胃癌前病变患者口腔中龋病和牙周病相关细菌以及口腔细菌多样性的研究
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摘要
龋病和牙周病是人类最常见的感染性疾病。龋病是一种发生在牙体硬组织的慢性感染性疾病,变异链球菌(Streptococcus mutans, S. mutans)和表兄链球菌(Streptococcus sobrinus, S. sobrinus)在龋齿中的检出率最高,被认为是最主要的致龋菌,能够利用糖产酸溶解牙釉质和牙本质中的无机物,因此龋病以硬组织脱矿、龋洞形成为主要特征。牙周病以牙龈炎症、牙周袋形成、牙槽骨吸收和牙齿松动为主要特征,流行病学研究表明牙周病与很多全身系统性疾病关系密切,这些系统性疾病包括：心血管疾病,胃炎及胃癌,艾滋病,早产低出生体重儿,糖尿病,呼吸道感染和骨质疏松症,得出牙周病是多种系统性疾病的危险因素之一的结论并不为过。细胞因子和炎性介质增多、直接感染、细菌抗原和自身抗原之间的交叉反应性／分子拟态能够引起系统性疾病,有报道,牙龈卟啉单胞菌(Porphyromonas gingivalis)其发展密切相关。而且,已经在心脏瓣膜病变、粥样斑块、早产高危孕妇的羊膜液以及早产孕妇的胎盘中检测到P. gingivalis等牙周病相关病原菌。另一种牙周主要病原菌伴放线放线杆菌(Actinobacillus actinomycetemcomitans)也在除口腔外的身体其他病变部位被检测到。尽管P.gingivalis和A. actinomycetemcomitans等牙周病相关细菌在系统性疾病中的作用尚不明确,但是这些细菌与全身疾病的关系这一方向值得探索。胃癌前病变(Precancerous lesions of gastric cancer, PLGC)是一个病理性概念,包括肠上皮化生(Intestinal Metaplasia, IM)和异型增生(Dysplasia, Dys),主要伴存于慢性萎缩性胃炎(Chronic Atrophic Gastritis, CAG),是从正常胃粘膜向胃癌转化过程中的一个重要阶段。这里我们主要研究龋病和牙周病及其主要病原菌与胃癌前病变的关系,并探索胃癌前病变患者口腔细菌的多样性。
第一部分
胃癌前病变患者的人口学特征和临床口腔状况评估
尽管过去的几十年来胃癌的发病率显著降低,它仍然是世界范围内第二大恶性肿瘤,在西方国家更是如此。胃癌的发病原因不明,可能与多种因素,如生活习惯、饮食种类、环境因素、遗传素质、精神因素等有关。幽门螺杆菌是胃部最常感染的细菌也是慢性胃炎最主要的致病菌。研究提示,龈下菌斑和牙周袋很可能是幽门螺杆菌的潜在聚集地。很多流行病学调查研究发现,自述牙齿丧失与胃癌的风险呈正相关。牙齿丧失最常见的原因是龋病或牙周疾病,在年轻人群中前者较多,对于年龄较长的人群,后者所占比重较大。但是尽管牙齿丧失在一定程度上是衡量牙周疾病的一个重要指标,它仍然不能作为一种直接的手段,我们需要牙周病的临床和微生物学指标以及牙齿脱落的病史等相关信息来探讨胃癌与其之间的关系。本部分研究从年龄、性别、种族、受教育程度、收入水平、婚姻状况和身高体重指数BMI等方面,评估研究人群的胃癌的人口统计学风险因素和口腔健康状况之间的关系。
方法：本实验选取New York Harbor Healthcare System (VANYHHS)和Bellevue Hospital Center胃肠科2009年3月30日至2011年4月27日之间,约定进行上消化道内窥镜检查的98名应试者。研究对象纳入标准为：30岁以上,无食管癌、胃癌、胃静脉曲张或门静脉高压性胃病。按病理诊断结果,将研究对象分为两组：对照组无任何症状或仅有浅表性胃炎；病例组患有胃癌前病变(包括慢性萎缩性胃炎、肠上皮化生或异型增生)。口腔检查时进行龋病和牙周病的临床状况评估。SPSS17.0统计学软件分析病例组和对照组人群的胃癌的人口统计学风险因素。
结果：胃癌前病变患者的龋病相关临床指标与对照组未见显著性差异。与健康对照组相比,胃癌前病变患者的牙周健康状况较差,病例组的牙周袋深度(Pocket depth, PD)的均值、附着丧失(Loss of attachment, LOA)均值、PD≥4mm的位点数所占的百分比、LOA≥3mm和LOA≥5mm的位点数所占的百分比以及探诊出血的位点数所占的百分比等所有牙周临床检查指标均高于对照组。
结论：胃癌前病变患者的牙周健康状况较差,龋病相关临床指标与健康对照组未见显著差异。
第二部分
胃癌前病变患者口腔中龋病和牙周病相关细菌的研究
有研究报道,慢性炎症是疾病进展初期的主要原因,由于牙周致病菌感染可以导致慢性系统性炎症,而后者又是胃癌发展过程中的重要的危险因素,我们推测,牙周主要致病菌的存在增加了胃癌发生发展的风险。为了验证此假说,我们进行了一项临床探索性的病例-对照研究,通过实时定量PCR检测唾液以及牙菌斑样本中的四种牙周病相关主要致病菌：牙龈卟啉单胞菌(Pg)；福赛斯坦纳菌(Tannerella forsythensis, Tf);牙密螺旋体(Treponema denticola, Td);伴放线放线杆菌(Aa)；两种龋病相关主要致病菌：变异链球菌(Sm)和表兄链球菌(Sb)。结合临床相关指标以及患者的人口统计学胃癌风险因子,探索口腔感染性疾病与胃癌的发生发展是否存在一定的相关性。
方法：研究对象同第一部分。口腔检查时采集唾液和牙菌斑样本,MasterPure DNA纯化试剂盒提取样本的细菌基因组DNA后进行实时定量PCR检测样本中的特异性细菌。以特异性细菌的标准菌株DNA作为阳性对照,以灭菌水作为阴性对照,通过标准DNA产生的标准曲线对样本中的目标细菌进行绝对定量。SPSS 17.0统计软件行临床指标、风险因素以及微生物水平之间的相关性统计学分析。
结果：研究人群唾液中牙周主要致病菌的DNA水平与牙菌斑中的DNA水平呈正相关。Pg, Td和Tf检出阳性的人群,平均牙周袋深度显著大于检出阴性的人群,中重度牙周袋(PD>4mm)和探诊出血的位点数也显著高于阴性人群。Pg、Td和Tf的DNA水平与牙周袋深度显著相关,其中Pg和Td的DNA水平还与被测人群的中重度牙周袋和探诊出血的位点数密切相关。龋病和牙周病主要致病菌的检出率,在胃癌前病变患者和健康对照组两组间有所不同。胃癌前病变患者的牙周病主要病原菌Pg和Td的阳性率高于对照组,唾液和龈下菌斑中四种牙周病原菌的DNA水平也略高于对照组,但统计学上无显著性差异。
