g/ml, and develop them in differentconditions.
The operation of extraorgan sensitivity test: the TSA plate cultivation is adopted, anaerobicculture (90%N_2 5%CO_2 37℃) lasted 18 hours. Reanimate the germs survived and inoculatethem at agar plate. Pick out a single coenobium from its surface and suspend it a respectiveliquid medium for cultivation (one 1mm coenobium per ml). When the density comes upto 10~(5-6)CFU/ml, it is ready for use.
Two sorts of sample liquid are imbibed. The final liquid is from the two-fold dilutionto the stock solution (1:1, 1:2,1:4......). In the extraorgan sensitivity test, microamountliquid dilution is taken to test minimal bactericidal concentration (MBC).
MBC Test: by using aseptic inoculation loop, pick out some culture in the microwellplate that cannot be seen by naked eyes. And inoculate them in the respective plates, andput them in proper conditions for incubation. After 48 hours, observe them again, MBC isthe minimum drug density where no bacteria grow.
MIT method tests cell multiplication: Cells from the 5th generation, digested byparenzyme of 0.25%, are inoculated in 96 shadow mask at the density of 1×105/ml, 100μlper mask. After 24 hours, remove the previous culture fluid. And then put into themconditioned medium 200μl.8 masks for each level of density. After cultivated for one day,three days, and five days respectively, put into each mask MTT (Smg/ml) 20μl. Aftercultivated for four hours under saturation condition of 37℃, 5%CO_2, remove the culturefluid, put into each mask DMSO150μl. Vibrate them for ten minutes, and test its OD atbiocatalyst scale meter, with the wave length 490nm. The nutrient medium with only 5%blood serum is the control group.
The staining method of Coomassie brilliant blue tests cell total protein: Cells from the5th generation, digested by parenzyme of 0.25%, are inoculated in 96 shadow mask at thedensity of 1×105/ml, 100μl per mask. After 24 hours, remove the previous culture fluid. And then put into them conditioned medium 200μl(excluding 1×10~(-2)g/ml). 5 masks foreach level of density. After cultivated for one day, three days, and five days respectively,put into each mask 100μl0.1%TritonX-100. Vibrate them for 30 minutes, and shift them toanother 96 shadow mask. Put into each mask the liquid of Coomassie brilliant blue 200μl,vibrate them for 10 minutes, and test the OD with spectro photometer at the wave length of595nm. The nutrient medium with only 5% blood serum is the control group.
Ultramicrostructure: Cells from the 5th generation, digested by parenzyme of 0.25%,are inoculated into a new culture flask. After 24 hours, remove the culture fluid. Put into itconditioned medium at 1×10~(-3)g/m for 5 days. 5%DMEM Medium with no drug extract isthe control group. On the 5th day, digest it with 0.25%parenzyme. And collect it in somecentrifuge tubes, centrifugalize them for 10 minutes at the speed of 1000r/min. Thenremove the clear supernatant liquid. Fix the cell aggregate with 2% glutaral for 2 hours,dehydrate it at room temperature with series acetone. Embed it with EPON812, observe itwith transmission electron microscope after extra thin section.
2. Clinical research
Prescriptional Extract is prepared into the following 4 groups: low-drug-density group(0.2g/ml, diluted with distilled water), high-drug-density group (0.4g/ml, diluted withdistilled water), and positive control group (half the density of KouTai), and negativecontrol group (distilled water). Double blind method is utilized for experimentalobservation. Those who provide collutory don't join for the check. The workers who arecarrying out the periodontal index inspection are the trained stomatological doctors. Andthose who provide children with collutory and supervise them are the kindergarten teachers.Before the experiment, stomatological doctors inspect the periodontal index of each child.And then the teachers arrange for them to rinse the mouth twice a day for two weeks. Eachtime, 10ml is used for mouthwash, and 2 minutes later, clean the mouth. One hour after thelast mouthwash, stomatologicaI doctors inspect the periodontal index again. In the wholeprocess of the experiment, the children don't change their daily life habit. Test theperiodontal index, including the plaque index (PLI), and the gingival index (GI)
3. Epidemiologic research
On the basis of the surveying method in the Basic Approaches to the Survey ofPeriodontal Health by the WHO, this research involves the following features asmulti-stage, stratification, equal capacity, and random sampling. 1584 people in JiangsuProvince are divided into two age-groups of 35-44 and 65-74. Half of them are men and from countryside.
Result
1.The drug extract has bactericidal effect on every sort of bacterium in theexperiment. It's MBC value on porphyrin unit-cell bacterium, actinobacillusactinomycetem comitans, fusobacterium nucleatum and streptococcus mutans arerespectively 7.8125,3.90625,7.8125 and 7.8125 mg/ml
2.The drug extract can noticeably improve the generation of HPLF at the density ofbetween 1×10~(-7)g/ml and 1×10~(-3)g/ml. Compared with the control group, it has significantdifference. And it functions the best at 1×10~(-3)g/ml. At the density of between 1×10~(-4)g/mland 1×10~(-3)g/ml, it can noticeably increase the composition of the total protein ofcollagenoblast. Compared with the control group, it has significant difference. Within thisrange, the function of drug extract shows a clear density and time-reliance effect.
3.Compared with the control group, the endocytoplasmic reticulum and the bioblast ofthe cells in the experimental group show a clear increase.
4.The use of gargarism that has such ingredients as honeysuckble and Tokyo violetherb can help reduce the periodontal index, which shows highly significant difference,compared with the control group.
5.(1)The detection rate and average segment of periodontal health of the citizens in therural area and the city are only 0.70% and 0.50.67.74% and 1.78 gingiva bleeding. 93.93%and 4.59 calculus dentalis. 32.07% and 0.56 for shallow periodontal pocket; 7.46% and0.10 for deep periodontal pocket.
(2) The detection rate and average segment of periodontal pocket in the age group of65-74 is higher than that in the age group of 35-44 (P〈0.01 )
(3) The detection rate and average segment of the periodontal attachment whosedeprivation scores are 2, 3 and 4 in the age group of 65-74 is higher than that in the agegroup of 35-44 (P〈0.01).
Conclusion
1. The prescriptional Exact at the density of 7.8125 mg/ml can effectively killperidontal bacterium without causing damage to the regional peridontally eubiosis
2. The prescriptional Exact can help the generation and protein synthesis of HPLF.
3. The prescriptional Exact can promote corpuscular metabolism and proteinaceouscomposition, which has the same result as the test in cell multiplication experiment and thetotal protein test.
4. The prescriptional collutory of honeysuckble and Tokyo violet herb can effectivelyremove Tokyo violet herb, and shows antibiosis and antiphlogosis.
5. Antiphlogosis is a common and frequently encountered ailment. Calculus dentalis isanother important issue that the dental hygiene is faced with. So that it is an obligation tointensify the prevention of periodontal disease, to offer dental hygiene service incommunity and to increase dental health of all the citizens, and further demonstrate thatthere is a great prospect to search for the medicine that can prevent and cure periodontaldisease and put the medicine into treatment.
牙周病是口腔常见病和多发病，是一种发生在牙周支持组织上以细菌为主的感染性疾病，是成年人口腔中病理性失牙最主要的原因。牙周可疑致病菌是牙周病的主要致病因素，是引起牙周组织慢性破坏性疾病的始动因子。消除致病因子和阻断牙周病的发生发展是预防和治疗牙周病的重要手段，如采用龈上下洁刮治术或药物治疗，控制引起牙周病的牙菌斑。长期以来，许多学者对中医药在牙周病的病因、治疗和预防方面都进行了大量的研究，特别是二十世纪八十年代以来，天然药物防治口腔疾病逐渐受到国内外学者的关注。本研究采用名中医干祖望教授推荐的经验方，制成中药复方提取液进行了相关的临床和实验研究，并且按照世界卫生组织《口腔健康调查基本方法》进行牙周健康状况的调查。
了解人群牙周健康流行状况，既为开展口腔预防保健提供了基线资料，又进一步说明研究开发防治牙周病药物具有十分重要的意义；探讨中药复方提取液减少牙茵斑的作用和寻找一个合适的用药方法；观察中药提取物对牙周病可疑致病菌的杀菌作用；观察中药复方提取液在不同浓度下对人牙周膜成纤维细胞(HPLF)增殖和总蛋白含量的变化以及观察中药复方提取液对HPLF形态和超微结构的影响。为研究该中药防治牙周病的机制提供实验依据。
方法
本课题研究分实验研究、临床研究和流行病学研究三个部分
1、实验研究
中药复方提取液：选用江苏省中医院名中医干祖望教授推荐的防治牙周病的方药(以金银花、紫花地丁、蚤休、芦根等组成)，中药材来源于江苏省中医院药剂科。将中药材加5倍量水提取3次，过滤合并滤液，90％乙醇沉淀，离心后去除沉淀，取上清液，浓缩至所需浓度1g／ml作为原液。原液于沸水中煮沸10min，备用。
实菌验株及培养基：选用国际标准菌株(由上海交通大学附属第九人民医院口腔医学研究所提供)：牙龈卟啉单胞菌(Porphyromonas gingivalis，Pg)ATCC 33277、具核梭杆菌(Fusobacterium rlucleatum，Fn)ATCC 25586、伴放线放线杆菌(Actinobacillus actinomycetemcomitans，Aa)ATCC Y4。用专性厌氧培养基(杭州天和微生物试剂有限公司提供)传代培养。变形链球菌(st reptOCOCCUS mutans，sm)NCTC Ingbritt。用(Ms)轻唾培养基(杭州天和微生物试剂有限公司提供)传代培养。
细胞培养：取正畸需要拔除的无龋、无牙周病的10～16岁青少年前磨牙，随即放置于含有100u／ml青霉素、100μg／ml链霉素预冷的DMEM培养基中，在超净工作台内，无菌条件下，刮取牙根中1／3的牙周膜组织，剪成1mm×1mm×1mm小块，平铺于25ml培养瓶中，加入含有20％血清、100u／ml青霉素、100μg／ml链霉素的DMEM培养基2ml，在CO_2培养箱中，37℃、5％CO_2饱和条件下培养4 h后，翻转培养瓶，继续培养。有细胞游出后，每隔3天换液，细胞长满瓶底壁后，以0.25％胰蛋白酶消化，传代。凡免疫组化角蛋白阴性、波形蛋白阳性的4～10代细胞用于实验。
配制系列浓度中药提取液：取1g／ml中药贮存液，用5％血清的DMEM培养基将其稀释成0g／ml(对照组)、1×10~(-7)g／ml、1×10~(-6)g／ml、1×10~(-5)g／ml、1×10~(-4)g／ml、1×10~(-3)g／ml、1×10~(12)g／ml不同浓度的条件培养基。
体外敏感试验操作程序(微量液体稀释法)：采用TSA血平板培养基。厌氧培养18小时(90％N_2 5％CO_2 37℃)。保存之细菌复苏，经生化鉴定后接种于相应琼脂平板。挑取琼脂表面的单个菌落混悬于相应液体培养基中(1个1mm菌落／ml)培养，培养相应时间后使菌液浓度呈10~(-6)CFU／ml备用。
分别吸取两种样品原液。药品液为原液对倍稀释(1：1，1：2，1：4……)。体外敏感试验采用微量液体稀释法检测MBC(最小杀菌浓度)。
