臺灣博碩士論文加值系統

背景：
　　牙周炎是一種發炎性疾病，會影響且破壞牙齒周遭支持組織，包括：牙齦、齒槽骨、牙周韌帶…等。形成牙周炎過程是複雜且多基因參與，且由多種環境因子和基因共同影響宿主免疫反應系統和致病菌的交互作用。Toll-like receptor的基因多型性會影響宿主對於牙周致病菌的先天免疫反應系統。本研究的目標是探討台灣漢民族TLR4的基因多型性和牙周炎之間的關聯性。

材料與方法：
抽取122位慢性牙周炎患者、84位侵犯性牙周炎患者及64位牙周健康者20ml靜脈血之白血球來萃取DNA。以Real Time TaqMan probe PCR技術檢測Toll-like receptor4 (rs2197914)T>C基因多型性。統計分析以卡方檢定分析基因型、性別、抽菸有無在各組間的差異，以邏輯回歸分析基因型和罹患牙周炎風險相關性。
同時檢驗研究樣本中基因型分布是否符合Hardy-Weinberg equilibrium定律。

結果：
TLR4基因型和座基因分布頻率在慢性牙周炎、侵犯性牙周炎和牙周健康者三個族群中並無統計學上顯著差異。以對數回歸分析總樣本基因型和牙周炎的關係，TT基因型罹患侵犯性牙周炎的風險是CT/CC的1.95倍，但未達統計學上顯著性(OR=1.95, p = 0.0884, 95%CI = 0.231-1.104)。但若區分出抽菸和未抽菸族群，在未抽菸者中，TT基因型罹患侵犯性牙周炎的風險為CT+CC基因型的2.66倍，達統計上顯著差異(OR=2.66, p = 0.0383, 95%CI = 0.144 - 0.929)。 在抽菸者中，基因型和牙周炎間的關聯性皆未達統計上差異。

結論：
本篇研究結果顯示在台灣漢民族未有抽菸習慣族群中，TLR4基因多型性中的TT基因型比CT/CC基因型有較高罹患侵犯性牙周炎的風險。然而，關於此TLR4基因多型性與牙周炎相關的作用機轉仍需更進一步研究討論。

Objectives:
Periodontitis is an inflammatory disease that affecting the periodontal connective supporting tissues. Gene polymorphisms of Toll-like receptor could affect the host’s ability to respond to microbial pathogens. This study aimed to investigate the possible association between TLR4 gene polymorphism and periodontal disease in a Taiwan Han population.
Material and methods:
Genomic DNA was obtained from the peripheral blood of 122 patients with chronic periodontitis (CP), 84 patients with (aggressive periodontitis (AgP) and 64 periodontally healthy subjects (HC). The genotype of TLR4 SNP was performed using the TaqMan SNP genotyping assay (Applied Biosystems Inc. (ABI), Foster City). The probe fluorescence signals were detected with a TaqMan Assay for Real-Time PCR (7500 Real Time PCR System, Applied Biosystems Inc. ABI). X2 analysis was used to test the deviation of genotype frequencies from Hardy Weinberg equilibrium. The data were analyzed by ANOVA, chi-square test and logistic regression analysis with JMP 6.0 (SAS, Cary, NC, USA).
Results:
There were no statistical differences in the frequencies of genotypes and alleles between CP, AgP and HC (p = 0.6811, X2 = 2.298). Genotypes of all study subjects were further divided into C-allele and non-C-allele carriers. The logistic regression analysis adjusted for gender, alcohol drinking, cigarette smoking, betel nut chewing and age. The association of allele carries between AgP and HC did not reach significance (OR=1.95, p = 0.0884, 95%CI = 0.231-1.104). Subjects were further stratified by smoking habit. The association of allele carries showed statistical differences between AgP and HC in non-smoking subjects (OR = 2.66, p = 0.0383, 95%CI = 0.144-0.929).
Conclusion:
The results of our study showed that TT genotype of TLR4 polymorphism was associated with an increase risk of aggressive periodontitis (AgP) in Taiwanese population without smoking habits. The genetic effect of TT genotype was masked by smoking habit in subjects of aggressive periodotitis. However, further research for the function of TLR4 polymorphism is necessary.

