CN102037360B - 口腔护理方法和系统 - Google Patents
口腔护理方法和系统 Download PDFInfo
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Abstract
本发明涉及评价口腔的生物群落和根据评价利用碱性氨基酸提供适合治疗的方法。
Description
本申请要求2008年2月9日提交的美国专利申请61/027,437的权益，也要求2008年2月9日提交的美国专利申请61/027,442和均在2008年2月8日提交的美国专利申请61/027,432、61/027,431、61/027,420和61/027,435的权益，这些申请的内容均通过引用结合到本文中。
发明领域
本发明涉及测定口腔中致龋菌和精氨酸分解菌(arginolyticbacterium)相对量的方法，例如使用含游离或盐形式的碱性氨基酸的组合物作为牙护理方案的部分。
发明背景
精氨酸和其他碱性氨基酸已被提出用于口腔护理，并相信其对抗蛀洞形成和牙过敏有显著的益处。市售的精氨酸基牙膏为含的和两者包含精氨酸和碳酸氢钙。
口腔中生物群落的类型一般在蛀洞发展和口腔健康中起重要作用。例如，已假设精氨酸有益效果的重要因素是，精氨酸和其他碱性氨基酸可由某些类型的细菌代谢，例如不致龋并且与致龋菌(例如变异链球菌(S.mutans))竞争牙齿上和口腔中位置的血链球菌(S.sanguis)。精氨酸分解菌可利用精氨酸和其他碱性氨基酸产生氨，从而提高它们的环境的pH，而致龋菌使糖代谢成乳酸，乳酸倾向于降低菌斑pH，并使牙齿脱矿质，最终导致蛀洞。
因此，具有监测口腔中生物群落(bioflora)类型，例如以决定最佳治疗和监测治疗患者有效性的有效方式是有用的。
发明概述
本发明提供评价口腔中生物群落的快速简单的方法。
在第一个实施方案中，本发明测定菌斑产氨量，以确定精氨酸分解菌的相对群体量(relative population)。
在另一个实施方案中，本发明测定菌斑乳酸量，以确定致龋菌的相对群体量。
在另一个实施方案中，本发明用聚合酶链式反应(PCR)(例如定量实时PCR)表征口腔中的生物群落，例如菌斑或唾液中的生物群落。
在另一个实施方案中，本发明用反转录酶PCR(RT-PCR)表征口腔中的生物群落，例如菌斑或唾液中的生物群落。
在另一个实施方案中，用抗体探针(例如，荧光抗体探针)表征口腔中的生物群落，例如菌斑或唾液中的生物群落。
例如，本发明对至少一种致龋菌(例如变异链球菌)和至少一种精氨酸分解菌(例如血链球菌)的量进行定量。
在另一个实施方案中，用前述方法之一评价患者，并由此确定治疗方案。
本发明的方法特别可用于检测菌斑生态学的潜在损伤变化，并允许在有可测量或显著的牙齿脱矿质或损伤之前校正性治疗。
因此，本发明提供促进口腔健康的方法，例如，以
a.减轻或抑制龋齿的形成，
b.减轻或抑制牙齿脱矿质和促进牙齿再矿化，
c.治疗、减轻或抑制早期牙釉质损伤的形成，
d.减轻牙齿过敏性，
e.减轻或抑制牙龈炎，
f.促进口腔中溃疡或伤口的痊愈，
g.减少产酸细菌的量，
h.增加精氨酸分解菌的相对量，
i.抑制口腔中形成微生物生物膜，
j.在糖攻击后将菌斑pH提高和/或保持在至少pH5.5的水平，
k.减少菌斑积累，
l.治疗、缓和或减轻口干，
m.使牙齿增白，
n.促进全身健康，包括心血管健康，例如，通过减小经由口腔组织全身感染的可能性，
o.使牙齿对致龋菌及其作用具有免疫力，
p.清洁牙齿和口腔，并且/或者
q.减轻牙齿腐蚀，
所述方法包括例如用任何前述方法测定口腔的生物群落，并且如果指示，则给予包含有效量碱性氨基酸或其盐(例如精氨酸)的口腔护理制品。
本发明进一步提供游离或盐形式的碱性氨基酸用于制备药物的用途，所述药物用于促进受试者的口腔健康，所述受试者的口腔生物群落包含根据本发明的方法测定的提高量的致龋菌和/或提高量的乳酸根和/或低量的精氨酸分解菌和/或低量的菌斑产氨量。
