牙周致病菌的特异检测、治疗及其内毒素刺激人牙周膜细胞差异表达基因的研究
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摘要
牙周膜细胞是牙周组织的主要组成部分，具有一系列重要的功
能，在牙周组织正常代谢和修复再生过程中起重要作用。在牙周致病菌的
刺激下，牙周膜细胞生长受到抑制，不能行使正常的功能，因此研究牙周致
病菌刺激下牙周膜细胞的基因表达谱对了解牙周致病菌的致病机制、寻找
防治措施具有重要意义。本研究利用基因芯片分析牙周致病菌内毒素（LPS）
刺激前后人牙周膜细胞的差异基因表达谱，为进一步研究牙周致病菌致病
机理、寻找防治方法提供分子水平的依据。
利用基因芯片研究表明：中间普氏菌LPS刺激人牙周膜细胞前后共有
14条差异表达基因，其中6条上调基因，8条下调基因。上调基因主要为蛋
白激酶和蛋白磷酸酶类基因，而下调基因主要为转录调控基因、凋亡相关
基因、受体基因。因此，基因芯片可有效地筛选差异表达基因，从而用于
牙周炎病因的研究。
为验证基因芯片的分析结果，我们采用原位杂交技术研究中间普氏菌
LPS刺激前后人牙周膜细胞IAP-2的表达，结果表明：体外培养的对照组第
7代人牙周膜细胞，IAP-2表达为阳性；经LPS刺激24h后，人牙周膜细胞
IAP-2表达为阴性，说明LPS可通过抑制牙周膜细胞IAP-2表达，诱导牙周
膜细胞的凋亡。原位杂交的结果与基因芯片结果一致。
二、目前认为牙周炎是一种有部位特异性的疾病，需要对牙周炎活动
性进行快速、准确、客观的诊断和位点特异性的治疗。本研究利用寡核苷
酸探针和Periocheck椅旁快速检测技术，观察牙周致病菌在慢性牙周炎位点
中的分布，评价两种微生物检测方法在牙周炎活动性监测中的意义。
第四军医大学博1：论义
1．利用寡核昔酸探针对慢性牙周炎位点进行检测发现：Pg、Bf、Td在 60
个牙周炎位点中检出率很高，分别为86．67o、85o、96．67O，而Aa检
出率为引．67％，说明 Pg、Bf、Td是慢性牙周炎发生、发展中的优势菌
群。另外，本研究还发现Pg、Bf、Td两两正相关，表明牙周袋内门能
存在Pg、Bf、Td三种微生物的复合体，彼此之间具有协同生长作用。
2．Perioclleck检测的慢性牙周炎位点肽酶水平显著高J‘健康位点，）jk酶水
平与牙周临床指标正相关，因此Periocheck检测可用于判断患病位点，
有较高的敏感性和特异性，可用于检测牙周炎位点的活动性。
3．Periocheck检测的龈下菌斑肽酶活性和寡核昔酸探针检测的Pg、Bf、
Td细菌数正相关，说明Periocheck椅旁检测的肽酶活性可以间接反映
三种牙周公认致病菌的感染。
4．Periocheck检测的龈下菌斑中肽酶活性和 GCFAST、GCFALP水平小
相关，说明肽酶活性所代表的 Pg、Bf、Td三种龈下微生物的水平和
GCFAST所代表的牙周组织破坏程度、GCF－ALP代表的早期牙龈炎症
具有一定的相关性，进一步说明口腔微生物在牙周病病理中的作用。
5．Periocheck椅旁检测与寡核昔酸探针方法比较：Periocheck椅旁快速检测
技术比较简易和快捷，而寡核昔酸＊＊A探针检测比较特异，两种方法
均可以用于牙周可疑致病菌的检测，对牙周治疗效果进行评价。
二、利用kri。。他ck椅旁快速检测和寡核昔酸探针评估习’周袋内周部使
用盐酸二甲胺四环素软膏的治疗效果，观察用药前后临床指标及牙周袋内
微生物的改变，并检测GCF．AST、GCFALP水平的变化，结果表明：
1．本研究结果表明牙周洁治和根面平整后，Periocheck转阴（A组）位点
各临床指标和实验室指标均有显著改善，疾病具有较好的转归，说明
Periocheck检测可用于评价牙周治疗效果，预测疾病的发展趋势。
3
第四军医大学博l：论文
2．牙周洁治和根面平整后，Periocheck阳性位点只有部分转阴，说明机械
性刮治不能完全控制牙周菌斑，对机械性刮治效果不明显的位点需要进
行局部化学药物治疗，有选择的抑制牙周袋内的相关致病菌。
3．B组位点在洁治和根面平整后Periocheck检测仍为卜），牙周袋内局部仗
用软酸H甲胺四环素软膏（B－2组、B－3组）后，其牙龈指数、菌斑指
数、牙周袋深度、附着丧失的改善较再刮治组闪刁组）明显，说明对机
械性治疗效果不敏感的位点，局部化学药物可取得较好的临床效果。
4．局部使用盐酸h甲胺四环素（B－2组、B－3组）与对照组（B1组）比较：
GCFALP、GCFAST水平均有显著下降，说明盐酸二甲胺四环素软禽
可以有效抑制骨吸收，减少牙周组织破坏?