结论：Pg、Tf、Td三者与牙周病的临床指标,尤其是牙周袋深度和探诊出血密切相关。与健康对照组相比,胃癌前病变患者Pg和Td的阳性检出率较高,四种牙周病原菌的DNA水平也较高。
第三部分
胃癌前病变患者口腔细菌多样性的研究
口腔中的微生物群体是目前所知的最为复杂的微生物群落之一。随着分子生物学检测技术的提高,目前已明确鉴别出口腔细菌群落或生物膜中包含至少500种微生物。口腔这一众多微生物栖息的场所,对于人类健康和疾病都有着至关重要的意义。口腔是消化道的开端,常常被成为"inner outside",解剖学上讲消化道由连续的器官构成,胃也属于消化系统,口腔和胃的微生物在某种程度上能够互相影响。一方面,口腔中的细菌可以通过摄入食物被带入胃内,另一方面,口腔菌群也有可能受到胃部疾病影响。例如,有些胃癌患者会伴发胃食管返流疾病(Gastroesophageal Reflux Disease, GERD),返流物能够造成上消化道系统的相应病变,改变其正常细菌群落,若发生呕吐症状,还可影响到口腔。因此,我们推测,胃癌前病变患者的口腔细菌多样性与健康人群不同。本研究采用聚合酶链反应-变性梯度凝胶电泳分析技术(polymerase chain reaction-based denaturing gradient gel electrophoresis technique, PCR-DGGE)作为手段分析胃癌前病变患者口腔中唾液和牙菌斑样本的细菌多样性,同时分析牙周病原菌的存在对口腔细菌多样性的影响。
方法：研究对象同第一部分。唾液和牙菌斑样本DNA的提取同第二部分。采用GeneAmp PCR SystemTM 9700进行巢式PCR。制备变形梯度凝胶,PCR产物和特异性DGGE参考Maker上样,在pH 8.5温度为58℃的1×Tris-acetate-EDTA (TAE)电泳缓冲液中恒压60 V电泳17小时。电泳结束后取下凝胶,在0.5μg/mL的EB溶液中染色12分钟,蒸馏水中漂洗6分钟后,AlphalmagerTM 3300 System采集DGGE图像,BioNumerics软件分析图像,SPSS17.0统计软件分析微生物学指标和细菌多样性之间的相关性。
结果：胃癌前病变患者的龈下菌斑样本的DGGE条带数为35.6,显著高于对照组的33.2(p<0.05),但两组间唾液样本的DGGE条带数量,未见显著性差异。整体研究人群的6个不同牙位点龈下菌斑之间的相似性达80.7%,而唾液和龈下菌斑的相似性只有78.0%。病例组唾液样本和龈下菌斑样本的相似程度(76.5%)略低于健康对照组(78.6)；病例组不同牙位龈下菌斑样本之间的相似程度却比对照组略高。不同牙位龈下菌斑之间的相似性越高,龈下菌斑样本的细菌多样性越高(p<0.001)。牙周病原菌在多牙位(≥2)检出阳性的人群,唾液样本DGGE条带数低于只有一个牙位检出阳性或全部牙位检出阴性的人群,龈下菌斑的多样性情况与此相反,Pg, Td和Tf多牙位检出阳性的人群,龈下菌斑的DGGE条带数均高于只有一个牙位检出阳性或全部牙位检出阴性的人群的人群。
结论：胃癌前病变患者口腔中龈下菌斑的细菌多样性略高于健康人群,同种来源样本(不同牙位龈下菌斑)细菌多样性的相似程度高于不同来源样本(唾液和龈下菌斑)。龈下菌斑的细菌多样性和各牙位龈下菌斑相似性高度相关。牙周病原菌在多位点检出阳性的人群,唾液细菌多样性有所下降,而龈下菌斑细菌多样性有所升高。
Dental caries and periodontal diseases are chronic biofilmbased diseases that occur in more than 90%(caries) and 35%(periodontal disease) in the adult population in the US. Mutans streptococci are generally considered to be the principal etiological agents of dental caries. S. mutans and S. sobrinus are the most commonly found bacteria in human plaque and are known to be associated with the development of dental caries. Periodontal diseases are characterized by gingival inflammation, periodontal pocket and attachment loss, which lead to tooth loss. In the past decades, increased epidemiological studies have presented evidence of positive association between periodontal diseases and the development of systemic diseases, including cardiovascular diseases, preterm delivery of low birth weight, diabetes mellitus, respiratory diseases, gastric cancer, AIDS and osteoporosis. However, the etiological details remain unclear. Nevertheless, more recent investigations have provided various results suggesting that periodontal infection and subsequent direct oral-hematogenous spread of bacteria are implicated in the development of various systemic diseases. Herein, we focus on the evaluation of the associations between the colonization of cariogenic and periodontal pathogens and their socio-demographic and clinical characteristics in individuals with gastric pre-cancerous lesions. Furthermore, we also evaluated the oral microbial diversity of the same group of individuals.