MBC测定：用无菌接种环挑取肉眼观察无细菌生长的微孔板中培养物，划线接种于相应平板，放置适宜培养条件下孵育。48h培养后观察，无细菌生长的最低药物浓度为MBC。
MTT法测定细胞增殖：取第5代细胞，用0.25％胰蛋白酶消化，以1×10~5／ml的细胞浓度接种到96孔板，每孔100μl，培养24h后，吸弃原有培养液，每孔加入条件培养液：200μl，每个浓度8孔。分别于培养1d、3d、5d后，每孔加入MTT(5mg／ml)20μl，37℃，5％CO_2饱和条件下培养4h，吸弃培养液，每孔加入DMSO150μl，振荡10min，于酶标仪测定OD值，波长为490nm。仅含有5％血清的培养基作为对照组。
考马斯亮蓝染色法测定细胞总蛋白：取第5代细胞，用0.25％胰蛋白酶消化，以1×10~5／ml的细胞浓度接种到96孔板，每孔100μl，培养24h后，吸弃原有培养液，每孔加入条件培养液200μl(不包括1×10~(-2)g／ml)，每个浓度5孔。分别于培养1d、3d、5d后，每孔加入100μl0.1％TritonX-100，室温下振荡30min，吸取20μl，转移到另一块96孔板，每孔加入考马斯亮蓝染液200μl，室温下振荡10min，用分光光度计在595nm波长处测定溶液的OD值。仅含有5％血清的培养基作为对照组。
超微结构：取第5代细胞，用0.25％胰蛋白酶消化接种到新的培养瓶中，培养24h后，吸弃原培养液，加入1×10~(-3)g／ml浓度条件培养液培养5d，以不含中药提取液的5％DMEM培养基作为对照组。第5天用0.25％胰蛋白酶消化，分别收集在离心管中，1000r／min，10min离心后，弃上清液。2％戊二醛固定细胞团块2h，室温下系列丙酮脱水，EPON812包埋，超薄切片后透射电镜观察。
2、临床研究
中药复方提取液同实验研究，并将其配制成中药低浓度组(0.2g／ml，用蒸馏水稀释)、中药高浓度组(0.4g／ml，用蒸馏水稀释)、阳性对照组(1／2浓度的口泰)、阴性对照组(蒸馏水)4个组分别编为A、B、C、D 4个组。采用双盲法进行实验观察，提供漱口液者不参加实验检查，进行牙周指数检查的人员是经过培训的口腔医生，直接给幼儿提供漱口液并进行监督的是幼儿园老师。实验开始前由口腔医生对幼儿进行牙周指数的检查，再由幼儿园老师安排幼儿漱口，要求每天含漱2次，每次用量10ml，含漱2分钟后吐干净，连续二周，最后一次漱口完后1hr由口腔医生检查牙周指数。整个实验过程中幼儿不改变平时的生活习惯。测量牙周指数，包括菌斑指数(plaqueindex，PLI)和牙龈指数(gingival index，GI)。
3、流行病学研究
参考世界卫生组织(WHO)《口腔健康调查基本方法》(第四版)调查方法，采用多阶段、分层、等容量、随机抽样的方法，调查江苏省35-44岁和65-74岁两个年龄组的城乡居民1584人，男女各半，城乡人数各半。
结果
1、中药复方提取液对各实验细菌均有杀菌作用，对牙周常见可疑致病菌牙龈卟啉单胞菌、伴放线放线杆菌、具核梭杆菌和常见致龋菌变形链球菌的MBC值分别为7.8125、3.90625、7.8125和7.8125 mg／ml。
2、中药复方提取液浓度在1×10~(-7)×10~(-3)g／ml，均能明显促进人牙周膜成纤维细胞增殖，与对照组比较，有显著性差异，且1×10~(-3)g／ml作用效应最大。中药复方提取液浓度在1×10~(-4)～1×10~(-3)g／ml，能够明显增加人牙周膜成纤维细胞总蛋白的合成，与对照组比较，有显著性差异。在此范围内，中药复方提取液的作用显示出一个明显的浓度依赖效应和时间依赖效应。
3、与对照组比较，用药组细胞的内质网、线粒体等明显增多。
4、应用含金银花、紫地丁等组成的含漱液漱口可降低牙周指数，与对照组之间有高度显著差异。
5、流行病学调查表明：(1)中老年城乡居民牙周健康的检出率和平均区段数仅为0.70％和0.50；牙龈出血检出率和平均区段数为67.74％和1.78；牙结石检出率和平均区段数为93.93％和4.59；浅牙周袋检出率和平均区段数为32.07％和0.56；深牙周袋检出率和平均区段数为7.46％和0.10。(2)65-74岁年龄组牙周袋检出率和平均区段数高于35-44岁年龄组(P〈0.01)。(3)牙周附着丧失记分为2、3、4的检出率和平均区段数均为65-74岁组高于35-44岁组(P〈0.01)。
结论
1、浓度为7.8125mg／ml的中药复方提取液在不破坏牙周局部生态平衡的情况下可有效杀灭牙周细菌。
2、中药复方提取液可促进人牙周膜成纤维细胞增殖及蛋白质合成。
3、中药复方提取液能促进细胞的代谢和蛋白质的合成，与细胞增殖实验和总蛋白测定结果一致。
4、金银花、紫地丁等组成的中药复方漱口液可有效去除牙菌斑，能够发挥药物的抗茵、消炎作用。
5、牙周病是口腔常见病、多发病，牙结石是人们口腔卫生面临的一个重要问题。因此，应加强牙周病的预防工作，开展社区口腔卫生服务，提高居民口腔健康水平；并且说明研究开发防治牙周病的药物具有广阔的应用前景。
Objective
Periodontal disease is a common ailment and frequently occurring illness in oral cavity.It is a bacterial infection disease occurring in periodontally supporting tissue, and it is themost important reason that causes pathological teeth-losing in adults. Suspiciousperiodontally pathogenic bacteria is the main causative agent of periodontal disease, and theinitiation factor to the chronically destructive disease in dental capsule. The chief way toprevent and cure periodontal disease is to eliminate the causative agent and obstruct itsformation and development. For instance, Curettage around gum or drug treatment cancontrol the bacterial plaque. For many years, many scholars have done considerable studyon the cause, treatment and precaution from the perspective of Chinese Medicine. Andsince 1980s, the use of naturally occurring drugs in preventing and treating oral disease hasincreasingly drawn the attention of the scholars both abroad and at home.
This study shows the clinical and experimental research with respect to theprescriptional extract from Chinese crude drug, which is based on the empirical formularecommended by Professor Gan Zuwang, a famous TCM doctor. And a survey onperiodontal health-status has been made on the basis of Basic Approaches to the Survey ofPeriodontal Health by the WHO.
To gain a profound understanding of the situation of periodontal health, so as to collectbasic information,and further demonstrate that searching for and improving the medicinethat can prevent and cure periodontal disease is very meaningful. To discover the functionof prescription extract on reducing bacterial plaque and find a proper medication method. To examine the bactericidal effect of the exact on relative pathogenic bacteria. To observethe influence of the exact with different density on the multiplication of HPLF and on thechange of total protein level. And to observe its effect on the form and theultramicrostructure of the HPLF. All these will help provide experimental data for the studyof preventing and treating mechanism for periodontal disease from the aspect of ChineseMedicine.
Methods
This study includes the following three parts: experimental research, clinical researchand epidemiologic research.
1.Experimental research
The prescriptional Exact: It is based on the empirical formula recommended byProfessor Gan Zuwang, which includes honeysuckble, herba, rhizome and phragmitis. Thecrude drug is from the pharmaceutical division of the Jiangsu Provincial Hospital of TCM.The crude drug in 5 times amount of time is extracted three times. All the colature isfiltered, then the 90% ethanol is added, and the precipitation is removed. And the clearsupernatant liquid is chosen, and is condensed to 1 g/ml, which is the stock solution that isneeded in the study. And the stock solution is prepared for use after being in boiling waterfor 10 minutes.
Experimental strain and the nutrient medium: They are of international standard,which include Porphyromonas gingivalis (ATCC 33277), Fusobacterium nucleatum (ATCC25586), and Actinobacillus actinomycetemcomitans (ATCC Y4). They are cultivated byserial subcultivation with profession anaerobian medium. Streptococcus mutans(NCTCIngbritt) is from serial subcultivation with saliva mudium.
Cell Culture: Take a bicuspid tooth for a youngster between 10 to 16 who has amisshapen problem but has no periodontal disease or odontosphacelism, and put it in thecooled DMEM mudium which has in it penicillin of 100U/ml and phytomycin of 100μg/ml.In the germ free condition, on the clean working table, one third of the tissue ofalveolodental ligament in the dental root is scraped and cut into small pieces of 1 mm×1 mm×1mm, then displayed in a culture flask of 25ml volume. Put into the flask 2 ml DMEMnutrient medium including 20% serum, 100U/ml penicillin and phytomycin 100μg/ml, andplace it into CO_2 incubato, with a fine condition of 37℃and 5%CO_2, for development. After 4 hours, reverse the flask for development. When cell emigration appears, the culturefluid is changed every 3 other days. When the flask bottom is full of cells, trypsinization,the density of 0.25%, helps its serial subcultivation. Those cells from the 4th generation tothe 10th generation, which are negative in ceratin and positive in vimentin, are used in theexperiment.
The preparation of the drug extract with different density: Dilute a lg/ml stocksolution with DMEM medium of 5% serum, into 0g/ml(control group), 1×10~(-7)g/ml,1×10~(-6)g/ml、1×104g/ml、1×10~(-4)g/ml、1×10~(-3)g/ml、1×10~(-2)g/ml, and develop them in differentconditions.
The operation of extraorgan sensitivity test: the TSA plate cultivation is adopted, anaerobicculture (90%N_2 5%CO_2 37℃) lasted 18 hours. Reanimate the germs survived and inoculatethem at agar plate. Pick out a single coenobium from its surface and suspend it a respectiveliquid medium for cultivation (one 1mm coenobium per ml). When the density comes upto 10~(5-6)CFU/ml, it is ready for use.
Two sorts of sample liquid are imbibed. The final liquid is from the two-fold dilutionto the stock solution (1:1, 1:2,1:4......). In the extraorgan sensitivity test, microamountliquid dilution is taken to test minimal bactericidal concentration (MBC).
MBC Test: by using aseptic inoculation loop, pick out some culture in the microwellplate that cannot be seen by naked eyes. And inoculate them in the respective plates, andput them in proper conditions for incubation. After 48 hours, observe them again, MBC isthe minimum drug density where no bacteria grow.
MIT method tests cell multiplication: Cells from the 5th generation, digested byparenzyme of 0.25%, are inoculated in 96 shadow mask at the density of 1×105/ml, 100μlper mask. After 24 hours, remove the previous culture fluid. And then put into themconditioned medium 200μl.8 masks for each level of density. After cultivated for one day,three days, and five days respectively, put into each mask MTT (Smg/ml) 20μl. Aftercultivated for four hours under saturation condition of 37℃, 5%CO_2, remove the culturefluid, put into each mask DMSO150μl. Vibrate them for ten minutes, and test its OD atbiocatalyst scale meter, with the wave length 490nm. The nutrient medium with only 5%blood serum is the control group.
The staining method of Coomassie brilliant blue tests cell total protein: Cells from the5th generation, digested by parenzyme of 0.25%, are inoculated in 96 shadow mask at thedensity of 1×105/ml, 100μl per mask. After 24 hours, remove the previous culture fluid. And then put into them conditioned medium 200μl(excluding 1×10~(-2)g/ml). 5 masks foreach level of density. After cultivated for one day, three days, and five days respectively,put into each mask 100μl0.1%TritonX-100. Vibrate them for 30 minutes, and shift them toanother 96 shadow mask. Put into each mask the liquid of Coomassie brilliant blue 200μl,vibrate them for 10 minutes, and test the OD with spectro photometer at the wave length of595nm. The nutrient medium with only 5% blood serum is the control group.