1. Periodontal ligament cells( PDLCs) are the predominant element of
periodontium and play a central role in the normal turnover, regeneration and
repair following injury to periodontiurn. Periodontal pathogen can inhibit the
growth of PDLCs and affect the normal function of cells. It is very important to
analyze the gene expression patterns in PDLCs stimulated by periodontal
pathogen LI扴 by DNA Chip to study the pathogenesis mechanism and to find
the prevention and treatment measurement.
By applying DNA Chip we analyzed the gene expression patterns in PDLCs
stimulated by Pi LPS. we identified 14 differentially expressed genes. including
6 upregulated and 8 downregulated genes, most of the genes upregulated were
involved in protein kinase and protein phosphatase, while downregulated genes
were related to signal transduction and transcription. apoptosis related gene
receptor gene.
We employed in situ hybridization method to observe the expression of lAP-
2 in PDLCs. The results showed that PDLCs were IAP-2 positive before
stimulation by Pi LPS and IAP-2 negative after stimulation by Pi LPS. The result
showed that Pi LPS could inhibit the IAP-2 expression of PDLCs and induce
apoptosis in PDLCs.
2. The periodontal diseases are now accepted as being site-specific, requiring
the development of more rapid, accurate diagnostic tests and site-specific therapy
based on the diagnosis of periodontal disease activity. The aim of the present
study was to evaluate the diagnostic value of oligonucleotide DNA probe and
5
Periocheck chair-side examination for periodontal pathogens and detect the
prevalence of specific periodontal pathogens in subgingival plaque.
A) Oligonucleotide DNA probes analysis showed the positive detection
frequency rates of Pg. Bf and Td in patients were 86.67%, 85% and 96.67%
respectively, Aa was detected in 31.67% diseased sites. Pg.. Bf and Td were
found to be prominent periodontopathic bacteria and their presence in
subgingival pocket can lead to development of periodontal disease. Pg.. Bf
and Td were found to be related with each other, maybe existed in microbial
complexes in subgingival plaque and coaggretate together.
B) Peptidase activity in subgingival plaque by Periocheck test was significantly
correlated with clinical parameters. peptidase activity in periodontitis sites
was higher than that of healthy control sites. The result suggested peptidase
activity detected by Periocheck test had a good sensitivity and specificity.
could be used to differentiate active and inactive periodontal diseased sites.
C) Peptidase activity in subgingival plaque by Periocheck test was significantly
correlated with Pg.. Bf and Td detected by oligonucleotide DNA probes. the
results implied that peptidase activity could indicate the infection of Pg.. Bf
andTd.
D) Peptidase activity in subgingival plaque was found to positively associated
with GCF-AST, GCF-ALP level, the relationship demonstrated specific
bacteria were involved in the progression of periodontitis. because the
peptidase activity was derived from Pg, Bf and Td, GCF-AST level could be
used as a marker of tissue destruction and GCF-ALP level might be used as a
marker of gingival inflammation.