Part One- The socio-demographic and clinical oral health characteristics in individuals with gastric precancerous lesions
Gastric cancer is the second most common malignancy worldwide. The etiology of gastric cancer is not clear. There are many risk factors associated with gastric cancer, including life style, dietary structure, environmental, genetic and mental factors. Helicobacte pylori (H. pylori) was one of the most investigated etiological bacteria that are closely linked to chronic gastritis, peptic ulcer, gastric cancer. A few studies have suggested that H. pylori exist in subgingival plaque and therefore these sites are considered reservoirs for H. pylori. Meanwhile, several epidemiologic studies have also suggested a positive association between self-reported tooth loss and the risk of gastric cancer. Tooth loss in older age is more likely to be caused by chronic periodontal disease, whereas in younger age is usually caused by dental caries. Although these studies suggested that tooth lossis associated with an increased risk of gastric cancer, there was no detailed caries and periodontal assessment completed in these studies. More comprehensive clinical and microbial measurements are needed to investigate their association. The present study evaluated the socio-demographic and clinical oral health characteristics in individuals with gastric precancerous lesions.
Methods The population of this study was consisted of 98 individuals who were scheduled for an upper endoscopy procedure at the gastrointestinal clinics in the Veterans Affairs New York Harbor Healthcare System and Bellevue Hospital Center from March 30,2009 to April 27,2011. All individuals were more than 30 years old and had no prior history of esophageal cancer, gastric cancer, gastric varices, or portal gastropathy. Based on biopsy diagnostics, these individuals were divided into two groups:Cases were those individuals with gastric precancerous lesion (chronic atrophic gastritis, intestinal metaplasia, or dysplasia). Controls were those individuals with no lesions or only superficial gastritis. Evaluation of the dental caries and periodontal status was conducted at the time of dental examination.
Results Compared with the individuals in the control group, the individuals with gastric precancerous lesions harbored worse periodontal condition. They had deeper pocket depth, loss of attachment and more percent of sites with Pocket depth (PD) >4mm, Loss of attachment (LOA)≥3mm and LOA≥5mm. The percent of bleeding sites in the case group was significant higher than in the control groups (p<0.05).