Ultramicrostructure: Cells from the 5th generation, digested by parenzyme of 0.25%,are inoculated into a new culture flask. After 24 hours, remove the culture fluid. Put into itconditioned medium at 1×10~(-3)g/m for 5 days. 5%DMEM Medium with no drug extract isthe control group. On the 5th day, digest it with 0.25%parenzyme. And collect it in somecentrifuge tubes, centrifugalize them for 10 minutes at the speed of 1000r/min. Thenremove the clear supernatant liquid. Fix the cell aggregate with 2% glutaral for 2 hours,dehydrate it at room temperature with series acetone. Embed it with EPON812, observe itwith transmission electron microscope after extra thin section.
2. Clinical research
Prescriptional Extract is prepared into the following 4 groups: low-drug-density group(0.2g/ml, diluted with distilled water), high-drug-density group (0.4g/ml, diluted withdistilled water), and positive control group (half the density of KouTai), and negativecontrol group (distilled water). Double blind method is utilized for experimentalobservation. Those who provide collutory don't join for the check. The workers who arecarrying out the periodontal index inspection are the trained stomatological doctors. Andthose who provide children with collutory and supervise them are the kindergarten teachers.Before the experiment, stomatological doctors inspect the periodontal index of each child.And then the teachers arrange for them to rinse the mouth twice a day for two weeks. Eachtime, 10ml is used for mouthwash, and 2 minutes later, clean the mouth. One hour after thelast mouthwash, stomatologicaI doctors inspect the periodontal index again. In the wholeprocess of the experiment, the children don't change their daily life habit. Test theperiodontal index, including the plaque index (PLI), and the gingival index (GI)
3. Epidemiologic research
On the basis of the surveying method in the Basic Approaches to the Survey ofPeriodontal Health by the WHO, this research involves the following features asmulti-stage, stratification, equal capacity, and random sampling. 1584 people in JiangsuProvince are divided into two age-groups of 35-44 and 65-74. Half of them are men and from countryside.
Result
1.The drug extract has bactericidal effect on every sort of bacterium in theexperiment. It's MBC value on porphyrin unit-cell bacterium, actinobacillusactinomycetem comitans, fusobacterium nucleatum and streptococcus mutans arerespectively 7.8125,3.90625,7.8125 and 7.8125 mg/ml
2.The drug extract can noticeably improve the generation of HPLF at the density ofbetween 1×10~(-7)g/ml and 1×10~(-3)g/ml. Compared with the control group, it has significantdifference. And it functions the best at 1×10~(-3)g/ml. At the density of between 1×10~(-4)g/mland 1×10~(-3)g/ml, it can noticeably increase the composition of the total protein ofcollagenoblast. Compared with the control group, it has significant difference. Within thisrange, the function of drug extract shows a clear density and time-reliance effect.
3.Compared with the control group, the endocytoplasmic reticulum and the bioblast ofthe cells in the experimental group show a clear increase.
4.The use of gargarism that has such ingredients as honeysuckble and Tokyo violetherb can help reduce the periodontal index, which shows highly significant difference,compared with the control group.
5.(1)The detection rate and average segment of periodontal health of the citizens in therural area and the city are only 0.70% and 0.50.67.74% and 1.78 gingiva bleeding. 93.93%and 4.59 calculus dentalis. 32.07% and 0.56 for shallow periodontal pocket; 7.46% and0.10 for deep periodontal pocket.
(2) The detection rate and average segment of periodontal pocket in the age group of65-74 is higher than that in the age group of 35-44 (P〈0.01 )
(3) The detection rate and average segment of the periodontal attachment whosedeprivation scores are 2, 3 and 4 in the age group of 65-74 is higher than that in the agegroup of 35-44 (P〈0.01).
Conclusion
1. The prescriptional Exact at the density of 7.8125 mg/ml can effectively killperidontal bacterium without causing damage to the regional peridontally eubiosis
2. The prescriptional Exact can help the generation and protein synthesis of HPLF.
3. The prescriptional Exact can promote corpuscular metabolism and proteinaceouscomposition, which has the same result as the test in cell multiplication experiment and thetotal protein test.
4. The prescriptional collutory of honeysuckble and Tokyo violet herb can effectivelyremove Tokyo violet herb, and shows antibiosis and antiphlogosis.
5. Antiphlogosis is a common and frequently encountered ailment. Calculus dentalis isanother important issue that the dental hygiene is faced with. So that it is an obligation tointensify the prevention of periodontal disease, to offer dental hygiene service incommunity and to increase dental health of all the citizens, and further demonstrate thatthere is a great prospect to search for the medicine that can prevent and cure periodontaldisease and put the medicine into treatment.
引文
1．全国牙病防治指导组．第二次全国口腔健康流行病学抽样调查[M]．北京：人民卫生出版社，1999．268-317．
2. Socransky S,Haffajee A.Evidence of bacterial aetiology:a historical perspective. Periodontol 2000, 1994; 5: 7-25.
3. Zambon JJ. Periodontol diseases: microbial factors. Ann Periodontol, 1996; 1: 879-925.
4. Darveau RP, Tanner A,Page RC. The microbial challenge in periodontitis. Periodontol 2000, 1997; 14: 12-32.
5. Jansen HJ, van der Hoeven JS. Protein degradation by Prevotella intermedia and Actinomyces meyeri supports the growth of non-protein-cleaving oral bacteria in serum.J Clin Periodontol, 1997;24:346-353.
6. Curtis MA,Kuramitsu HK,Lantz M,et al.Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis.J Periodont Res, 1999;34:464-472.
7．曹采方．牙周病学[M]．北京：人民卫生出版社，2000．160
8. Jun Isaka, Atsushi Ohazama,Makoto Kobayashi,et al.Participation of periodontal ligament cells with regeneration of alveolar bone[J].J periodontal,2001,72(3):314-323
9. Wishmura F, Terranova VP.Comparative studying of the chemotactia responses of periodontal ligament cells and gingival fibroblasts to polypeptide growth factors[J].J Dent Res. 1996,75(4):986-992
10．徐治鸿．实用中医口腔病学[M]．天津：天津科技翻译出版公司，1991．93
11．裴霞，华红，徐志鸿．中医药在牙周疾病的临床应用于．现代口腔医学杂志，2001，15(2)：151-152．
12．徐志鸿，孙小平，华红等．固齿健周煎剂对老年小鼠的抗氧化作用．中华口腔医学杂志，1998，33(4)：219．
13．宋红，赵瑞芳，周以钧．固齿膏对牙周炎龈沟液中IL-8的影响．中华口腔医学杂志，1997，32(6)：372．
14．徐竺婷，李鸣宇．10种中药有效成分抑制口腔主要致病菌比较[J]．中国现代应用药学杂志，2000，17(4)：277-279
15．朱彩莲，李鸣宇．17种中药提取物对6种牙周可疑致病菌的体外抑菌实验[J]．上海口腔医学，2006，15(4)：434-436．
16．叶显纯，孙文忠，金岚，等．中药临床手册[M]．上海：上海科学技术出版社，1993．
17. Wu-Yuan CD,Chen CY, Wu RT.Gallotannins inhibit growth,water-insoluble glucan synthesis,and aggregation of mutans streptococci[J].J Dent Res,1988,67(1):51]
18．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[J]．牙体牙髓牙周病学杂志，2002，12(5)：255-257
19．王静，唐荣银，王志良等．五倍子水提取物对胶原酶活性的影响[J]．临床口腔医学杂志，2006，22(8)：451-453．
20．张红梅，朱秀丽，陈强等．五倍子对牙龈卟啉单胞菌胰酶样蛋白酶活性的抑制作用[J]．牙体牙髓牙周病学杂志，2004，14(1)：29-31
21．周汝俊，陈涟如，黄敬耀，等．黄甲棒缓释剂的研究[J]．中华口腔医学杂志，1996，31(3)：159
22．王茜，樊明文，边专等．熊果酸的提取及其对牙周病原菌的作用[J]．中华口腔医学 杂志，2002，37(5)：388-390．
23．李廷谦．循证医学与中西医结合现状及设想[J]．中国中西医结合杂志，2002，22(1)：8
[1] Feng Z,Weinberg A.Role of bacteria in health and disease of periodontal tissues [J].Periodontology,2006,40(1):50-76.
[2] 沈家平，朱维建，陆平成．中药复方漱口液漱口前后牙周指数的变化[J]．南京中医药大学学报，2006，22(2)：118-119
[3] National Committee for Clinical Laboratory Standards.Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically [M]. Wayne,Pennsylvania:National Committee for Clinical Laboratory Standards.NCCLS Document M7-A5,2000.
[4] 樊明文主编．牙体牙髓病学[M]．北京：人民卫生出版社，2000．22-25．
[5] Bragd L,Dahlen G, Wisstrm M,et al.The capability of Actinobacillus actinomycetemcomitans,Bacteroides gingivalis,Bacteroides intermedius to indicate progressive periodontitis,a retrospective study[J].J Clin Periodontol,1987,14:95
[6] Grossi S,Genco R,Machtei E,et al.Assessment of risk for periodontal disease.I.Risk indicators for alveolar bone loss[J].J Periodontol, 1995,66:23
[7] Mayrand D,Holt SC.Biology of asaocharolytic black-pigmented bacteroides species [J].Microbiol Rev, 1998,52(1):134
[8] Zambon JJ,Christersson LA,Slots J.Actinobacillus action myycete mcomitans in human periodontaldisease.Prevalence in patient groups and distribution of biotypes within families[J].J Periodontol, 1983,54:707.