中文摘要-----------------------------------------------------------1
Abstract-----------------------------------------------------------3
第一章 前言-------------------------------------------------------5
第一節 研究背景-----------------------------------------------5
第二節 研究目的-----------------------------------------------7
第二章 文獻回顧---------------------------------------------------8
第一節 牙周炎的定義-------------------------------------------8
第二節 牙周炎的分類-------------------------------------------8
１、 慢性牙周炎--------------------------------------------9
２、 侵犯性牙周炎-----------------------------------------10
第三節 牙周病的病因------------------------------------------10
第四節 基因多型性在基因表現中扮演的角色----------------------12
第五節 Toll-like receptor 4 和牙周炎的關係-------------------14
１、 Toll-like receptor簡介-----------------------------14
２、 Toll-like receptor 4的角色和訊息傳遞機制-----------15
３、 人類Toll-like receptor 4的基因變異-----------------17
４、 基因多型性在不同人種中和牙周炎的關係----------------17
第三章 材料和方法------------------------------------------------20
第一節 研究對象來源------------------------------------------20
第二節 採樣和DNA純化----------------------------------------22
第三節 Real-time PCR-----------------------------------------24
第四節 統計分析----------------------------------------------25
第四章 結果------------------------------------------------------27
第一節 總樣本的統計分析結果----------------------------------27
第二節 未抽菸樣本之統計分析結果------------------------------30
第三節 抽菸樣本之統計分析結果--------------------------------33
第五章 討論------------------------------------------------------37
第六章 結論------------------------------------------------------47
參考文獻----------------------------------------------------------48
附表--------------------------------------------------------------54