Conclusion There is association between gastric precancer and the periodontal condition.
Part Two- The colonization of cariogenic and periodontal diseases pathogens in individuals with gastric precancerous
lesions
There is strong evidence that chronic inflammation is largely responsible for the early stages of disease progression. Because periodontal infection can lead to chronic systemic inflammation, which is an important risk factor in the development of gastric cancer, we hypothesize that major periodontal pathogens are associated with an increased risk for gastric cancer. To test this hypothesis, we conduct an exploratory hospital-based case-control study. The colonization of four periodontal pathogens, Porphyromonas gingivalis (Pg); Tannerella forsythensis (Tf); Treponema denticola (Td) and Actinobacillus actinomycetemcomitans (Aa) which are etiologically linked with destructive periodontal diseases; S. mutans (Sm) and S. sobrinus (Sb) which are cariogenic pathogens were measured in both saliva and subgingival plaque samples using the real-time quantitative PCR methods with species-specific DNA primers. We analyzed the microbial data together with the socio-demographic risk factors and clinical oral health characteristics, to see if the association exists between oral infectious diseases and gastric cancer.
Methods The population of this study was the same as part one. Non-stimulated whole saliva and plaque samples were collected from each subject at the time of dental examination. Bacterial genomic DNA of the saliva and plaque was isolated using the MasterPure DNA purification kit. Standardization of the template DNA for real-time qPCR was established first so that the final results can be compared. The species-specific PCR primers for the four periodontal pathogens were used in this study. All reactions were carried out in duplicate, and the final analysis was based on the mean of the two qPCR reactions.
Results The DNA level of periodontal pathogens in saliva was significantly associated with that in plaque (p<0.01). Subjects detected positive for Pg, Td and Tf had deeper pocket depth and higher percent of sites with PD>4mm and bleeding sites (p<0.05). The DNA level of Pg, Td and Tf in subgingival plaque was also significantly correlated with the percent of sites with PD>4mm and bleeding sites. The detection rates of Pg and Td were higher in the subjects with gastric precancerous lesion than the subjects in the healthy control groups. The detection rates of Aa and Tf were lower in the subjects with gastric precancerous lesion than the subjects in the control groups. The DNA level of four tested periodontal pathogens in both saliva and plaque was higher in case group than in control group, eventhough the difference didn't reach the statistic level.
Conclusion Pg, Td and Tf were significantly associated with the clinical symptom, especially the pocket depth and bleeding on probing. The detection rates of cariogenic and periodontal pathogens were different in case and control groups. Subjects with gastric precancerous lesion were more likely to test positive for Pg and Td and had higher DNA level of four periodontal pathogens compared with control groups.
Part Three-Oral microbial diversity in individuals with gastric precancerous lesions
The bacterial community in the oral cavity is one of the most complex mixtures of bacteria known. Recent developments of molecular biological techniques for microbial identification have identified more than 500 microbial species. The oral cavity as a home to the microbial community plays an important role in human health and disease. Oral cavity is the entrance of the digestive tract, which is often regarded as the'inner outside'. The stomach also belongs to the digestive system. Their bacteria can interfere with each other to some extent. Oral bacteria can be taken to stomach along with the intake food, while some gastric cancer patients have GRED which might affect some bacteria in the oral cavity. Based on the information above, we hypothesize that the oral microbial diversity differs in individuals with gastric cancer compared with individuals without gastric cancer. To test this hypothesis, we conducted an exploratory hospital-based case-control study. In the present study, polymerase chain reaction-based denaturing gradient gel electrophoresis technique (PCR-DGGE) was used for the analysis of the microbial populations obtained from both saliva and subgingival plaque samples from all subjects, including individuals with gastric precancerous lesions and healthy controls. We also compared the variation of the oral bacterial population between the two study groups and among the colonization sites; and to determine if periodontal bacterial colonization has effect on oral microbial diversity.
Methods A total of 98 subjects were enrolled in this study as part one. Bacterial samples were taken from all 98 subjects at the time of dental examination performed. Bacterial genomic DNA of the plaque or saliva was isolated as part two. Nested PCR was performed with use of the GeneAmp PCR SystemTM 9700. PCR products and species-specific DGGE reference markers were directly loaded in each lane, and electrophoresis was performed at a constant 60 V at 58℃for 17 hrs in 1×TAE, pH 8.5 buffer. After electrophoresis, the gels were rinsed and stained for 12 min in water containing 0.5μg/mL ethidium bromide, followed by 6 min of de-staining in water. DGGE profile images were digitally captured and recorded using the AlphalmagerTM 3300 System. DGGE profiles were analyzed using the BioNumerics Software.