[9] Haffajee AD,Socransky SS.Microbial etiological agents of destructive periodontal diseases.periodontol 2000,1994.5:78-111
[10] 朱彩莲，李鸣宇．17种中药提取物对6种牙周可疑致病菌的体外抑菌实验[J]．上海口腔医学，2006，15(4)：434-436
[11] 李鸣宇，刘正．茶多酚对口腔咽喉致病菌作用的体外实验[J]．上海第二医科大学学报，1999，19：41-43．
[12] 黄吉燕，朱聘倬，刘力．三种中药对牙周致病厌氧菌抑制作用的体外实验[J]．上海中医药杂志，2006，40(5)：62-63．
[13] 黄霞，谭红，黄定明等．天然药物对牙龈卟啉单胞菌的体外抗菌研究[J]．中国微生态学杂志，2005，17(5)：346-347．
[14] 徐竺婷，李鸣宇．10种中药有效成分抑制口腔主要致病菌比较[J]．中国现代应用药学杂志，2000，17(4)：277-279
[15] 高学敏主编．中药学[M]．北京：人民卫生出版社，2000．595-601．
[1] 徐燕，蒋勇，李颂等．烟草对牙周成纤维细胞影响的实验观察[J]．中华口腔医学 杂志，2003，38(5)：367-369
[2] 司徒镇强，吴军正．细胞培养[M]．西安：世界图书出版公司西安公司，1996
[3] 李谨，蒋春梅，孙卫斌．人牙周膜成纤维细胞体外培养条件的优化[J]．临床口腔医学杂志，2003，19(6)：333-335
[4] 冯雪．体外培养牙周膜成纤维细胞矿化能力的实验研究[J]．华西口腔医学杂志，1998，4294-295
[5] Rose GC,et al.Human periodontal ligament cells in vitro[J].J periodont Res, 1987,22:20-28
[6] Mariotti A,et al.Characterization of fibroblasts derived from human periodontal ligament and gingival fibroblast[J].J periodontal, 1990,61:103-114
[7] Murphy KG, et al. Human periodontal ligament cells in vitro:cell culture passage effect on collagen gel contraction[J].J periodont Res,1987,22:342-348
[8] Liu HW, Yacobi R, Savion N.A collagenous cementum derived attachment protein is a marker for progenitors of mineralized tissue-forming cell lineage of the periodontal ligament.J Bone Miner Res, 1997,12(10): 1691-1699
[9] Jun Isaka, Atsushi Ohazama,Makoto Kobayashi,et al.Participation of periodontal ligament cells with regeneration of alveolar bone.J periodontol,2001,72(3):314-323
[10] Wishmura F, Terranova VP. Comparative studying of the chemotactia responses of periodontal ligament cells and gingival fibroblasts to polypeptide growth factors.J Dent Res,1996,75(4):986-992
[11] 章锦才，吉庆勇，肖卓然．白细胞介素-1对牙龈成纤维细胞增殖和前列腺素2产生的影响．口腔医学纵横杂志，1992，8(4)：199-200
[12] Chung CP, Park JB,Bae KH.Pharmacological effects of methanolic extract from the root of scutetlaria baicalensis and its flavonoids on human gingival fibroblasts.Planta Med,1995,61 (2): 150-153
[13] 曹正国，李成章，刘小平．中药黄芩苷对人牙周膜细胞增殖和总蛋白含量的影响[J]．口腔医学研究：2003，19(1)：10-12
[14] 李成章，曹正国，樊明文等．黄芩苷对人牙周韧带细胞超微结构的影响[J]．牙体牙髓牙周病学杂志，2003，13(2)：76-77。
[15] 许彦枝，邹慧儒，王小玲等．中药双黄补对人牙周膜细胞总蛋白含量和超微结构的影响[J]．实用口腔医学杂志，2005，21(3)：374-377
[16] 章魁华，于世风，主编．实验口腔病理学[M]．北京：人民卫生出版社，2002．308-312
[17] Palmon A,Roos H,Edel J,et al.Inverse dose-and time-dependent effect of basic fibroblast growth factor on the gene expression of collagen type I and matrix metalloproteinase-1 by periodontal ligament cells in culture.J Periodontol,2000,71 (6):974-980
[18] 沈家平，朱维建，陆平成．中药复方漱口液漱口前后牙周指数的变化[J]．南京中医药大学学报，2006，22(2)：118-119
[19] King GN,Cochran DL.Factors that modulate the effects of bone morphogenetic protein-induced periodontal regeneration;a critical review.J Periodontal,2002,73(8):925-936
[20] 司晓辉，刘正．转化生长因子β对人牙周膜成纤维细胞的生物学作用[J]．中华口腔医学杂志，2001，36(1)：23-26
[1] Chung CP, Park JB,Bae KH.Pharmacological effects of methanolic extract from the root of scutellaria baicalensis and its flavonoids on human gingival fibroblasts.Planta Med, 1995,61 (2): 150-153
[2] 李成章，曹正国，樊明文等．黄芩苷对人牙周韧带细胞超微结构的影响[J]．牙体牙髓牙周病学杂志,2003，13(2)：76-77
[3] 司徒镇强，吴军正．细胞培养[M]．西安：世界图书出版公司西安公司，1996
[4] 李谨，蒋春梅，孙卫斌．人牙周膜成纤维细胞体外培养条件的优化[J]．临床口腔医学杂志，2003，19(6)：333-335
[5] 冯雪．体外培养牙周膜成纤维细胞矿化能力的实验研究[J]．华西口腔医学杂志，1998，4294-259
[6] Rose GC,et al.Human periodontal ligament cells in vitro[J].J periodont Res,1987,22:20-28
[7] Mariotti A,et al.Characterization of fibroblasts derived from human periodontal ligament and gingival fibroblast [J].J periodontal, 1990,61:103-114
[8] Murphy KG, et al. Human periodontal ligament cells in vitro:cell culture passage effect on collagen gel contraction[J].J periodont Res, 1987,22:342-348
[9] 徐燕，蒋勇，李颂等．烟草对牙周成纤维细胞影响的实验观察[J]．中华口腔医学杂志,2003，38(5)：367-369
[1] 刘宏伟，徐治鸿，俞帆．咀嚼黄芩口胶前后牙周指数的变化[J]．现代口腔医学杂志,2000,14(6)：405-406．
[2] 孔令义，杨智，闵知大．对中药现代化中有效成份研究的思考[J]．中草药，1998,29(5)：354．
[3] 卞金有主编．预防口腔医学[M]．北京：人民卫生出版社，2003．32．33．
[4] 樊明文主编．口腔生物学．北京：人民卫生出版社，1996．181-198，214-230．
[5] Haffajee AD,Socransky SS.Microbial etiological agents of destructive periodontal diseases.periodontol 2000,1994.5:78-111
[6] 李鸣宇，刘正．茶多酚对口腔咽喉致病菌作用的体外实验[J]．上海第二医科大学学报，1999，19：41-43．
[7] Ooshima T, Minami T, Matsumoto M,et al.Comparison of the Cariostatic Effects between Regimens to Administer Oolong Tea Polyphenols in SPF Rats.Caries Res, 1998;32(2):75
[8] 黄冰冰，樊明文，杨祥良等．桔梗、甘草及其组成的复方对口腔病原菌生长影响的体外实验[J]．实用口腔医学杂志，2003，19(2)：148-150．
[1] World Health Organization.Oral Health Surveys:Basic Methods 4thed Geneva: WHO, 1997.
[2] 全国牙病防治指导组．第二次全国口腔健康流行病学抽样调查[M]．北京：人民卫生出版社，1999．268-317．
[3] 卞金有主编．预防口腔医学[M]．北京：人民卫生出版社，2003，第4版，37．
[4] 韩晓兰，颜雨春，蒋勇等．安徽省城乡人群口腔健康状况[J]．疾病控制杂志，2003，7(4)：352-353．
[5] Pilot T, Miyazaki H.Global results:15 years of CPITN epidemiology[J].Int Dent J, 1994,44:553.
[6] Pilot Y,Lu ZY,Lin ZQ,et al. Periodontal conditions in 35-44-year-old factory workers in Shanghai[J]. Community Dent Oral Epidemiol, 1989,17:216.
[7] 廖旭辉，林焕彩，卢展民等．广东省中老年人的牙周健康状况[J]．中山医科大学学报，2000，21(4)：314-315．
[8] Miyazaki H,Ohtani L,Abe N,et al.Periodontal conditions in elder age cohorts aged 65 years and eider in Japan,measured by CPITN and loss of attachment[J].Community Dent Health, 1995,12:216.
[9] Petersen P E, Peng B, Tai B J.Oral health status and oral health behaviour of middle-aged and elderly people in P R China. Int Dent J. 1997,47(5):305-312.
[10] 胡德渝．中国人口结构与口腔疾病的改变趋势[J]．华西口腔医学杂志，2000，18(2)：126-128．
[11] 周红玲，朱文昊，初里楠等．北京市西城区口腔健康调查资料的统计与分析[J]．北京口腔医学，2004，12(3)：160-162．
[1] Socransky S,Haffajee A.Evidence of bacterial aetiology:a historical perspective [J].Periodontol 2000,1994;5:7-25.
[2] Harris NO.Primary Preventive Dentistry. Fifthedition. America:Stamford, Connecticut, 1999
[3] Kinae DF. Causation and pathogenesis of periodontal disease. Pefiodontol 2000. 2001; 25: 8-20.
[4] Albandar JM, Brunelle JA, Kingman A. Destructive periodontal disease in adults 30 yers of age and older inthe United States. 1981—1984. J Periodontol, 1999; 70: 13-29.
[5] Brown LJ, Loe H. Prevalence, extent, severity and progression of periodontal disease. Periodontol 2000, 1993; 2: 57-71.
[6] Papapanou PN, Wennstrrm JL Grondhal K. Periodontal status in relation to age and tooth type. A cross-sectional radiographic study. J Clin Periodontol, 1988; 15: 469-478.
[7] Lindhe J, Okamoto H, Yoneyama T, el al. Periodontal loser sites in untreated adult subjects, J Clin Periodontol, 1989; 16: 671-678.
[8] Jenkins WMM, Kinane DF. The"high risk"group in periodontitis. Br Dent J, 1989; 167: 168-171.
[9] 卞金有主编．预防口腔医学．第四版．北京：人民卫生出版社，2003；146-150
[10] Axesson P, Lindhe J, Nystrrm B. On the prevention of caries and periodontal disease. Results of a 15-year longitudinal study in adults. J Clin Pefiodontol, 1991: 18: 182-189.
[11] Van der Weijden GA, Timmerrnan ME, Danser MM, et al. Effect of Pre-experimental maintenance care duration on the development of gingivitis in a partial mouth experimental gingivitis model. J Periodontal Res, 1994; 29: 168-173.
[12] 曹采方主编．牙周病学．第二版．北京：人民卫生出版社，2004；35-74。
[13] 陈莉丽，章锦才等．《口腔厌氧菌与牙周病》．北京：人民卫生出版社1998年6月．
[14] Socransky S, Haffajee A. Evidence of bacterial aetiology: a historical perspective. Periodontol 2000, 1994; 57-25.
[15] Zambon JJ. Periodontal diseases: microbial factors. Ann Periodontol, 1996; 1: 879-925.
[16] Haffajee A, Dzink J, Socransky S. Effect of modified Widman flap surgery and systemic tetracycline on the subgingival microbiota of periodontal lesions. J Clin Periodontal, 1988; 15: 255-262.
[17] van Winkelhoff AJ, van der Velden U, de Graaff J. Microbial succession in recolonizing deep periodontal pockets after single course of supra-and subgingival debridement. J Clin Periodontal. 1988: 15: 116-122.
[18]Bragd L, Dahlden G, Wisstrom M, Slots J. The capability of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, Bacteroides intermedius to indicate progressive periodontitis; a retrospective study. J Clin Periodontal, 1987; 14: 95-99.
[19]Mahanonda R, Seymour G, Powell L, et al. Effect of initial treatment of chronic inflammatory periodontal disease on the frequency of peripheral blood T-lymphocytes specific to periodontopathic bacteria. Oral Microbiol Immunol, 1991; 6: 221-227.
[20] Haffajee A, Socransky S, Dzink J, et al. Clinical, microbiological and immunological features of subjects with refractory periodontal diseases. J Clin Periodontal, 1988; 15: 390-398.
[21] Dzink J, Socransky S, Ebersole J, et al. ELISA and conventional techniques for identification of black-pigmented Bacteroides isolated from periodontal pockets. J Periodontal Res, 1983; 18: 369-374.
[22]Grossi S, Genco R, Machtei E, el al. Assessment of risk for periodontal disease. I. Risk indicators for alveolar bone loss. J Periodontal, 1995; 66:23-29.
[23] Lai C-H, Listgarten M, Shirakawa M, Bacteroides forsythus in adult gingivitis and periodontitis. Oral Microbiol Immunol, 1987: 2: 152-157.
[24]Carlos J, Wolfe M, Zambon J, et al. Periodontal disease in adolescents: some clinical and microbiological correlates of attachment loss. J dent Res, 1988; 66: 1510-1514.
[25] Zambon JJ,Christersson LA,Slots J. Actinobacillus actinomycetemcomitans in human periodontal disease. Prevalence in patient groups and distribution of biotypes and serotypes within families. J Periodontal, 1983; 54: 707-711.
[26]Dzink J, Socransky S, Haffajee A. The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases.J Clin Periodontal, 1988; 15: 316-323.
[27]Nakazawa F,Zambon JJ,Reynolds HS,et al.Serological studies of oral Bacteroides intermedius.Infect Immun,1988,56:1647 1651.