1.Williams RC: Periodontal disease. N Engl J Med 1990, 322(6):373-382.
2.Shaw JH: Causes and control of dental caries. N Engl J Med 1987, 317(16):996-1004.
3.Kinane DF, Hart TC: Genes and gene polymorphisms associated with periodontal disease. Crit Rev Oral Biol Med 2003, 14(6):430-449.
4.Haffajee AD, Socransky SS: Microbial etiological agents of destructive periodontal diseases. Periodontol 2000 1994, 5:78-111.
5.Bergstrom J: Cigarette smoking as risk factor in chronic periodontal disease. Community Dent Oral Epidemiol 1989, 17(5):245-247.
6.Aderem A, Ulevitch RJ: Toll-like receptors in the induction of the innate immune response. Nature 2000, 406(6797):782-787.
7.Akira S, Yamamoto M, Takeda K: Role of adapters in Toll-like receptor signalling. Biochem Soc Trans 2003, 31(Pt 3):637-642.
8.Yoshimura A, Kaneko T, Kato Y, Golenbock DT, Hara Y: Lipopolysaccharides from periodontopathic bacteria Porphyromonas gingivalis and Capnocytophaga ochracea are antagonists for human toll-like receptor 4. Infect Immun 2002, 70(1):218-225.
9.Dixon DR, Bainbridge BW, Darveau RP: Modulation of the innate immune response within the periodontium. Periodontol 2000 2004, 35:53-74.
10.Loos BG, John RP, Laine ML: Identification of genetic risk factors for periodontitis and possible mechanisms of action. J Clin Periodontol 2005, 32 Suppl 6:159-179.
11.Folwaczny M, Glas J, Torok HP, Limbersky O, Folwaczny C: Toll-like receptor (TLR) 2 and 4 mutations in periodontal disease. Clin Exp Immunol 2004, 135(2):330-335.
12.Flemmig TF: Periodontitis. Ann Periodontol 1999, 4(1):32-38.
13.Gold SI: Periodontics. The past. Part (I). Early sources. J Clin Periodontol 1985, 12(2):79-97.
14.Gottlieb A: A Constructive Suggestion. Cal State J Med 1920, 18(12):420.
15.Armitage GC: Development of a classification system for periodontal diseases and conditions. Ann Periodontol 1999, 4(1):1-6.
16.Brown LJ, Loe H: Prevalence, extent, severity and progression of periodontal disease. Periodontol 2000 1993, 2:57-71.
17.Novak MJ, Novak KF: Early-onset periodontitis. Curr Opin Periodontol 1996, 3:45-58.
18.Tonetti MS, Mombelli A: Early-onset periodontitis. Ann Periodontol 1999, 4(1):39-53.
19.Page RC, Offenbacher S, Schroeder HE, Seymour GJ, Kornman KS: Advances in the pathogenesis of periodontitis: summary of developments, clinical implications and future directions. Periodontol 2000 1997, 14:216-248.
20.Page RC: The pathobiology of periodontal diseases may affect systemic diseases: inversion of a paradigm. Ann Periodontol 1998, 3(1):108-120.
21.Bartold PM, Cantley MD, Haynes DR: Mechanisms and control of pathologic bone loss in periodontitis. Periodontol 2000 2010, 53:55-69.
22.Reynolds JJ, Meikle MC: Mechanisms of connective tissue matrix destruction in periodontitis. Periodontol 2000 1997, 14:144-157.
23.Stephens JC, Schneider JA, Tanguay DA, Choi J, Acharya T, Stanley SE, Jiang R, Messer CJ, Chew A, Han JH et al: Haplotype variation and linkage disequilibrium in 313 human genes. Science 2001, 293(5529):489-493.
24.Takashiba S, Naruishi K: Gene polymorphisms in periodontal health and disease. Periodontol 2000 2006, 40:94-106.
25.Nielsen R: Population genetic analysis of ascertained SNP data. Hum Genomics 2004, 1(3):218-224.
26.Jasinska A, Michlewski G, de Mezer M, Sobczak K, Kozlowski P, Napierala M, Krzyzosiak WJ: Structures of trinucleotide repeats in human transcripts and their functional implications. Nucleic Acids Res 2003, 31(19):5463-5468.
27.Reich DE, Lander ES: On the allelic spectrum of human disease. Trends Genet 2001, 17(9):502-510.
28.Rogers MJ, Gordon S, Benford HL, Coxon FP, Luckman SP, Monkkonen J, Frith JC: Cellular and molecular mechanisms of action of bisphosphonates. Cancer 2000, 88(12 Suppl):2961-2978.
29.Barton A, John S: Approaches to identifying genetic predictors of clinical outcome in rheumatoid arthritis. Am J Pharmacogenomics 2003, 3(3):181-191.
30.Akira S, Hemmi H: Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 2003, 85(2):85-95.
31.Medzhitov R, Janeway C, Jr.: Innate immunity. N Engl J Med 2000, 343(5):338-344.
32.Kawai T, Akira S: TLR signaling. Cell Death Differ 2006, 13(5):816-825.
33.Takeda K, Akira S: Toll-like receptors in innate immunity. Int Immunol 2005, 17(1):1-14.
34.Hashimoto C, Hudson KL, Anderson KV: The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell 1988, 52(2):269-279.
35.Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA: The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 1996, 86(6):973-983.
36.Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF: A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci U S A 1998, 95(2):588-593.