Results Subjects with gastric precancerous lesions had more DGGE bands (35.6) in pooled plaque sample, compared with healthy population (33.2), while there was no significant difference in saliva sample. The similarity of diversity between saliva and pooled plaque of the study population was 78.0%, lower than that among 6 subgingival plaques, which was 80.7%. The similarity of diversity between saliva and pooled plaque of the subjects with gastric precancerous lesions was lower compared with the control group. However, the similarity among 6 subgingival plaques of the case group was higher than that of the control group. The more similar among the 6 subgingival plaques the subjects had, the more microbial diversity in their pooled plaque they would have. Subjects had at least two teeth with none-zero value of the periodontal pathogens had less microbial diversity in saliva but more in pooled subgingival plaque.
Conclusion Subjects with gastric precancerous lesions had more microbial diversity in pooled subgingival plaque, compared with healthy population. The similarity of same kind of sample (among plaques) was higher than different ones (saliva vs. plaque). There was highly correlation between diversity in subgingival plaque and the similarity among the subgingival plaques. The population had more tested positive teeth with periodontal pathogens had a decreased diversity in saliva but increased diversity in pooled subgingival plaque.
第一部分
胃癌前病变患者的人口学特征和临床口腔状况评估
尽管过去的几十年来胃癌的发病率显著降低,它仍然是世界范围内第二大恶性肿瘤,在西方国家更是如此。胃癌的发病原因不明,可能与多种因素,如生活习惯、饮食种类、环境因素、遗传素质、精神因素等有关。幽门螺杆菌是胃部最常感染的细菌也是慢性胃炎最主要的致病菌。研究提示,龈下菌斑和牙周袋很可能是幽门螺杆菌的潜在聚集地。很多流行病学调查研究发现,自述牙齿丧失与胃癌的风险呈正相关。牙齿丧失最常见的原因是龋病或牙周疾病,在年轻人群中前者较多,对于年龄较长的人群,后者所占比重较大。但是尽管牙齿丧失在一定程度上是衡量牙周疾病的一个重要指标,它仍然不能作为一种直接的手段,我们需要牙周病的临床和微生物学指标以及牙齿脱落的病史等相关信息来探讨胃癌与其之间的关系。本部分研究从年龄、性别、种族、受教育程度、收入水平、婚姻状况和身高体重指数BMI等方面,评估研究人群的胃癌的人口统计学风险因素和口腔健康状况之间的关系。
方法：本实验选取New York Harbor Healthcare System (VANYHHS)和Bellevue Hospital Center胃肠科2009年3月30日至2011年4月27日之间,约定进行上消化道内窥镜检查的98名应试者。研究对象纳入标准为：30岁以上,无食管癌、胃癌、胃静脉曲张或门静脉高压性胃病。按病理诊断结果,将研究对象分为两组：对照组无任何症状或仅有浅表性胃炎；病例组患有胃癌前病变(包括慢性萎缩性胃炎、肠上皮化生或异型增生)。口腔检查时进行龋病和牙周病的临床状况评估。SPSS17.0统计学软件分析病例组和对照组人群的胃癌的人口统计学风险因素。
结果：胃癌前病变患者的龋病相关临床指标与对照组未见显著性差异。与健康对照组相比,胃癌前病变患者的牙周健康状况较差,病例组的牙周袋深度(Pocket depth, PD)的均值、附着丧失(Loss of attachment, LOA)均值、PD≥4mm的位点数所占的百分比、LOA≥3mm和LOA≥5mm的位点数所占的百分比以及探诊出血的位点数所占的百分比等所有牙周临床检查指标均高于对照组。
结论：胃癌前病变患者的牙周健康状况较差,龋病相关临床指标与健康对照组未见显著差异。
第二部分
胃癌前病变患者口腔中龋病和牙周病相关细菌的研究
有研究报道,慢性炎症是疾病进展初期的主要原因,由于牙周致病菌感染可以导致慢性系统性炎症,而后者又是胃癌发展过程中的重要的危险因素,我们推测,牙周主要致病菌的存在增加了胃癌发生发展的风险。为了验证此假说,我们进行了一项临床探索性的病例-对照研究,通过实时定量PCR检测唾液以及牙菌斑样本中的四种牙周病相关主要致病菌：牙龈卟啉单胞菌(Pg)；福赛斯坦纳菌(Tannerella forsythensis, Tf);牙密螺旋体(Treponema denticola, Td);伴放线放线杆菌(Aa)；两种龋病相关主要致病菌：变异链球菌(Sm)和表兄链球菌(Sb)。结合临床相关指标以及患者的人口统计学胃癌风险因子,探索口腔感染性疾病与胃癌的发生发展是否存在一定的相关性。
方法：研究对象同第一部分。口腔检查时采集唾液和牙菌斑样本,MasterPure DNA纯化试剂盒提取样本的细菌基因组DNA后进行实时定量PCR检测样本中的特异性细菌。以特异性细菌的标准菌株DNA作为阳性对照,以灭菌水作为阴性对照,通过标准DNA产生的标准曲线对样本中的目标细菌进行绝对定量。SPSS 17.0统计软件行临床指标、风险因素以及微生物水平之间的相关性统计学分析。
结果：研究人群唾液中牙周主要致病菌的DNA水平与牙菌斑中的DNA水平呈正相关。Pg, Td和Tf检出阳性的人群,平均牙周袋深度显著大于检出阴性的人群,中重度牙周袋(PD>4mm)和探诊出血的位点数也显著高于阴性人群。Pg、Td和Tf的DNA水平与牙周袋深度显著相关,其中Pg和Td的DNA水平还与被测人群的中重度牙周袋和探诊出血的位点数密切相关。龋病和牙周病主要致病菌的检出率,在胃癌前病变患者和健康对照组两组间有所不同。胃癌前病变患者的牙周病主要病原菌Pg和Td的阳性率高于对照组,唾液和龈下菌斑中四种牙周病原菌的DNA水平也略高于对照组,但统计学上无显著性差异。
结论：Pg、Tf、Td三者与牙周病的临床指标,尤其是牙周袋深度和探诊出血密切相关。与健康对照组相比,胃癌前病变患者Pg和Td的阳性检出率较高,四种牙周病原菌的DNA水平也较高。
第三部分
胃癌前病变患者口腔细菌多样性的研究