[28]Ashimoto A,Chen C,Bakker I,et al.Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivalis and advanced periodontal lesions,Oral Microbiol Immunol, 1996,11:266 273.
[29]Socransky SS,Haffajee AD.Effect of therapy on periodontal infections.J Periodontol,1993,64:754 759.
[30] Kigure T, Saito A, Seida K, et al. Distribution of Porphyromonas gingival and Treponema denticola in human subgingival plaque at different periodontal pocket depths examined by immunohistochemical methods.J Periodontal Res, 1995; 30: 332.
[31]Bragd L,Dahlon G,Wisstrom M,et al.The capability of Actinobacillus actionmycetemco mitans,Bacteroides gingivalis,Bacteroides intermedius to indicate progressive periodontitis;a retrospective study[J].J Clin Periodontol,1987,14:95
[32]Grossi S,Genco R,Machtei E,et al.Assessment of risk for periodontal ldisease.I.Risk indicators for alveolar bone loss[J].J Periodontol,1995,66:23
[33]Mayrand D,Holt SC.Biology of asaccharolytic black-pig-mented bacteroides species[J].Microbiol Rev,1998,52(1):134.
[34] Curtis MA,Kuramitsu HK,Lantz M,et al.Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis[J].J Periodont Res, 1999,34:464
[35] Bretz WA,Lopatin DE,Loesche WJ.Benzoyl-argininenapthylamide(BANA) hydrolysis by Treponema denticola and/or Bateroides gingivalis in periodontal plaques[J].Oral Microbiology and Immunology, 1990,5:275
[36]Tanner AC,Socransky SS,Goodson JM.Microbiota of periodontal pockets losing crestal alveolar bone.J Periodontal Res, 1984,19:279 291.
[37] Schwartz Z, Goultschin J, Dean DD, et al. Mechanisms of alveolar bone destruction in periodontitis. Periodontal 2000, 1997; 14: 158-172.
[38]Kornman KS, Page RC, Tonetti MS. The host response to the microbial challenge in periodontitis: assembling the players. Periodontol 2000, 1997; 14: 33-53.
[39]Reynolds JJ, Meikle MC. Mechanisms of connective tissue matrix destruction in periodontitis. Periodontal 2000, 1997; 14:144-157.
[40] Darveau RP,Tanner A, Page RC. The microbial challenge in periodontitis. Periodontal 2000, 1997; 14: 12-32.
[41]Satio S,Ngan P,Satio M.et al. Interactive effects between cytokines on PGE production by human periodontal ligament fibroblasts in vitro[J].J dent Res,1990,69(8):1456
[42]Nojima N,Kobayashi M,Shinonome M,et al. Fibroblastic cells derived from bovine periodontal ligaments have the phenotypes of osteoblasts[J].J periodontal Res,1990,25(3):179
[43] Gazi MI, Cox sw, Clark DT et al. Comparison of host tissue and bacterial dipeptidyl peptidases in human gingival crevicular fluid by analytical isoelectric focusing. Arch Oral boil, 1995; 40: 731-736.
[44] Eley BM, Cox SW. Bacterial protease in gingival crevicular fluid before and after periodontal treatment. Br Dent J, 1995; 178: 133-139.
[45] Wikstrom M, Potempa J, Polanowski A, et al. Detection of Porphyromonas gingivalis in gingival exudates by a dipeptide—enhanced trypsin—like activity. J Periodonto 1.1994; 65: 47-55.
[46] Graves DT, Cochran D.The contribution of interleukin-1 and tumor necrosis factor to periodontal tissue destruction. J Periodontol,2003,74(3):391-401.
[47] Oates TW, Graves DT, Cochran DL. Clinical,radiographic and biochemical assessment of IL-1/TNF-alpha antagonist inhibition of bone loss in experimental periodontitis. J Clin Periodontol,2002,29(2): 137 143
[48] Assuma R,Oates T, Cochran D,et al IL-1 and TNF antagonists nhibitthe inflammatory response and bone loss in experimental periodontitis. J Immunol, 1998,160(1):403 409
[49] Masaka T, Hayashi J,Ishikanwa I.Soluble CD 14-dependent intercellular adhesion molecular-induction by Porphyrononas gingivalis lipoplysaccharide in human gingival fibroblasts[J].J periodontol,1999,70(7):772
[50] Brandon H Joe,James L Borke,Meral Keskintepe,et al.Interleukin-1b regulation of adhesion molecules on human gingival and periodontal ligament fibroblasts[J].J periodontol,2001,72(7):865
[51] Socransky S,Haffaj ee A.Evidence of aetiology:a historical perspective [J].Periodontol,2000,1994,5:7.
[52] Zambon JJ.Periodontal diseases:microbial factors [J].Ann Periodontol, 1996,1:879.
[53] Gillette W.Re:Antibiotics and periodontal diseasa[J].J Periodontol,1999,67(7):726.
[54] Bollen CM,et al.Microbiology response to mechanical treatment in combination with adjunctive therapy [J].J Periodontol, 1999,67(11) 1143.
[55] van Winkellfoff AJ,et al.Systemic antibiotic therapy in sensre periodontitis[J].Curr Opin Periodontol, 1997,4:35.
[56] 朱彩莲，李鸣宇．17种中药提取物对6种牙周可疑致病菌的体外抑菌实验[J]．上海口腔医学，2006，15(4)：434-436
1．全国牙病防治指导组．全国第二次全国口腔健康流行病学调查[M]．北京，人民卫生出版社，1999．
2．徐治鸿．实用中医口腔病学[M]．天津；天津科技翻译出版公司，1991．93
3．裴霞，华红，徐志鸿．中医药在牙周病的临床应用．现代口腔医学杂志，2001，15 (2)：151-152
4．乔守正．中医对牙周病的一些认识．北京口腔医学，1998，6(1)：20
5．徐治鸿．实用中医口腔病学[M]．天津：天津科技翻译出版公司，1991．93
6．徐治鸿，孙小平，华红，等．固齿健周煎剂对老年小鼠的抗氧化作用[J]．中华口腔医学杂志，1998，33(4)：219
7．樊明文，凌均棨，程祥荣，等．固齿增骨散的研制和初步临床应用[J]．口腔医学纵横杂志，1994，10(4)：195
8．谢吴，苏霞，黄志强，等．固齿散对牙周炎抗炎作用的治疗观察[J]．口腔医学纵横杂志，1998，14(1)：14
9．樊明文，彭彬，罗玲荆．固齿散对体外培养人牙龈成纤维细胞的影响[J]．中华口腔医学杂志，1995，30(5)：298
10．赵瑞芳，周以钧，黄明霞．青少年牙周炎患者服用固齿膏的观察．中华口腔医学杂志，1988，23(6)：368
11．周黎安，赵瑞芳，周以钧．青少年牙周炎患者服用固齿膏的疗效观察．牙体牙骨牙周病学杂志，1993，3(4)：217
12．宋红，赵瑞芳，周以钧．固齿膏对牙周炎龈沟液中IL-8的影响[J]．中华口腔医学杂志，1997，32(6)：372
13．1张举之，杨新雪，仝月华．固齿丸治疗青少年牙周炎的临床研究[J]．华西口腔医学杂志，1985，3(1)：17
14．张举之．固齿丸治疗牙周病机理探讨[J]．华西口腔医学杂志，1990，8(3)：198
15．张举之．固齿丸合并螺旋霉素治疗牙周病的临床研究[J]．中国中西医结合杂志，1992，12(2)：83
16．欧炯光，张举之．固齿丸对青少年牙周炎患者周围血多形核白细胞趋化和吞噬功能的作用[J]．中华口腔医学杂志，1991，26(1)：51
17．吴娅菲，张举之．用暗视野显微镜监测牙周病的治疗[J]．中华口腔医学杂志，1990，25(1)：48
18．丁一，张举之．女性牙周炎患者服用固齿丸前后唾液孕酮雌二醇水平的变化[J]．华西口腔医学杂志，1990，8(3)：204
19．胡琳，张举之，肖邦良．固齿丸对动物实验性牙周炎酶组织化学改变的影响[J]．中华口腔医学杂志，1992，27(2)：84。8孟琳，张举之，郑光静，等．固齿丸抗自由基损伤研究[J]．华西口腔医学杂志，1990，8(3)：208
20．朱聘倬．清胃散治疗急性牙周炎疗效观察[J]．上海中医药杂志，2000，34(7)：37
21．黄颐玉，李林可，岑锴．固齿增骨散治疗慢性牙周炎疗效观察[J]．北京中医药大学 学报，2000，23(4)：60
22．丁一，张举之．牙周炎患者唾液孕酮、雌二醇、睾酮水平的初步观察[J]．中华口腔医学杂志，1991，26(5)：287
23. Sooriyamoorthy M,Gower DB.Hormonal influences on gingival tissue: relationship to periodontal disease[J]. J Clin Periodont, 1989,16:201
24. Aufdemorte TB,Sheridan PJ.Nuclear uptake of sex steroids in gingival of the baboon[J].J periodontal, 1981,52(8):430
25. Parkar MH,Newman HN,Olsen I.Polymerase chain reaction analysis of oestrogen and androgen receptor expression in human gingival and periodontal tissus.Arch Oral Bioi,1996,41(10):979
26. Van Dyke TE, Horoszewicz HU, Cianciola LJ, et al. Neutrophil chemotaxis dysfunction in human periodontitis[J]. Infect Immun, 1980, 27(1): 124
27．班红艳，周秀青，关晶等．补肾合剂对牙周炎大鼠外周血中雌二醇及睾酮含量的影响[J]．北京口腔医学，2004，12(4)：195-197
28. Kopeikin VN,Kushlinskii NE,Semenov Ⅱu,et al,Estrogen receptors in the tissues of the margina lperiodontium of patient with hronic generalized periodontitis. Stomatologiia(Mosk), 1995,74(4):13
29．邬继东，游士奇，屠嫩斐．应用性激素类避孕药妇女牙周情况的配对调查．中华口腔医学杂志，1992，27(3)：151
30．徐治鸿，彭先忠，马慧敏，等．牙周病患者肾虚辨证与部分内分泌激素的关系．中华口腔医学杂志，1995，30(5)：301
31．关晶，周秀青，班红艳等．补肾合剂对牙周炎大鼠外周血中肿瘤坏死因子α含量的影响[J]．北京口腔医学，2005，13(1)：13-15．
32．徐竺婷，李鸣宇．10种中药有效成分抑制口腔主要致病菌比较[J]。中国现代应用药学杂志，2000，17(4)：277-279
33．朱彩莲，李鸣宇．17种中药提取物对6种牙周可疑致病菌的体外抑菌实验[J]．上海口腔医学，2006，15(4)：434-436．
34．褚敏，梁景平，朱彩莲．茶多酚、鞣酸抗细菌生长、粘附能力的研究[J]．现代口腔医学杂志，2001，15(2)：127-128
35．杨明华，武影，沈海雁．大黄葸醌衍生物对人牙周膜细胞作用的体外实验研究[J]．牙体牙髓牙周病学杂志，2002，12(7)：355
36．叶显纯，孙文忠，金岚，等．中药临床手册[M]．第1版．上海：上海科学技术出版社，1993．
37．难波恒雄．传统中药预防龋齿的研究(Ⅳ)[J]．国外医学中医中药分册，1986，2(1)：26
38. Wolinsky LE,Sote EO.Isolation of natural plaque-inhibiting substances from Nigerian chewing sticks [J]. Caries Res, 1984,18 (3):216
39 Wu-Yuan CD,Chen CY, Wu RT.Gallotannins inhibit growth,water-insoluble glucan synthesis,and aggregation of mutans streptococci [J].J Dent Res, 1988,67(1):51]
40．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[J]．牙体牙髓牙周病学杂志，2002，12(5)：255-257
41 王静，唐荣银，王志良等．五倍子水提取物对胶原酶活性的影响[J]．临床口腔医学杂志，2006，22(8)：451-453．
42．张红梅，朱秀丽，陈强等．五倍子对牙龈卟啉单胞菌胰酶样蛋白酶活性的抑制作用[J]．牙体牙髓牙周病学杂志，2004，14(1)：29-31
43．周汝俊，陈涟如，黄敬耀，等．黄甲棒缓释剂的研究[J]．中华口腔医学杂志，1996，31(3)：159
44．李红，李继遥等．五倍子对牙菌斑生物膜细菌生长代谢的研究[J]．中国中药杂志，2005，31(21)：1685．
45．王茜，樊明文，边专等．熊果酸的提取及其对牙周病原菌的作用[J]．中华口腔医学杂志，2002，37(5)：388．
46. Asadi SG;Asadi ZG. Chewing sticks and the oral hygiene habits of the adult Pakistani population[J].Int Dent J, 1997;47:275-278.