E) To evaluate the diagnostic value of oligonucleotide DNA probe and
能，在牙周组织正常代谢和修复再生过程中起重要作用。在牙周致病菌的
刺激下，牙周膜细胞生长受到抑制，不能行使正常的功能，因此研究牙周致
病菌刺激下牙周膜细胞的基因表达谱对了解牙周致病菌的致病机制、寻找
防治措施具有重要意义。本研究利用基因芯片分析牙周致病菌内毒素（LPS）
刺激前后人牙周膜细胞的差异基因表达谱，为进一步研究牙周致病菌致病
机理、寻找防治方法提供分子水平的依据。
利用基因芯片研究表明：中间普氏菌LPS刺激人牙周膜细胞前后共有
14条差异表达基因，其中6条上调基因，8条下调基因。上调基因主要为蛋
白激酶和蛋白磷酸酶类基因，而下调基因主要为转录调控基因、凋亡相关
基因、受体基因。因此，基因芯片可有效地筛选差异表达基因，从而用于
牙周炎病因的研究。
为验证基因芯片的分析结果，我们采用原位杂交技术研究中间普氏菌
LPS刺激前后人牙周膜细胞IAP-2的表达，结果表明：体外培养的对照组第
7代人牙周膜细胞，IAP-2表达为阳性；经LPS刺激24h后，人牙周膜细胞
IAP-2表达为阴性，说明LPS可通过抑制牙周膜细胞IAP-2表达，诱导牙周
膜细胞的凋亡。原位杂交的结果与基因芯片结果一致。
二、目前认为牙周炎是一种有部位特异性的疾病，需要对牙周炎活动
性进行快速、准确、客观的诊断和位点特异性的治疗。本研究利用寡核苷
酸探针和Periocheck椅旁快速检测技术，观察牙周致病菌在慢性牙周炎位点
中的分布，评价两种微生物检测方法在牙周炎活动性监测中的意义。
第四军医大学博1：论义
1．利用寡核昔酸探针对慢性牙周炎位点进行检测发现：Pg、Bf、Td在 60
个牙周炎位点中检出率很高，分别为86．67o、85o、96．67O，而Aa检
出率为引．67％，说明 Pg、Bf、Td是慢性牙周炎发生、发展中的优势菌
群。另外，本研究还发现Pg、Bf、Td两两正相关，表明牙周袋内门能
存在Pg、Bf、Td三种微生物的复合体，彼此之间具有协同生长作用。
2．Perioclleck检测的慢性牙周炎位点肽酶水平显著高J‘健康位点，）jk酶水
平与牙周临床指标正相关，因此Periocheck检测可用于判断患病位点，
有较高的敏感性和特异性，可用于检测牙周炎位点的活动性。
3．Periocheck检测的龈下菌斑肽酶活性和寡核昔酸探针检测的Pg、Bf、
Td细菌数正相关，说明Periocheck椅旁检测的肽酶活性可以间接反映
三种牙周公认致病菌的感染。
4．Periocheck检测的龈下菌斑中肽酶活性和 GCFAST、GCFALP水平小
相关，说明肽酶活性所代表的 Pg、Bf、Td三种龈下微生物的水平和
GCFAST所代表的牙周组织破坏程度、GCF－ALP代表的早期牙龈炎症
具有一定的相关性，进一步说明口腔微生物在牙周病病理中的作用。
5．Periocheck椅旁检测与寡核昔酸探针方法比较：Periocheck椅旁快速检测
技术比较简易和快捷，而寡核昔酸＊＊A探针检测比较特异，两种方法
均可以用于牙周可疑致病菌的检测，对牙周治疗效果进行评价。
二、利用kri。。他ck椅旁快速检测和寡核昔酸探针评估习’周袋内周部使
用盐酸二甲胺四环素软膏的治疗效果，观察用药前后临床指标及牙周袋内
微生物的改变，并检测GCF．AST、GCFALP水平的变化，结果表明：
1．本研究结果表明牙周洁治和根面平整后，Periocheck转阴（A组）位点
各临床指标和实验室指标均有显著改善，疾病具有较好的转归，说明
Periocheck检测可用于评价牙周治疗效果，预测疾病的发展趋势。
3
第四军医大学博l：论文
2．牙周洁治和根面平整后，Periocheck阳性位点只有部分转阴，说明机械
性刮治不能完全控制牙周菌斑，对机械性刮治效果不明显的位点需要进
行局部化学药物治疗，有选择的抑制牙周袋内的相关致病菌。
3．B组位点在洁治和根面平整后Periocheck检测仍为卜），牙周袋内局部仗
用软酸H甲胺四环素软膏（B－2组、B－3组）后，其牙龈指数、菌斑指
数、牙周袋深度、附着丧失的改善较再刮治组闪刁组）明显，说明对机
械性治疗效果不敏感的位点，局部化学药物可取得较好的临床效果。
4．局部使用盐酸h甲胺四环素（B－2组、B－3组）与对照组（B1组）比较：
GCFALP、GCFAST水平均有显著下降，说明盐酸二甲胺四环素软禽
可以有效抑制骨吸收，减少牙周组织破坏?