口腔中的微生物群体是目前所知的最为复杂的微生物群落之一。随着分子生物学检测技术的提高,目前已明确鉴别出口腔细菌群落或生物膜中包含至少500种微生物。口腔这一众多微生物栖息的场所,对于人类健康和疾病都有着至关重要的意义。口腔是消化道的开端,常常被成为"inner outside",解剖学上讲消化道由连续的器官构成,胃也属于消化系统,口腔和胃的微生物在某种程度上能够互相影响。一方面,口腔中的细菌可以通过摄入食物被带入胃内,另一方面,口腔菌群也有可能受到胃部疾病影响。例如,有些胃癌患者会伴发胃食管返流疾病(Gastroesophageal Reflux Disease, GERD),返流物能够造成上消化道系统的相应病变,改变其正常细菌群落,若发生呕吐症状,还可影响到口腔。因此,我们推测,胃癌前病变患者的口腔细菌多样性与健康人群不同。本研究采用聚合酶链反应-变性梯度凝胶电泳分析技术(polymerase chain reaction-based denaturing gradient gel electrophoresis technique, PCR-DGGE)作为手段分析胃癌前病变患者口腔中唾液和牙菌斑样本的细菌多样性,同时分析牙周病原菌的存在对口腔细菌多样性的影响。
方法：研究对象同第一部分。唾液和牙菌斑样本DNA的提取同第二部分。采用GeneAmp PCR SystemTM 9700进行巢式PCR。制备变形梯度凝胶,PCR产物和特异性DGGE参考Maker上样,在pH 8.5温度为58℃的1×Tris-acetate-EDTA (TAE)电泳缓冲液中恒压60 V电泳17小时。电泳结束后取下凝胶,在0.5μg/mL的EB溶液中染色12分钟,蒸馏水中漂洗6分钟后,AlphalmagerTM 3300 System采集DGGE图像,BioNumerics软件分析图像,SPSS17.0统计软件分析微生物学指标和细菌多样性之间的相关性。
结果：胃癌前病变患者的龈下菌斑样本的DGGE条带数为35.6,显著高于对照组的33.2(p<0.05),但两组间唾液样本的DGGE条带数量,未见显著性差异。整体研究人群的6个不同牙位点龈下菌斑之间的相似性达80.7%,而唾液和龈下菌斑的相似性只有78.0%。病例组唾液样本和龈下菌斑样本的相似程度(76.5%)略低于健康对照组(78.6)；病例组不同牙位龈下菌斑样本之间的相似程度却比对照组略高。不同牙位龈下菌斑之间的相似性越高,龈下菌斑样本的细菌多样性越高(p<0.001)。牙周病原菌在多牙位(≥2)检出阳性的人群,唾液样本DGGE条带数低于只有一个牙位检出阳性或全部牙位检出阴性的人群,龈下菌斑的多样性情况与此相反,Pg, Td和Tf多牙位检出阳性的人群,龈下菌斑的DGGE条带数均高于只有一个牙位检出阳性或全部牙位检出阴性的人群的人群。
结论：胃癌前病变患者口腔中龈下菌斑的细菌多样性略高于健康人群,同种来源样本(不同牙位龈下菌斑)细菌多样性的相似程度高于不同来源样本(唾液和龈下菌斑)。龈下菌斑的细菌多样性和各牙位龈下菌斑相似性高度相关。牙周病原菌在多位点检出阳性的人群,唾液细菌多样性有所下降,而龈下菌斑细菌多样性有所升高。
Dental caries and periodontal diseases are chronic biofilmbased diseases that occur in more than 90%(caries) and 35%(periodontal disease) in the adult population in the US. Mutans streptococci are generally considered to be the principal etiological agents of dental caries. S. mutans and S. sobrinus are the most commonly found bacteria in human plaque and are known to be associated with the development of dental caries. Periodontal diseases are characterized by gingival inflammation, periodontal pocket and attachment loss, which lead to tooth loss. In the past decades, increased epidemiological studies have presented evidence of positive association between periodontal diseases and the development of systemic diseases, including cardiovascular diseases, preterm delivery of low birth weight, diabetes mellitus, respiratory diseases, gastric cancer, AIDS and osteoporosis. However, the etiological details remain unclear. Nevertheless, more recent investigations have provided various results suggesting that periodontal infection and subsequent direct oral-hematogenous spread of bacteria are implicated in the development of various systemic diseases. Herein, we focus on the evaluation of the associations between the colonization of cariogenic and periodontal pathogens and their socio-demographic and clinical characteristics in individuals with gastric pre-cancerous lesions. Furthermore, we also evaluated the oral microbial diversity of the same group of individuals.
Part One- The socio-demographic and clinical oral health characteristics in individuals with gastric precancerous lesions