47. A1 lafi T, Ababneh H.The effect of the extract of the miswak(chewing sticks) used in Jordan and the Middle East on oral bacteria[J]. Dent J, 1995;45(3):218-222.
48．曹采方．牙周病学[M]．北京：人民卫生出版社，2000．30
49．刘正。口腔生物学[M]．第2版，北京：人民卫生出版社，2003
50. Feng Z,Weinberg A.Role of bacteria in health and disease of periodontal tissues[J].periodontalogy,2006,40(1)50-76.
50．李红，李继遥等．五倍子对牙菌斑生物膜细菌生长代谢的研究[J]．中国中药杂志，2005，31(21)：1685．
52．王茜，樊明文，边专等．熊果酸的提取及其对牙周病原菌的作用[J]．中华口腔医学杂志，2002，37(5)：388-390．
53．李廷谦．循证医学与中西医结合现状及设想[J]．中国中西医结合杂志，2002，22(1)：8
54．孔令义，杨智，闵知大．对中药现代化中有效成份研究的思考[J]．中草药，1998， 29 (5): 354.
55. Cao CF,Sun XP.Herbal medicine for periodontal disease[J].Intern Dent J, 1998,48:316.
2. Socransky S,Haffajee A.Evidence of bacterial aetiology:a historical perspective. Periodontol 2000, 1994; 5: 7-25.
3. Zambon JJ. Periodontol diseases: microbial factors. Ann Periodontol, 1996; 1: 879-925.
4. Darveau RP, Tanner A,Page RC. The microbial challenge in periodontitis. Periodontol 2000, 1997; 14: 12-32.
5. Jansen HJ, van der Hoeven JS. Protein degradation by Prevotella intermedia and Actinomyces meyeri supports the growth of non-protein-cleaving oral bacteria in serum.J Clin Periodontol, 1997;24:346-353.
6. Curtis MA,Kuramitsu HK,Lantz M,et al.Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis.J Periodont Res, 1999;34:464-472.
7．曹采方．牙周病学[M]．北京：人民卫生出版社，2000．160
8. Jun Isaka, Atsushi Ohazama,Makoto Kobayashi,et al.Participation of periodontal ligament cells with regeneration of alveolar bone[J].J periodontal,2001,72(3):314-323
9. Wishmura F, Terranova VP.Comparative studying of the chemotactia responses of periodontal ligament cells and gingival fibroblasts to polypeptide growth factors[J].J Dent Res. 1996,75(4):986-992
10．徐治鸿．实用中医口腔病学[M]．天津：天津科技翻译出版公司，1991．93
11．裴霞，华红，徐志鸿．中医药在牙周疾病的临床应用于．现代口腔医学杂志，2001，15(2)：151-152．
12．徐志鸿，孙小平，华红等．固齿健周煎剂对老年小鼠的抗氧化作用．中华口腔医学杂志，1998，33(4)：219．
13．宋红，赵瑞芳，周以钧．固齿膏对牙周炎龈沟液中IL-8的影响．中华口腔医学杂志，1997，32(6)：372．
14．徐竺婷，李鸣宇．10种中药有效成分抑制口腔主要致病菌比较[J]．中国现代应用药学杂志，2000，17(4)：277-279
15．朱彩莲，李鸣宇．17种中药提取物对6种牙周可疑致病菌的体外抑菌实验[J]．上海口腔医学，2006，15(4)：434-436．
16．叶显纯，孙文忠，金岚，等．中药临床手册[M]．上海：上海科学技术出版社，1993．
17. Wu-Yuan CD,Chen CY, Wu RT.Gallotannins inhibit growth,water-insoluble glucan synthesis,and aggregation of mutans streptococci[J].J Dent Res,1988,67(1):51]
18．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[J]．牙体牙髓牙周病学杂志，2002，12(5)：255-257
19．王静，唐荣银，王志良等．五倍子水提取物对胶原酶活性的影响[J]．临床口腔医学杂志，2006，22(8)：451-453．
20．张红梅，朱秀丽，陈强等．五倍子对牙龈卟啉单胞菌胰酶样蛋白酶活性的抑制作用[J]．牙体牙髓牙周病学杂志，2004，14(1)：29-31
21．周汝俊，陈涟如，黄敬耀，等．黄甲棒缓释剂的研究[J]．中华口腔医学杂志，1996，31(3)：159
22．王茜，樊明文，边专等．熊果酸的提取及其对牙周病原菌的作用[J]．中华口腔医学 杂志，2002，37(5)：388-390．
23．李廷谦．循证医学与中西医结合现状及设想[J]．中国中西医结合杂志，2002，22(1)：8
[1] Feng Z,Weinberg A.Role of bacteria in health and disease of periodontal tissues [J].Periodontology,2006,40(1):50-76.
[2] 沈家平，朱维建，陆平成．中药复方漱口液漱口前后牙周指数的变化[J]．南京中医药大学学报，2006，22(2)：118-119
[3] National Committee for Clinical Laboratory Standards.Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically [M]. Wayne,Pennsylvania:National Committee for Clinical Laboratory Standards.NCCLS Document M7-A5,2000.
[4] 樊明文主编．牙体牙髓病学[M]．北京：人民卫生出版社，2000．22-25．
[5] Bragd L,Dahlen G, Wisstrm M,et al.The capability of Actinobacillus actinomycetemcomitans,Bacteroides gingivalis,Bacteroides intermedius to indicate progressive periodontitis,a retrospective study[J].J Clin Periodontol,1987,14:95
[6] Grossi S,Genco R,Machtei E,et al.Assessment of risk for periodontal disease.I.Risk indicators for alveolar bone loss[J].J Periodontol, 1995,66:23
[7] Mayrand D,Holt SC.Biology of asaocharolytic black-pigmented bacteroides species [J].Microbiol Rev, 1998,52(1):134
[8] Zambon JJ,Christersson LA,Slots J.Actinobacillus action myycete mcomitans in human periodontaldisease.Prevalence in patient groups and distribution of biotypes within families[J].J Periodontol, 1983,54:707.
[9] Haffajee AD,Socransky SS.Microbial etiological agents of destructive periodontal diseases.periodontol 2000,1994.5:78-111
[10] 朱彩莲，李鸣宇．17种中药提取物对6种牙周可疑致病菌的体外抑菌实验[J]．上海口腔医学，2006，15(4)：434-436
[11] 李鸣宇，刘正．茶多酚对口腔咽喉致病菌作用的体外实验[J]．上海第二医科大学学报，1999，19：41-43．
[12] 黄吉燕，朱聘倬，刘力．三种中药对牙周致病厌氧菌抑制作用的体外实验[J]．上海中医药杂志，2006，40(5)：62-63．
[13] 黄霞，谭红，黄定明等．天然药物对牙龈卟啉单胞菌的体外抗菌研究[J]．中国微生态学杂志，2005，17(5)：346-347．
[14] 徐竺婷，李鸣宇．10种中药有效成分抑制口腔主要致病菌比较[J]．中国现代应用药学杂志，2000，17(4)：277-279
[15] 高学敏主编．中药学[M]．北京：人民卫生出版社，2000．595-601．
[1] 徐燕，蒋勇，李颂等．烟草对牙周成纤维细胞影响的实验观察[J]．中华口腔医学 杂志，2003，38(5)：367-369
[2] 司徒镇强，吴军正．细胞培养[M]．西安：世界图书出版公司西安公司，1996
[3] 李谨，蒋春梅，孙卫斌．人牙周膜成纤维细胞体外培养条件的优化[J]．临床口腔医学杂志，2003，19(6)：333-335
[4] 冯雪．体外培养牙周膜成纤维细胞矿化能力的实验研究[J]．华西口腔医学杂志，1998，4294-295
[5] Rose GC,et al.Human periodontal ligament cells in vitro[J].J periodont Res, 1987,22:20-28
[6] Mariotti A,et al.Characterization of fibroblasts derived from human periodontal ligament and gingival fibroblast[J].J periodontal, 1990,61:103-114
[7] Murphy KG, et al. Human periodontal ligament cells in vitro:cell culture passage effect on collagen gel contraction[J].J periodont Res,1987,22:342-348
[8] Liu HW, Yacobi R, Savion N.A collagenous cementum derived attachment protein is a marker for progenitors of mineralized tissue-forming cell lineage of the periodontal ligament.J Bone Miner Res, 1997,12(10): 1691-1699
[9] Jun Isaka, Atsushi Ohazama,Makoto Kobayashi,et al.Participation of periodontal ligament cells with regeneration of alveolar bone.J periodontol,2001,72(3):314-323
[10] Wishmura F, Terranova VP. Comparative studying of the chemotactia responses of periodontal ligament cells and gingival fibroblasts to polypeptide growth factors.J Dent Res,1996,75(4):986-992
[11] 章锦才，吉庆勇，肖卓然．白细胞介素-1对牙龈成纤维细胞增殖和前列腺素2产生的影响．口腔医学纵横杂志，1992，8(4)：199-200
[12] Chung CP, Park JB,Bae KH.Pharmacological effects of methanolic extract from the root of scutetlaria baicalensis and its flavonoids on human gingival fibroblasts.Planta Med,1995,61 (2): 150-153
[13] 曹正国，李成章，刘小平．中药黄芩苷对人牙周膜细胞增殖和总蛋白含量的影响[J]．口腔医学研究：2003，19(1)：10-12
[14] 李成章，曹正国，樊明文等．黄芩苷对人牙周韧带细胞超微结构的影响[J]．牙体牙髓牙周病学杂志，2003，13(2)：76-77。
[15] 许彦枝，邹慧儒，王小玲等．中药双黄补对人牙周膜细胞总蛋白含量和超微结构的影响[J]．实用口腔医学杂志，2005，21(3)：374-377
[16] 章魁华，于世风，主编．实验口腔病理学[M]．北京：人民卫生出版社，2002．308-312
[17] Palmon A,Roos H,Edel J,et al.Inverse dose-and time-dependent effect of basic fibroblast growth factor on the gene expression of collagen type I and matrix metalloproteinase-1 by periodontal ligament cells in culture.J Periodontol,2000,71 (6):974-980
[18] 沈家平，朱维建，陆平成．中药复方漱口液漱口前后牙周指数的变化[J]．南京中医药大学学报，2006，22(2)：118-119
[19] King GN,Cochran DL.Factors that modulate the effects of bone morphogenetic protein-induced periodontal regeneration;a critical review.J Periodontal,2002,73(8):925-936
[20] 司晓辉，刘正．转化生长因子β对人牙周膜成纤维细胞的生物学作用[J]．中华口腔医学杂志，2001，36(1)：23-26
[1] Chung CP, Park JB,Bae KH.Pharmacological effects of methanolic extract from the root of scutellaria baicalensis and its flavonoids on human gingival fibroblasts.Planta Med, 1995,61 (2): 150-153
[2] 李成章，曹正国，樊明文等．黄芩苷对人牙周韧带细胞超微结构的影响[J]．牙体牙髓牙周病学杂志,2003，13(2)：76-77
[3] 司徒镇强，吴军正．细胞培养[M]．西安：世界图书出版公司西安公司，1996
[4] 李谨，蒋春梅，孙卫斌．人牙周膜成纤维细胞体外培养条件的优化[J]．临床口腔医学杂志，2003，19(6)：333-335
[5] 冯雪．体外培养牙周膜成纤维细胞矿化能力的实验研究[J]．华西口腔医学杂志，1998，4294-259
[6] Rose GC,et al.Human periodontal ligament cells in vitro[J].J periodont Res,1987,22:20-28
[7] Mariotti A,et al.Characterization of fibroblasts derived from human periodontal ligament and gingival fibroblast [J].J periodontal, 1990,61:103-114
[8] Murphy KG, et al. Human periodontal ligament cells in vitro:cell culture passage effect on collagen gel contraction[J].J periodont Res, 1987,22:342-348
[9] 徐燕，蒋勇，李颂等．烟草对牙周成纤维细胞影响的实验观察[J]．中华口腔医学杂志,2003，38(5)：367-369
[1] 刘宏伟，徐治鸿，俞帆．咀嚼黄芩口胶前后牙周指数的变化[J]．现代口腔医学杂志,2000,14(6)：405-406．
[2] 孔令义，杨智，闵知大．对中药现代化中有效成份研究的思考[J]．中草药，1998,29(5)：354．
[3] 卞金有主编．预防口腔医学[M]．北京：人民卫生出版社，2003．32．33．
[4] 樊明文主编．口腔生物学．北京：人民卫生出版社，1996．181-198，214-230．
[5] Haffajee AD,Socransky SS.Microbial etiological agents of destructive periodontal diseases.periodontol 2000,1994.5:78-111
[6] 李鸣宇，刘正．茶多酚对口腔咽喉致病菌作用的体外实验[J]．上海第二医科大学学报，1999，19：41-43．
[7] Ooshima T, Minami T, Matsumoto M,et al.Comparison of the Cariostatic Effects between Regimens to Administer Oolong Tea Polyphenols in SPF Rats.Caries Res, 1998;32(2):75
[8] 黄冰冰，樊明文，杨祥良等．桔梗、甘草及其组成的复方对口腔病原菌生长影响的体外实验[J]．实用口腔医学杂志，2003，19(2)：148-150．
[1] World Health Organization.Oral Health Surveys:Basic Methods 4thed Geneva: WHO, 1997.