1. Periodontal ligament cells( PDLCs) are the predominant element of
periodontium and play a central role in the normal turnover, regeneration and
repair following injury to periodontiurn. Periodontal pathogen can inhibit the
growth of PDLCs and affect the normal function of cells. It is very important to
analyze the gene expression patterns in PDLCs stimulated by periodontal
pathogen LI扴 by DNA Chip to study the pathogenesis mechanism and to find
the prevention and treatment measurement.
By applying DNA Chip we analyzed the gene expression patterns in PDLCs
stimulated by Pi LPS. we identified 14 differentially expressed genes. including
6 upregulated and 8 downregulated genes, most of the genes upregulated were
involved in protein kinase and protein phosphatase, while downregulated genes
were related to signal transduction and transcription. apoptosis related gene
receptor gene.
We employed in situ hybridization method to observe the expression of lAP-
2 in PDLCs. The results showed that PDLCs were IAP-2 positive before
stimulation by Pi LPS and IAP-2 negative after stimulation by Pi LPS. The result
showed that Pi LPS could inhibit the IAP-2 expression of PDLCs and induce
apoptosis in PDLCs.
2. The periodontal diseases are now accepted as being site-specific, requiring
the development of more rapid, accurate diagnostic tests and site-specific therapy
based on the diagnosis of periodontal disease activity. The aim of the present
study was to evaluate the diagnostic value of oligonucleotide DNA probe and
5
Periocheck chair-side examination for periodontal pathogens and detect the
prevalence of specific periodontal pathogens in subgingival plaque.
A) Oligonucleotide DNA probes analysis showed the positive detection
frequency rates of Pg. Bf and Td in patients were 86.67%, 85% and 96.67%
respectively, Aa was detected in 31.67% diseased sites. Pg.. Bf and Td were
found to be prominent periodontopathic bacteria and their presence in
subgingival pocket can lead to development of periodontal disease. Pg.. Bf
and Td were found to be related with each other, maybe existed in microbial
complexes in subgingival plaque and coaggretate together.
B) Peptidase activity in subgingival plaque by Periocheck test was significantly
correlated with clinical parameters. peptidase activity in periodontitis sites
was higher than that of healthy control sites. The result suggested peptidase
activity detected by Periocheck test had a good sensitivity and specificity.
could be used to differentiate active and inactive periodontal diseased sites.
C) Peptidase activity in subgingival plaque by Periocheck test was significantly
correlated with Pg.. Bf and Td detected by oligonucleotide DNA probes. the
results implied that peptidase activity could indicate the infection of Pg.. Bf
andTd.
D) Peptidase activity in subgingival plaque was found to positively associated
with GCF-AST, GCF-ALP level, the relationship demonstrated specific
bacteria were involved in the progression of periodontitis. because the
peptidase activity was derived from Pg, Bf and Td, GCF-AST level could be
used as a marker of tissue destruction and GCF-ALP level might be used as a
marker of gingival inflammation.