Gastric cancer is the second most common malignancy worldwide. The etiology of gastric cancer is not clear. There are many risk factors associated with gastric cancer, including life style, dietary structure, environmental, genetic and mental factors. Helicobacte pylori (H. pylori) was one of the most investigated etiological bacteria that are closely linked to chronic gastritis, peptic ulcer, gastric cancer. A few studies have suggested that H. pylori exist in subgingival plaque and therefore these sites are considered reservoirs for H. pylori. Meanwhile, several epidemiologic studies have also suggested a positive association between self-reported tooth loss and the risk of gastric cancer. Tooth loss in older age is more likely to be caused by chronic periodontal disease, whereas in younger age is usually caused by dental caries. Although these studies suggested that tooth lossis associated with an increased risk of gastric cancer, there was no detailed caries and periodontal assessment completed in these studies. More comprehensive clinical and microbial measurements are needed to investigate their association. The present study evaluated the socio-demographic and clinical oral health characteristics in individuals with gastric precancerous lesions.
Methods The population of this study was consisted of 98 individuals who were scheduled for an upper endoscopy procedure at the gastrointestinal clinics in the Veterans Affairs New York Harbor Healthcare System and Bellevue Hospital Center from March 30,2009 to April 27,2011. All individuals were more than 30 years old and had no prior history of esophageal cancer, gastric cancer, gastric varices, or portal gastropathy. Based on biopsy diagnostics, these individuals were divided into two groups:Cases were those individuals with gastric precancerous lesion (chronic atrophic gastritis, intestinal metaplasia, or dysplasia). Controls were those individuals with no lesions or only superficial gastritis. Evaluation of the dental caries and periodontal status was conducted at the time of dental examination.
Results Compared with the individuals in the control group, the individuals with gastric precancerous lesions harbored worse periodontal condition. They had deeper pocket depth, loss of attachment and more percent of sites with Pocket depth (PD) >4mm, Loss of attachment (LOA)≥3mm and LOA≥5mm. The percent of bleeding sites in the case group was significant higher than in the control groups (p<0.05).
Conclusion There is association between gastric precancer and the periodontal condition.
Part Two- The colonization of cariogenic and periodontal diseases pathogens in individuals with gastric precancerous
lesions
There is strong evidence that chronic inflammation is largely responsible for the early stages of disease progression. Because periodontal infection can lead to chronic systemic inflammation, which is an important risk factor in the development of gastric cancer, we hypothesize that major periodontal pathogens are associated with an increased risk for gastric cancer. To test this hypothesis, we conduct an exploratory hospital-based case-control study. The colonization of four periodontal pathogens, Porphyromonas gingivalis (Pg); Tannerella forsythensis (Tf); Treponema denticola (Td) and Actinobacillus actinomycetemcomitans (Aa) which are etiologically linked with destructive periodontal diseases; S. mutans (Sm) and S. sobrinus (Sb) which are cariogenic pathogens were measured in both saliva and subgingival plaque samples using the real-time quantitative PCR methods with species-specific DNA primers. We analyzed the microbial data together with the socio-demographic risk factors and clinical oral health characteristics, to see if the association exists between oral infectious diseases and gastric cancer.