本发明进一步提供促进口腔美容的方法(其中此促进美容可包括例如使牙齿更白和/或减轻口臭)，所述方法包括用本发明的方法测定口腔的生物群落，并且如果指示存在提高量的致龋菌和/或提高量的乳酸根和/或存在低量的精氨酸分解菌和/或低量的菌斑产氨量，则给予包含游离或盐形式的碱性氨基酸的口腔护理制品。
发明详述
菌斑代谢-产氨
牙菌斑使精氨酸转化成氨的能力是精氨酸分解活性的标志。某些细菌具有使精氨酸转化成氨的能力，正如某些细菌能够使糖转化成酸一样。有益的是，提高精氨酸分解物种的相对浓度，因为这些细菌产生不利于致龋菌增殖的条件，致龋菌偏爱酸性条件，并增加龋齿风险。期需日常使用精氨酸使菌斑生态学转移，这种转移以常见的糖消耗产生有利于产酸细菌的条件的类似方式有利于精氨酸分解菌。氨是一种能够中和酸并帮助维持中性菌斑pH的碱。中性pH条件更有利于非病原菌。产氨量测定法测定了能够使精氨酸转化成氨的所有细菌的作用。这与实时PCR方法(以下进一步描述)形成对比，后一种方法测定选择性精氨酸分解菌的浓度，并且不在代谢活性(活)细菌与非活性(死)细菌作出区分。
氨检测试剂盒为市售的，例如购自Diagnostic Chemicals Limited(Oxford，CT)，以测定产氨量。定量和测定的原理是，已知氨与α-酮戊二酸根和还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)反应生成L-谷氨酸根和NADP。此反应由谷氨酸脱氢酶(GLDH)催化。由于NADPH氧化而导致的在340nm处吸光度减小与氨浓度成比例。在预定的治疗方案后收集菌斑样品。在一些应用中，从釉质或安装在保持器(retainer)上的HAP样品收获菌斑。在其他应用中，直接从牙齿收获菌斑。
由乳酸量检测菌斑生态学
正如氨量测定法作为测定精氨酸分解菌量的代表一样，乳酸作为测定致龋菌量的代表。受试者在不经早晨口腔清洁和不经夜晚前饮食的情况下取得菌斑。将它们用10％蔗糖溶液清洗2分钟。8分钟后，通过刮擦牙齿表面收集菌斑。将菌斑样品收集在预称重管中于冰上，并测定菌斑重量。分析包括，将冰冷水加到已知量的菌斑样品中，然后将样品加热到80℃持续5分钟，以杀死细菌并且释放所有的酸，随后使样品在冰水中冷却另外5分钟。然后将样品离心，并过滤上清液。乳酸根浓度用毛细管电泳测定。
由定量实时PCR检测菌斑生态学
定量实时PCR(聚合酶链式反应)为DNA定量的高灵敏方法。从牙菌斑分离的细菌DNA用于对细菌总量定量，因为DNA的量与存在的细菌量有直接关系。实时PCR被政府机构如疾病控制中心(Center forDisease Control)和FDA承认为一种强有力的灵敏技术。利用很多口腔细菌的已知基因组序列，可设计探针检测口腔细菌或特定细菌如变异链球菌或血链球菌的总量。从菌斑或唾液样品分离的DNA由聚合酶链式反应扩增。DNA的量随PCR反应各循环成指数增加。此技术被称为“实时”，因为通过使用荧光报告分子实时跟踪反应。在本发明的一个实施方案中，用SYBR Green作为报告分子。此分子在与双链DNA配位(coordination)时强烈发出荧光。通过设定荧光阈并用不同浓度的DNA标准品测定达到阈所需的循环数，可实现定量。存在的DNA越多，达到阈所需的DNA循环数越少。市售实时PCR仪器可购自很多制造商，如Roche Diagnostics。
菌斑样品从釉质或羟基磷灰石样品以已知的恒定表面积收获。菌斑收集标准化很关键，因为存在的DNA的量与收集多少菌斑有直接关系。不适当的是，用菌斑质量作为由标准化实时PCR测定的总细菌的手段，因为两个量显著相关。结果报告为μgDNA/ml。可对DNA浓度Ln(DNA浓度)进行统计。对于总细菌，用受试者和治疗作为因子完成双因子ANOVA。如果检测差异为95％置信水平，则认为差异显著。对于特定细菌，如变异链球菌或血链球菌，用总细菌作为协变量进行双因子ANCOVA。由于与总细菌群体量相关，特定细菌的总量是菌斑生态学健康的更相关标志。