E) To evaluate the diagnostic value of oligonucleotide DNA probe and
引文
1 Chappie ILC. Periodontal disease diagnosis: Current status and future developments. J Dent 1997,25(1 ):3-15
2 Goodson MS, Tanner ACR, Haffajee AD, et al. Patterns of progression and regression of advanced destructive periodontal disease activity.J Clin Periodontal 1982, 9: 472-481
3 Page RC, Beck JD. Risk assessment for periodontal diseases. Int Dent J 1997, 47:61-87
4 Gabriel M, Thomas EVD. Periodontal diagnosis: current status and future directions. Int Dent J 1998,48(Supplement 1) :275-281
5 Nieminen A, Nordlund L,Uitto VJ.The effect of treatment on the activity of salivary proteases and glycosidases in adults with advanced periodontitis, J Periodontol, 1993,64,297-301
6 Ingaman T, Sorsa T, Konttinen YT, et al. Salivary collagenase, elastase and trypsin like proteases as biochemical markersof periodontal disease destruction in adult and localized juvenile periodontitis. Oral Micro and Immun, 1993,8,298-305
7 Over C,Yamalic N,Yazuayilmaz E, et al. Myeloperoxidase activity in peripheral blood, neutrophil crevicular fluid and whole saliva of patients with periodontal disease. J Nihon University Scool of Dentistry, 1993,35,235-240
8 Picarelli A,Porcelli D, Porcelli G, et al.Salivary kallikrein and kininase activities in periodontal disease. Advan in Experimenal Medicine and Biology, 1986, 898,433-437
9 Umeda M, Contreres A,Chen C,et al. The utility of whole saliva to detect the oral presence of periodontopathic bacteria. J Periodontol, 1998,69:828-833
10 Rosenberg M, Kozlovsky A, Gelernter I,et al. Self-estimation of oral malodor. J Dent Res,1995,74:1577-1582．
11 Kaufman E, Lamster IB. Analysis of saliva for periodontal diagnosis. J Clin Periodontal 2000,27:453-465
12 Eftimiadi C, Sakellari D, Diabart S, et al. Detection of crevicular fluid antibody to subgingival species using checkerboard immunoblotting. J Dent Res 1995, 74:226
13 Okada H, Murakami S, Kitamura M, Nozaki T,et al. Diagnostic strategies of periodontitis based on the molecular mechanisms of periodontal tissue destruction. Oral Dis 1996 Mar;2(1) :87-95
14 Roberts FA, Hockett RD, Bucy RP, et al. Quantitative assessment of inflammatory cytokine gene expression in chronic adult periodontitis. Oral Microbiol Immunol 1997 Oct; 12:336-344
15 Yamazaki K, Nakajima T, Kubota Y,et al. Cytokine messenger RNA expression in chronic inflammatory periodontal disease.Oral Microbiol Immunol 1997 Oct;12(5) :281-7
16 Takeichi O, Haber J, Kawai T, et al. Cytokine profiles of T-lymphocytes from gingival tissues with pathological pocketing. J Dent Res 2000 ;79(8) : 1548-55
17 Smith GLF. Diagnosis of periodontal disease activity by detection of key microbial antigens. J Clin Periodontal 1994,21:615-620
18 Listgarten MA. Microbiological testing in the diagnosis of periodontal disease. J Periodontol 1992:63:332-337
19 Zambon JJ. Principles of evaluation of the diagnostic value of subgingival bacteria. Ann Periodontol 1997:2:138-148
20 MeCulloch CAG. Host enzymes in gingival crevicular fluid as diagnostic indicators of periodontitis. J Clin Periodontal 1994, 21:497-506
21 Gibbs CH, Hirschfeld JW, Lee JG, et al. Description and evaluation of a new computerized periodontal probe-the Florida probe. J Clin Periodontal 1988,15:137-144
22 Kung RTV, Ochs B, Goodson JM. Temperatures as periodontal diagnostic. J Clin Periodontal 1990, 17:557-563
23 Jeffcoat MK. Radiographic methods for the detection of progressive alveolar bone loss. J Periodont 1992, 63:367-372
24 Eley BM,Cox SW. Advances in periodontal diagnosis 8． Commercial diagnostic kits based on GCF proteolytic and hydrolytic enzyme levels. Br Dent J, 1995 ,178:373-376
25 张贤华,吴织芬。牙周病的椅旁快速检测。国外医学口腔分册,1998, 25(6) :351-353
26 Persson GR, Alves ME, Chambers DA, et al. A multicenter clinical trial of PerioGard in distinguishing between diseased and healthy periodontal sites. (1) . Study design, methodology and therapeutic outcome. J Clin Periodontol 1995 Oct;22(10) :794-803
27 Wong MY, Lu CL, Liu CM, et al. Relationship of the subgingival microbiota to a chairside test for aspartate aminotransferase in gingival crevicular fluid. J Periodontol 1999 Jan;70(1) :57-62
28 Hart TC. Genetic risk factors for early-onset periodontitis. J Periodontal 1996, 67:355
29 Hart TC, Kornman KS. Genetic factors in the pathogenesis of periodontitis. Periodontology 2000, 1997,14:202
30 Komman KS, Crane A, Wang HY, et al. The interleukin-1 genotype as a severity factor in adult periodontal disease. J Clin Periodontol, 1997,24:72
2 Goodson MS, Tanner ACR, Haffajee AD, et al. Patterns of progression and regression of advanced destructive periodontal disease activity.J Clin Periodontal 1982, 9: 472-481
3 Page RC, Beck JD. Risk assessment for periodontal diseases. Int Dent J 1997, 47:61-87
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