[2] 全国牙病防治指导组．第二次全国口腔健康流行病学抽样调查[M]．北京：人民卫生出版社，1999．268-317．
[3] 卞金有主编．预防口腔医学[M]．北京：人民卫生出版社，2003，第4版，37．
[4] 韩晓兰，颜雨春，蒋勇等．安徽省城乡人群口腔健康状况[J]．疾病控制杂志，2003，7(4)：352-353．
[5] Pilot T, Miyazaki H.Global results:15 years of CPITN epidemiology[J].Int Dent J, 1994,44:553.
[6] Pilot Y,Lu ZY,Lin ZQ,et al. Periodontal conditions in 35-44-year-old factory workers in Shanghai[J]. Community Dent Oral Epidemiol, 1989,17:216.
[7] 廖旭辉，林焕彩，卢展民等．广东省中老年人的牙周健康状况[J]．中山医科大学学报，2000，21(4)：314-315．
[8] Miyazaki H,Ohtani L,Abe N,et al.Periodontal conditions in elder age cohorts aged 65 years and eider in Japan,measured by CPITN and loss of attachment[J].Community Dent Health, 1995,12:216.
[9] Petersen P E, Peng B, Tai B J.Oral health status and oral health behaviour of middle-aged and elderly people in P R China. Int Dent J. 1997,47(5):305-312.
[10] 胡德渝．中国人口结构与口腔疾病的改变趋势[J]．华西口腔医学杂志，2000，18(2)：126-128．
[11] 周红玲，朱文昊，初里楠等．北京市西城区口腔健康调查资料的统计与分析[J]．北京口腔医学，2004，12(3)：160-162．
[1] Socransky S,Haffajee A.Evidence of bacterial aetiology:a historical perspective [J].Periodontol 2000,1994;5:7-25.
[2] Harris NO.Primary Preventive Dentistry. Fifthedition. America:Stamford, Connecticut, 1999
[3] Kinae DF. Causation and pathogenesis of periodontal disease. Pefiodontol 2000. 2001; 25: 8-20.
[4] Albandar JM, Brunelle JA, Kingman A. Destructive periodontal disease in adults 30 yers of age and older inthe United States. 1981—1984. J Periodontol, 1999; 70: 13-29.
[5] Brown LJ, Loe H. Prevalence, extent, severity and progression of periodontal disease. Periodontol 2000, 1993; 2: 57-71.
[6] Papapanou PN, Wennstrrm JL Grondhal K. Periodontal status in relation to age and tooth type. A cross-sectional radiographic study. J Clin Periodontol, 1988; 15: 469-478.
[7] Lindhe J, Okamoto H, Yoneyama T, el al. Periodontal loser sites in untreated adult subjects, J Clin Periodontol, 1989; 16: 671-678.
[8] Jenkins WMM, Kinane DF. The"high risk"group in periodontitis. Br Dent J, 1989; 167: 168-171.
[9] 卞金有主编．预防口腔医学．第四版．北京：人民卫生出版社，2003；146-150
[10] Axesson P, Lindhe J, Nystrrm B. On the prevention of caries and periodontal disease. Results of a 15-year longitudinal study in adults. J Clin Pefiodontol, 1991: 18: 182-189.
[11] Van der Weijden GA, Timmerrnan ME, Danser MM, et al. Effect of Pre-experimental maintenance care duration on the development of gingivitis in a partial mouth experimental gingivitis model. J Periodontal Res, 1994; 29: 168-173.
[12] 曹采方主编．牙周病学．第二版．北京：人民卫生出版社，2004；35-74。
[13] 陈莉丽，章锦才等．《口腔厌氧菌与牙周病》．北京：人民卫生出版社1998年6月．
[14] Socransky S, Haffajee A. Evidence of bacterial aetiology: a historical perspective. Periodontol 2000, 1994; 57-25.
[15] Zambon JJ. Periodontal diseases: microbial factors. Ann Periodontol, 1996; 1: 879-925.
[16] Haffajee A, Dzink J, Socransky S. Effect of modified Widman flap surgery and systemic tetracycline on the subgingival microbiota of periodontal lesions. J Clin Periodontal, 1988; 15: 255-262.
[17] van Winkelhoff AJ, van der Velden U, de Graaff J. Microbial succession in recolonizing deep periodontal pockets after single course of supra-and subgingival debridement. J Clin Periodontal. 1988: 15: 116-122.
[18]Bragd L, Dahlden G, Wisstrom M, Slots J. The capability of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, Bacteroides intermedius to indicate progressive periodontitis; a retrospective study. J Clin Periodontal, 1987; 14: 95-99.
[19]Mahanonda R, Seymour G, Powell L, et al. Effect of initial treatment of chronic inflammatory periodontal disease on the frequency of peripheral blood T-lymphocytes specific to periodontopathic bacteria. Oral Microbiol Immunol, 1991; 6: 221-227.
[20] Haffajee A, Socransky S, Dzink J, et al. Clinical, microbiological and immunological features of subjects with refractory periodontal diseases. J Clin Periodontal, 1988; 15: 390-398.
[21] Dzink J, Socransky S, Ebersole J, et al. ELISA and conventional techniques for identification of black-pigmented Bacteroides isolated from periodontal pockets. J Periodontal Res, 1983; 18: 369-374.
[22]Grossi S, Genco R, Machtei E, el al. Assessment of risk for periodontal disease. I. Risk indicators for alveolar bone loss. J Periodontal, 1995; 66:23-29.
[23] Lai C-H, Listgarten M, Shirakawa M, Bacteroides forsythus in adult gingivitis and periodontitis. Oral Microbiol Immunol, 1987: 2: 152-157.
[24]Carlos J, Wolfe M, Zambon J, et al. Periodontal disease in adolescents: some clinical and microbiological correlates of attachment loss. J dent Res, 1988; 66: 1510-1514.
[25] Zambon JJ,Christersson LA,Slots J. Actinobacillus actinomycetemcomitans in human periodontal disease. Prevalence in patient groups and distribution of biotypes and serotypes within families. J Periodontal, 1983; 54: 707-711.
[26]Dzink J, Socransky S, Haffajee A. The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases.J Clin Periodontal, 1988; 15: 316-323.
[27]Nakazawa F,Zambon JJ,Reynolds HS,et al.Serological studies of oral Bacteroides intermedius.Infect Immun,1988,56:1647 1651.
[28]Ashimoto A,Chen C,Bakker I,et al.Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivalis and advanced periodontal lesions,Oral Microbiol Immunol, 1996,11:266 273.
[29]Socransky SS,Haffajee AD.Effect of therapy on periodontal infections.J Periodontol,1993,64:754 759.
[30] Kigure T, Saito A, Seida K, et al. Distribution of Porphyromonas gingival and Treponema denticola in human subgingival plaque at different periodontal pocket depths examined by immunohistochemical methods.J Periodontal Res, 1995; 30: 332.
[31]Bragd L,Dahlon G,Wisstrom M,et al.The capability of Actinobacillus actionmycetemco mitans,Bacteroides gingivalis,Bacteroides intermedius to indicate progressive periodontitis;a retrospective study[J].J Clin Periodontol,1987,14:95
[32]Grossi S,Genco R,Machtei E,et al.Assessment of risk for periodontal ldisease.I.Risk indicators for alveolar bone loss[J].J Periodontol,1995,66:23
[33]Mayrand D,Holt SC.Biology of asaccharolytic black-pig-mented bacteroides species[J].Microbiol Rev,1998,52(1):134.
[34] Curtis MA,Kuramitsu HK,Lantz M,et al.Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis[J].J Periodont Res, 1999,34:464
[35] Bretz WA,Lopatin DE,Loesche WJ.Benzoyl-argininenapthylamide(BANA) hydrolysis by Treponema denticola and/or Bateroides gingivalis in periodontal plaques[J].Oral Microbiology and Immunology, 1990,5:275
[36]Tanner AC,Socransky SS,Goodson JM.Microbiota of periodontal pockets losing crestal alveolar bone.J Periodontal Res, 1984,19:279 291.
[37] Schwartz Z, Goultschin J, Dean DD, et al. Mechanisms of alveolar bone destruction in periodontitis. Periodontal 2000, 1997; 14: 158-172.
[38]Kornman KS, Page RC, Tonetti MS. The host response to the microbial challenge in periodontitis: assembling the players. Periodontol 2000, 1997; 14: 33-53.
[39]Reynolds JJ, Meikle MC. Mechanisms of connective tissue matrix destruction in periodontitis. Periodontal 2000, 1997; 14:144-157.
[40] Darveau RP,Tanner A, Page RC. The microbial challenge in periodontitis. Periodontal 2000, 1997; 14: 12-32.