在本发明的具体实施方案中，将变异链球菌作为致龋性的标志测定，选择变异链球菌是因为它是龋齿开始中相关的公认的风险因子。虽然在龋过程中涉及其他产酸细菌，但已知变异链球菌特别在致龋过程的开始和早期阶段起重要作用。在本发明的一个实施方案中，将血链球菌选择为转变成较健康的菌斑生态学的标志，因为血链球菌是已知显示高水平精氨酸分解活性(使精氨酸转化成氨的能力)的细菌。
由RT-PCR检测菌斑生态学
反转录PCR测定样品中的RNA转录物。分离RNA，用反转录酶使转录物转化成cDNA，cDNA用PCR扩增。RT-PCR的优点是用于测定口腔细菌的基于DNA的方法不能测定那些物种的生存力。由于口腔细菌最常发现于生物膜群落，在杀死后死亡细菌的DNA可长时间保持于生物膜结构内。其他方法，如基于荧光的生存力测定(LiveDead试剂盒，Molecular Probes)可检测生物体是否具有受损的膜，但不直接检测特定物种。
因此，反转录实时PCR是对复杂群落内存在的口腔细菌的特定物种的活生物体定量的方法。mRNA具有相对较短的半衰期，因此指示最近活性的细菌。我们已对延伸因子tuf研究出物种特异性引物。此基因不显著受生长期、培养基或环境条件调节，因此使对细菌检测数的假影响(spurious effect)减到最低限度。利用伴放线放线杆菌(Aggregatibacteractinomycetemcomitans)作为试验生物体，可在少到20％存在的生物体存活时检测活细菌和EtOH杀死的细菌混合群体的生存力差异。另外，该方法允许在包含多达六个不同细菌物种的混合物种群体中可靠地鉴定伴放线放线杆菌的存在。计算的细菌浓度与基于对相同培养物的OD610的估计值密切相关(r＝0.96，＜1％差异)。此测定代表研究口腔复杂环境内特定生物体的生态学的手段。由于更多的基因序列数据变得可用，因而可对多种口腔细菌开发引物。
由荧光抗体探针检测细菌量
通过使用单克隆抗体，用龋诊断试剂盒检测唾液中致龋型细菌(例如变异链球菌)和/或非致龋型细菌(例如血链球菌)的量。所用特定抗体对细菌物种是特异的，并且具有连接到抗体的荧光染料。通过测定发射的荧光量，可检测细菌量。
实施例
实施例1
实时PCR测定总菌斑细菌量
使用不同的牙膏制剂，用上述步骤测定受试者的总菌斑细菌量(微克细菌DNA/ml)：
总细菌DNA 变异链球菌DNA 血链球菌DNA
250ppm氟化物制 6.091 0.09622 1.126
剂(对照)
1450ppm氟化物制 6.018 0.09903 1.107
剂
具有2％精氨酸碳 3.781 0.05998 1.291
酸氢盐和1450ppm
氟化物的制剂
精氨酸-氟化物制剂有效减小总菌斑负载量和变异链球菌(致龋)菌斑负载量，同时提高血链球菌(精氨酸分解)负载量。
实施例2-产氨量
使用不同的牙膏制剂，用上述方法测定受试者的产氨量：
氨量(ppm)
250ppm氟化物制剂 1.97
(对照)
1450ppm氟化物制剂 1.79
具有2％精氨酸碳酸 2.77
氢盐和1450ppm氟化
物的制剂
利用含精氨酸的制剂，受试者的菌斑产氨量显著更高。
实施例3-乳酸量
如上所述使用毛细管电泳，测定受试者的菌斑乳酸量，显示在蔗糖存在下乳酸根显著增加。
实施例4-实时PCR/RT-PCR
本发明将实时PCR检测细菌物种的原理与用信使RNA(mRNA)作为细胞内生物活性指示物组合。在从细菌样品纯化mRNA后，用反转录实时PCR检测和定量简单或复杂环境内的特定细菌。本发明涵盖引物序列和mRNA鉴定方法及其应用。
活细胞内DNA的一个功能是编码蛋白质的合成。DNA编码其相应mRNA链，然后后者用作指令用于装配完成的蛋白。与DNA不同，mRNA有很短的半衰期(秒至分)，并且仅存在于活或刚被杀死的细胞中。虽然DNA以固定数拷贝存在于细胞中，但是mRNA量通常响应细胞存在的条件而变化。不同蛋白质的表达可响应温度变化、生长培养基、生长期和其他环境条件而被上调或下调。因此，如果不仔细选择靶基因，就可能将环境条件下的变动误读为群体生存力的变动。为避免这些影响，本发明用延伸因子tu即基因tuf作为靶序列。此序列以前已用作标记物，因为已观察到在不同试验条件下tuf表达改变很小或不改变。