Methods The population of this study was the same as part one. Non-stimulated whole saliva and plaque samples were collected from each subject at the time of dental examination. Bacterial genomic DNA of the saliva and plaque was isolated using the MasterPure DNA purification kit. Standardization of the template DNA for real-time qPCR was established first so that the final results can be compared. The species-specific PCR primers for the four periodontal pathogens were used in this study. All reactions were carried out in duplicate, and the final analysis was based on the mean of the two qPCR reactions.
Results The DNA level of periodontal pathogens in saliva was significantly associated with that in plaque (p<0.01). Subjects detected positive for Pg, Td and Tf had deeper pocket depth and higher percent of sites with PD>4mm and bleeding sites (p<0.05). The DNA level of Pg, Td and Tf in subgingival plaque was also significantly correlated with the percent of sites with PD>4mm and bleeding sites. The detection rates of Pg and Td were higher in the subjects with gastric precancerous lesion than the subjects in the healthy control groups. The detection rates of Aa and Tf were lower in the subjects with gastric precancerous lesion than the subjects in the control groups. The DNA level of four tested periodontal pathogens in both saliva and plaque was higher in case group than in control group, eventhough the difference didn't reach the statistic level.
Conclusion Pg, Td and Tf were significantly associated with the clinical symptom, especially the pocket depth and bleeding on probing. The detection rates of cariogenic and periodontal pathogens were different in case and control groups. Subjects with gastric precancerous lesion were more likely to test positive for Pg and Td and had higher DNA level of four periodontal pathogens compared with control groups.
Part Three-Oral microbial diversity in individuals with gastric precancerous lesions
The bacterial community in the oral cavity is one of the most complex mixtures of bacteria known. Recent developments of molecular biological techniques for microbial identification have identified more than 500 microbial species. The oral cavity as a home to the microbial community plays an important role in human health and disease. Oral cavity is the entrance of the digestive tract, which is often regarded as the'inner outside'. The stomach also belongs to the digestive system. Their bacteria can interfere with each other to some extent. Oral bacteria can be taken to stomach along with the intake food, while some gastric cancer patients have GRED which might affect some bacteria in the oral cavity. Based on the information above, we hypothesize that the oral microbial diversity differs in individuals with gastric cancer compared with individuals without gastric cancer. To test this hypothesis, we conducted an exploratory hospital-based case-control study. In the present study, polymerase chain reaction-based denaturing gradient gel electrophoresis technique (PCR-DGGE) was used for the analysis of the microbial populations obtained from both saliva and subgingival plaque samples from all subjects, including individuals with gastric precancerous lesions and healthy controls. We also compared the variation of the oral bacterial population between the two study groups and among the colonization sites; and to determine if periodontal bacterial colonization has effect on oral microbial diversity.
Methods A total of 98 subjects were enrolled in this study as part one. Bacterial samples were taken from all 98 subjects at the time of dental examination performed. Bacterial genomic DNA of the plaque or saliva was isolated as part two. Nested PCR was performed with use of the GeneAmp PCR SystemTM 9700. PCR products and species-specific DGGE reference markers were directly loaded in each lane, and electrophoresis was performed at a constant 60 V at 58℃for 17 hrs in 1×TAE, pH 8.5 buffer. After electrophoresis, the gels were rinsed and stained for 12 min in water containing 0.5μg/mL ethidium bromide, followed by 6 min of de-staining in water. DGGE profile images were digitally captured and recorded using the AlphalmagerTM 3300 System. DGGE profiles were analyzed using the BioNumerics Software.
Results Subjects with gastric precancerous lesions had more DGGE bands (35.6) in pooled plaque sample, compared with healthy population (33.2), while there was no significant difference in saliva sample. The similarity of diversity between saliva and pooled plaque of the study population was 78.0%, lower than that among 6 subgingival plaques, which was 80.7%. The similarity of diversity between saliva and pooled plaque of the subjects with gastric precancerous lesions was lower compared with the control group. However, the similarity among 6 subgingival plaques of the case group was higher than that of the control group. The more similar among the 6 subgingival plaques the subjects had, the more microbial diversity in their pooled plaque they would have. Subjects had at least two teeth with none-zero value of the periodontal pathogens had less microbial diversity in saliva but more in pooled subgingival plaque.
Conclusion Subjects with gastric precancerous lesions had more microbial diversity in pooled subgingival plaque, compared with healthy population. The similarity of same kind of sample (among plaques) was higher than different ones (saliva vs. plaque). There was highly correlation between diversity in subgingival plaque and the similarity among the subgingival plaques. The population had more tested positive teeth with periodontal pathogens had a decreased diversity in saliva but increased diversity in pooled subgingival plaque.
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