[41]Satio S,Ngan P,Satio M.et al. Interactive effects between cytokines on PGE production by human periodontal ligament fibroblasts in vitro[J].J dent Res,1990,69(8):1456
[42]Nojima N,Kobayashi M,Shinonome M,et al. Fibroblastic cells derived from bovine periodontal ligaments have the phenotypes of osteoblasts[J].J periodontal Res,1990,25(3):179
[43] Gazi MI, Cox sw, Clark DT et al. Comparison of host tissue and bacterial dipeptidyl peptidases in human gingival crevicular fluid by analytical isoelectric focusing. Arch Oral boil, 1995; 40: 731-736.
[44] Eley BM, Cox SW. Bacterial protease in gingival crevicular fluid before and after periodontal treatment. Br Dent J, 1995; 178: 133-139.
[45] Wikstrom M, Potempa J, Polanowski A, et al. Detection of Porphyromonas gingivalis in gingival exudates by a dipeptide—enhanced trypsin—like activity. J Periodonto 1.1994; 65: 47-55.
[46] Graves DT, Cochran D.The contribution of interleukin-1 and tumor necrosis factor to periodontal tissue destruction. J Periodontol,2003,74(3):391-401.
[47] Oates TW, Graves DT, Cochran DL. Clinical,radiographic and biochemical assessment of IL-1/TNF-alpha antagonist inhibition of bone loss in experimental periodontitis. J Clin Periodontol,2002,29(2): 137 143
[48] Assuma R,Oates T, Cochran D,et al IL-1 and TNF antagonists nhibitthe inflammatory response and bone loss in experimental periodontitis. J Immunol, 1998,160(1):403 409
[49] Masaka T, Hayashi J,Ishikanwa I.Soluble CD 14-dependent intercellular adhesion molecular-induction by Porphyrononas gingivalis lipoplysaccharide in human gingival fibroblasts[J].J periodontol,1999,70(7):772
[50] Brandon H Joe,James L Borke,Meral Keskintepe,et al.Interleukin-1b regulation of adhesion molecules on human gingival and periodontal ligament fibroblasts[J].J periodontol,2001,72(7):865
[51] Socransky S,Haffaj ee A.Evidence of aetiology:a historical perspective [J].Periodontol,2000,1994,5:7.
[52] Zambon JJ.Periodontal diseases:microbial factors [J].Ann Periodontol, 1996,1:879.
[53] Gillette W.Re:Antibiotics and periodontal diseasa[J].J Periodontol,1999,67(7):726.
[54] Bollen CM,et al.Microbiology response to mechanical treatment in combination with adjunctive therapy [J].J Periodontol, 1999,67(11) 1143.
[55] van Winkellfoff AJ,et al.Systemic antibiotic therapy in sensre periodontitis[J].Curr Opin Periodontol, 1997,4:35.
[56] 朱彩莲，李鸣宇．17种中药提取物对6种牙周可疑致病菌的体外抑菌实验[J]．上海口腔医学，2006，15(4)：434-436
1．全国牙病防治指导组．全国第二次全国口腔健康流行病学调查[M]．北京，人民卫生出版社，1999．
2．徐治鸿．实用中医口腔病学[M]．天津；天津科技翻译出版公司，1991．93
3．裴霞，华红，徐志鸿．中医药在牙周病的临床应用．现代口腔医学杂志，2001，15 (2)：151-152
4．乔守正．中医对牙周病的一些认识．北京口腔医学，1998，6(1)：20
5．徐治鸿．实用中医口腔病学[M]．天津：天津科技翻译出版公司，1991．93
6．徐治鸿，孙小平，华红，等．固齿健周煎剂对老年小鼠的抗氧化作用[J]．中华口腔医学杂志，1998，33(4)：219
7．樊明文，凌均棨，程祥荣，等．固齿增骨散的研制和初步临床应用[J]．口腔医学纵横杂志，1994，10(4)：195
8．谢吴，苏霞，黄志强，等．固齿散对牙周炎抗炎作用的治疗观察[J]．口腔医学纵横杂志，1998，14(1)：14
9．樊明文，彭彬，罗玲荆．固齿散对体外培养人牙龈成纤维细胞的影响[J]．中华口腔医学杂志，1995，30(5)：298
10．赵瑞芳，周以钧，黄明霞．青少年牙周炎患者服用固齿膏的观察．中华口腔医学杂志，1988，23(6)：368
11．周黎安，赵瑞芳，周以钧．青少年牙周炎患者服用固齿膏的疗效观察．牙体牙骨牙周病学杂志，1993，3(4)：217
12．宋红，赵瑞芳，周以钧．固齿膏对牙周炎龈沟液中IL-8的影响[J]．中华口腔医学杂志，1997，32(6)：372
13．1张举之，杨新雪，仝月华．固齿丸治疗青少年牙周炎的临床研究[J]．华西口腔医学杂志，1985，3(1)：17
14．张举之．固齿丸治疗牙周病机理探讨[J]．华西口腔医学杂志，1990，8(3)：198
15．张举之．固齿丸合并螺旋霉素治疗牙周病的临床研究[J]．中国中西医结合杂志，1992，12(2)：83
16．欧炯光，张举之．固齿丸对青少年牙周炎患者周围血多形核白细胞趋化和吞噬功能的作用[J]．中华口腔医学杂志，1991，26(1)：51
17．吴娅菲，张举之．用暗视野显微镜监测牙周病的治疗[J]．中华口腔医学杂志，1990，25(1)：48
18．丁一，张举之．女性牙周炎患者服用固齿丸前后唾液孕酮雌二醇水平的变化[J]．华西口腔医学杂志，1990，8(3)：204
19．胡琳，张举之，肖邦良．固齿丸对动物实验性牙周炎酶组织化学改变的影响[J]．中华口腔医学杂志，1992，27(2)：84。8孟琳，张举之，郑光静，等．固齿丸抗自由基损伤研究[J]．华西口腔医学杂志，1990，8(3)：208
20．朱聘倬．清胃散治疗急性牙周炎疗效观察[J]．上海中医药杂志，2000，34(7)：37
21．黄颐玉，李林可，岑锴．固齿增骨散治疗慢性牙周炎疗效观察[J]．北京中医药大学 学报，2000，23(4)：60
22．丁一，张举之．牙周炎患者唾液孕酮、雌二醇、睾酮水平的初步观察[J]．中华口腔医学杂志，1991，26(5)：287
23. Sooriyamoorthy M,Gower DB.Hormonal influences on gingival tissue: relationship to periodontal disease[J]. J Clin Periodont, 1989,16:201
24. Aufdemorte TB,Sheridan PJ.Nuclear uptake of sex steroids in gingival of the baboon[J].J periodontal, 1981,52(8):430
25. Parkar MH,Newman HN,Olsen I.Polymerase chain reaction analysis of oestrogen and androgen receptor expression in human gingival and periodontal tissus.Arch Oral Bioi,1996,41(10):979
26. Van Dyke TE, Horoszewicz HU, Cianciola LJ, et al. Neutrophil chemotaxis dysfunction in human periodontitis[J]. Infect Immun, 1980, 27(1): 124
27．班红艳，周秀青，关晶等．补肾合剂对牙周炎大鼠外周血中雌二醇及睾酮含量的影响[J]．北京口腔医学，2004，12(4)：195-197
28. Kopeikin VN,Kushlinskii NE,Semenov Ⅱu,et al,Estrogen receptors in the tissues of the margina lperiodontium of patient with hronic generalized periodontitis. Stomatologiia(Mosk), 1995,74(4):13
29．邬继东，游士奇，屠嫩斐．应用性激素类避孕药妇女牙周情况的配对调查．中华口腔医学杂志，1992，27(3)：151
30．徐治鸿，彭先忠，马慧敏，等．牙周病患者肾虚辨证与部分内分泌激素的关系．中华口腔医学杂志，1995，30(5)：301
31．关晶，周秀青，班红艳等．补肾合剂对牙周炎大鼠外周血中肿瘤坏死因子α含量的影响[J]．北京口腔医学，2005，13(1)：13-15．
32．徐竺婷，李鸣宇．10种中药有效成分抑制口腔主要致病菌比较[J]。中国现代应用药学杂志，2000，17(4)：277-279
33．朱彩莲，李鸣宇．17种中药提取物对6种牙周可疑致病菌的体外抑菌实验[J]．上海口腔医学，2006，15(4)：434-436．
34．褚敏，梁景平，朱彩莲．茶多酚、鞣酸抗细菌生长、粘附能力的研究[J]．现代口腔医学杂志，2001，15(2)：127-128
35．杨明华，武影，沈海雁．大黄葸醌衍生物对人牙周膜细胞作用的体外实验研究[J]．牙体牙髓牙周病学杂志，2002，12(7)：355
36．叶显纯，孙文忠，金岚，等．中药临床手册[M]．第1版．上海：上海科学技术出版社，1993．
37．难波恒雄．传统中药预防龋齿的研究(Ⅳ)[J]．国外医学中医中药分册，1986，2(1)：26
38. Wolinsky LE,Sote EO.Isolation of natural plaque-inhibiting substances from Nigerian chewing sticks [J]. Caries Res, 1984,18 (3):216
39 Wu-Yuan CD,Chen CY, Wu RT.Gallotannins inhibit growth,water-insoluble glucan synthesis,and aggregation of mutans streptococci [J].J Dent Res, 1988,67(1):51]
40．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[J]．牙体牙髓牙周病学杂志，2002，12(5)：255-257
41 王静，唐荣银，王志良等．五倍子水提取物对胶原酶活性的影响[J]．临床口腔医学杂志，2006，22(8)：451-453．
42．张红梅，朱秀丽，陈强等．五倍子对牙龈卟啉单胞菌胰酶样蛋白酶活性的抑制作用[J]．牙体牙髓牙周病学杂志，2004，14(1)：29-31
43．周汝俊，陈涟如，黄敬耀，等．黄甲棒缓释剂的研究[J]．中华口腔医学杂志，1996，31(3)：159
44．李红，李继遥等．五倍子对牙菌斑生物膜细菌生长代谢的研究[J]．中国中药杂志，2005，31(21)：1685．
45．王茜，樊明文，边专等．熊果酸的提取及其对牙周病原菌的作用[J]．中华口腔医学杂志，2002，37(5)：388．
46. Asadi SG;Asadi ZG. Chewing sticks and the oral hygiene habits of the adult Pakistani population[J].Int Dent J, 1997;47:275-278.
47. A1 lafi T, Ababneh H.The effect of the extract of the miswak(chewing sticks) used in Jordan and the Middle East on oral bacteria[J]. Dent J, 1995;45(3):218-222.
48．曹采方．牙周病学[M]．北京：人民卫生出版社，2000．30
49．刘正。口腔生物学[M]．第2版，北京：人民卫生出版社，2003
50. Feng Z,Weinberg A.Role of bacteria in health and disease of periodontal tissues[J].periodontalogy,2006,40(1)50-76.
50．李红，李继遥等．五倍子对牙菌斑生物膜细菌生长代谢的研究[J]．中国中药杂志，2005，31(21)：1685．
52．王茜，樊明文，边专等．熊果酸的提取及其对牙周病原菌的作用[J]．中华口腔医学杂志，2002，37(5)：388-390．
53．李廷谦．循证医学与中西医结合现状及设想[J]．中国中西医结合杂志，2002，22(1)：8
54．孔令义，杨智，闵知大．对中药现代化中有效成份研究的思考[J]．中草药，1998， 29 (5): 354.
55. Cao CF,Sun XP.Herbal medicine for periodontal disease[J].Intern Dent J, 1998,48:316.
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