实时PCR利用在遗传物质聚合酶链式反应(PCR)扩增背后的基础化学，并使其与荧光标记的实时检测结合，作为各个扩增循环后存在的既定基因序列拷贝数定量的机制。这些方法中最简单的是利用SYBR Green I，一种专门嵌入双链DNA(dsDNA)的荧光探针。因此，SYBR Green荧光增加量与更大dsDNA浓度相关。当用特定基因序列引发的PCR反应中包含此染料时，荧光增加相应于靶基因的拷贝数增加。随后，可使信号跨预定强度阈所处的循环数与初始物质中基因序列的浓度相关。
实时PCR技术的发展已使得能够快速并以高精确度检测和定量特定生物物种。细菌物种定量的常规方法依赖于针对编码16s核糖体亚基的DNA的可变区的引物的开发。此亚基对细菌复制是关键的，因此，其序列不容易变异。检测对特定物种特异的16s rDNA序列可有助于检测和计数复杂环境内的单一细菌物种。
根据来自公共数据库(National Center for BiotechnologyInformation andthe Los Alamos Oral Pathogens Database)的tuf基因序列设计引物。用DNA StarLasergene程序MegAlign模块进行序列比对。为了使物种特异性的可能性达到最大程度，用此比对结果选择更大趋异的区域。根据从Roche Diagnostics LightCycler Probe Design软件得到的分析信息选择引物序列。本发明涵盖的引物不仅包括经过设计和试验的那些引物，而且还包括针对口腔病原体中此基因区域的所有引物。
使用适合的RNA分离试剂盒或其他RNA分离方法，从样品分离总RNA。可使用用于RNA分离的任何优选方法。用适合的DNA酶处理试剂处理纯化的RNA两次。此步骤使RNA制备物内的任何污染性DNA降解，并防止获得假阳性。然后使经分离的RNA反转录，以产生互补DNA(cDNA)分子。扩增所得cDNA，并用SYBR Green检测。作为DNA完全去除的品质对照，可在无反转录步骤下进行实时PCR反应。在无反转录反应下得到的PCR产物必定是污染性DNA的结果。
通过对分离自含已知量活细菌的培养物的RNA样品进行实时反转录PCR反应，产生标准曲线。将各已知样品的二阶导数最大值(second derivative maxinum value)对其已知细菌细胞浓度作图。然后，可将从未知细菌样品分离的RNA的扩增曲线的二阶导数最大值与标准曲线比较，以确定样品群体内活生物体的浓度。此数据是阻止抗菌剂和活性分子对口腔环境生态学的作用的有价值的信息。
设计以下引物对，以扩增来自伴放线放线杆菌(Aggregatibacter(Actinobacillus)actinomycetemcomitans)的tuf基因的228碱基对区域：
引物名 序列 Tm(℃) ％GC ΔG 退火温度
(kcal/mol)
正向引物 5’-AAGCGCGTGGTATCAC-3’ 49.05 56.25 -27.56 55℃
反向引物 5’-TGTAAGGAACACCTA-3’ 31.52 40.00 -20.15 55℃
表1.为伴放线放线杆菌中tuf的mRNA表达定量而设计的引物性质
用这些引物扩增从伴放线放线杆菌纯培养物和含有及不含有伴放线放线杆菌的混合群体两者分离的RNA。结果显示于图1的曲线图中，特定地讲，为荧光(F1)和循环数之间的关系。在图1中，“水”表示阴性对照，“Aa”为纯伴放线放线杆菌培养物的阳性对照。“混合物1”从含中间普雷沃氏菌(Prevoltella intermedia)、表兄链球菌(Streptococcussobrinus)、口腔链球菌(Streptococcus oralis)和粘放线菌(Actinomyces viscosus)的群体纯化，因此，对于用这些引物扩增应为阴性。“混合物3”来自含伴放线放线杆菌、牙龈卟啉单胞菌(Porphyromonas gingivalis)、格氏链球菌(Streptococcus gordonii)、变异链球菌(Streptococcus mutans)和血链球菌(Streptococcus sanquinis)的群体，并且对于扩增伴放线放线杆菌应为阳性。
此图表明，虽然含伴放线放线杆菌的混合物以类似于阳性对照的曲线扩增，但缺乏它的混合物遵循与水对照相同的扩增曲线，表明这些引物能够精确检测来自RNA物质混合物内的伴放线放线杆菌。
这些引物只精确检测和定量活伴放线放线杆菌生物体的能力如下确定。通过悬浮于80％乙醇15分钟，杀死已知浓度的伴放线放线杆菌细胞。然后将细菌通过离心沉淀，并重新悬浮于新鲜的脑心浸液(Brain Heart Infusion)肉汤生长培养基。将乙醇杀死的细菌在37℃过夜孵育，并检查生长，以确定没有活生物体保留。然后将乙醇杀死的细菌与活生物体以规定比例混合，并进行反转录，随后进行实时PCR。这些样品的扩增显示于图2中，图2显示来自活和死伴放线放线杆菌的混合物的RNA的扩增。
尽管事实上用作此反应的模板的所有群体包含相同总数的生物体，但在含更多活生物体的样品中观察到较早扩增，表明此测定能够检测活菌和死菌的混合物内的活生物体。另外，如图3所示，这些样品的解链曲线表明，单一相同产物在所有样品中扩增，这证明测定的高特异性。图3显示由伴放线放线杆菌的纯培养物和混合培养物扩增的产物的解链峰分析。这些曲线的重叠表明单一产物正由所有样品扩增。表2显示在选择的标准曲线样品中生物体的预期数目和计算数目的比较。
样品
预期活CFU
计算活CFU
100％ 5.00x107 5.34x107
50％ 2.50x107 2.28x107
40％ 2.00x107 2.54x107
30％ 1.50x107 1.18x107
20％ 1.00x107 1.07x107
10％ 5.00x106 4.06x106
5％ 2.50x106 2.96x106
表2.在选择的标准曲线样品中生物体的预期数目和计算数目的比较。
根据活的开始培养物和杀死的开始培养物的已知浓度，计算各群体中活生物体的近似数，并将其与各扩增曲线的二阶导数最大值结合用于产生标准曲线。结果显示于图4中，图4显示由已知浓度的活伴放线放线杆菌和死伴放线放线杆菌扩增产生的标准曲线和标准曲线的线性回归。回归线的r2值为0.96。
线性回归线的r2值表示回归公式对观察值的拟合的接近。r2接近1.00表明观察值紧密匹配回归线。对于以上实施例，标准曲线的r2值为0.96，表明线中观察的总差异为约96％是由于样品的实际测定差异，并且可用此标准曲线计算未知群体中活生物体的浓度。
实际上，在单一试验中，根据此标准曲线计算的活生物体的浓度显著不同于RNA分离前所加的实际浓度，并且相差＜20％。这些数据表明，此测定代表检测和定量复杂生物体群体内特定物种的活生物体的快速精确方法。这代表了用于分析对口腔微生物生态学的治疗效果的潜在强有力工具。
Claims (5)
1.一种用于评价口腔的生物群落的试剂盒，所述试剂盒包括测定精氨酸分解菌量的试剂和测定致龋菌量的试剂，其中通过测定菌斑产氨量评价精氨酸分解菌量，并且通过测定乳酸根产量评价致龋菌量。
2.权利要求1的试剂盒，其中用定量实时PCR(定量RT-PCR)和/或荧光抗体探针定量测定至少一种致龋菌和/或至少一种精氨酸分解菌的量。
3.权利要求1的试剂盒，其中精氨酸分解菌包括血链球菌。
4.权利要求1的试剂盒，其中致龋菌包括变异链球菌。
5.权利要求1的试剂盒，其中所述试剂盒检测在有可测量或显著的牙齿脱矿质或损伤之前的菌斑生态学的潜在损伤变化。
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| EP2648683B1 (en) | 2010-12-09 | 2016-04-20 | Colgate-Palmolive Company | Liquid cleaning compositions containing long-chain fatty alcohols |
| CA2825427A1 (en) | 2011-02-11 | 2012-08-16 | Colgate-Palmolive Company | Methods of analyzing plaque |
| US10206959B2 (en) | 2013-02-14 | 2019-02-19 | University Of Florida Research Foundation, Inc. | Probiotic arginolytic oral compositions and methods of making and using probiotic arginolytic oral compositions |
| WO2014127066A1 (en) * | 2013-02-14 | 2014-08-21 | University Of Florida Research Foundation, Inc. | Probiotic arginolytic oral compositions and methods of making and using probiotic arginolytic oral compositions |
| CN104721063B (zh) | 2013-12-19 | 2018-05-08 | 高露洁-棕榄公司 | 包含氧化锌和柠檬酸锌的洁齿剂组合物 |
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| CA3053680A1 (en) * | 2017-03-03 | 2018-09-07 | The Procter & Gamble Company | Calcium fluorescent probes to assess oral care composition efficacy in biofilm |
| CA3123251A1 (en) * | 2018-12-26 | 2020-07-02 | Colgate-Palmolive Company | Prebiotic role of arginine on beneficial oral bacteria |
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| EP2225562A2 (en) | 2010-09-08 |
| CA2711265A1 (en) | 2009-08-13 |
| JP2014138586A (ja) | 2014-07-31 |
| CN102037360A (zh) | 2011-04-27 |
| WO2009100262A2 (en) | 2009-08-13 |
| AU2009212318B2 (en) | 2012-12-13 |
| WO2009100262A3 (en) | 2009-11-05 |
| US9376722B2 (en) | 2016-06-28 |
| JP2011510683A (ja) | 2011-04-07 |
| RU2010137332A (ru) | 2012-03-20 |
| RU2476890C2 (ru) | 2013-02-27 |
| US20110085990A1 (en) | 2011-04-14 |
| EP2225562B1 (en) | 2019-06-26 |
| EP2225562A4 (en) | 2016-04-27 |
| MX2010005211A (es) | 2010-09-30 |
| AU2009212318A1 (en) | 2009-08-13 |
| ZA201003680B (en) | 2015-08-26 |
| MY155568A (en) | 2015-10-30 |
| CO6300814A2 (es) | 2011-07-21 |
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