37.Medzhitov R, Preston-Hurlburt P, Janeway CA, Jr.: A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997, 388(6640):394-397.
38.Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C et al: Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998, 282(5396):2085-2088.
39.Schumann RR, Leong SR, Flaggs GW, Gray PW, Wright SD, Mathison JC, Tobias PS, Ulevitch RJ: Structure and function of lipopolysaccharide binding protein. Science 1990, 249(4975):1429-1431.
40.Kim HM, Park BS, Kim JI, Kim SE, Lee J, Oh SC, Enkhbayar P, Matsushima N, Lee H, Yoo OJ et al: Crystal structure of the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. Cell 2007, 130(5):906-917.
41.Akira S, Takeda K: Toll-like receptor signalling. Nat Rev Immunol 2004, 4(7):499-511.
42.Miggin SM, O''Neill LA: New insights into the regulation of TLR signaling. J Leukoc Biol 2006, 80(2):220-226.
43.Smirnova I, Poltorak A, Chan EK, McBride C, Beutler B: Phylogenetic variation and polymorphism at the toll-like receptor 4 locus (TLR4). Genome Biol 2000, 1(1):RESEARCH002.
44.Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN, Jones M, Frees K, Watt JL, Schwartz DA: TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet 2000, 25(2):187-191.
45.A haplotype map of the human genome. Nature 2005, 437(7063):1299-1320.
46.Eto M, Watanabe K, Ishii K: A racial difference in apolipoprotein E allele frequencies between the Japanese and Caucasian populations. Clin Genet 1986, 30(5):422-427.
47.Schroder NW, Meister D, Wolff V, Christan C, Kaner D, Haban V, Purucker P, Hermann C, Moter A, Gobel UB et al: Chronic periodontal disease is associated with single-nucleotide polymorphisms of the human TLR-4 gene. Genes Immun 2005, 6(5):448-451.
48.Fukusaki T, Ohara N, Hara Y, Yoshimura A, Yoshiura K: Evidence for association between a Toll-like receptor 4 gene polymorphism and moderate/severe periodontitis in the Japanese population. J Periodontal Res 2007, 42(6):541-545.
49.Berdeli A, Emingil G, Han Saygan B, Gurkan A, Atilla G, Kose T, Baylas H: TLR2 Arg753Gly, TLR4 Asp299Gly and Thr399Ile gene polymorphisms are not associated with chronic periodontitis in a Turkish population. J Clin Periodontol 2007, 34(7):551-557.
50.Izakovicova Holla L, Buckova D, Fassmann A, Roubalikova L, Vanek J: Lack of association between chronic periodontitis and the Toll-like receptor 4 gene polymorphisms in a Czech population. J Periodontal Res 2007, 42(4):340-344.
51.Zhu G, Li C, Cao Z, Corbet EF, Jin L: Toll-like receptors 2 and 4 gene polymorphisms in a Chinese population with periodontitis. Quintessence Int 2008, 39(3):217-226.
52.Schroder NW, Schumann RR: Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious disease. Lancet Infect Dis 2005, 5(3):156-164.
53.Lin YC, Chang YM, Yu JM, Yen JH, Chang JG, Hu CJ: Toll-like receptor 4 gene C119A but not Asp299Gly polymorphism is associated with ischemic stroke among ethnic Chinese in Taiwan. Atherosclerosis 2005, 180(2):305-309.
54.Noguchi E, Nishimura F, Fukai H, Kim J, Ichikawa K, Shibasaki M, Arinami T: An association study of asthma and total serum immunoglobin E levels for Toll-like receptor polymorphisms in a Japanese population. Clin Exp Allergy 2004, 34(2):177-183.
55.Loe H, Anerud A, Boysen H, Morrison E: Natural history of periodontal disease in man. Rapid, moderate and no loss of attachment in Sri Lankan laborers 14 to 46 years of age. J Clin Periodontol 1986, 13(5):431-445.
56.Medzhitov R: Origin and physiological roles of inflammation. Nature 2008, 454(7203):428-435.
57.Michalowicz BS, Aeppli DP, Kuba RK, Bereuter JE, Conry JP, Segal NL, Bouchard TJ, Jr., Pihlstrom BL: A twin study of genetic variation in proportional radiographic alveolar bone height. J Dent Res 1991, 70(11):1431-1435.
58.Kornman KS: Mapping the pathogenesis of periodontitis: a new look. J Periodontol 2008, 79(8 Suppl):1560-1568.
59.Loos BG, Leppers-Van de Straat FG, Van de Winkel JG, Van der Velden U: Fcgamma receptor polymorphisms in relation to periodontitis. J Clin Periodontol 2003, 30(7):595-602.
60.Suzuki T, Kobayashi M, Isatsu K, Nishihara T, Aiuchi T, Nakaya K, Hasegawa K: Mechanisms involved in apoptosis of human macrophages induced by lipopolysaccharide from Actinobacillus actinomycetemcomitans in the presence of cycloheximide. Infect Immun 2004, 72(4):1856-1865.
61.Darveau RP, Pham TT, Lemley K, Reife RA, Bainbridge BW, Coats SR, Howald WN, Way SS, Hajjar AM: Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both toll-like receptors 2 and 4. Infect Immun 2004, 72(9):5041-5051.
62.Laine ML, Morre SA, Murillo LS, van Winkelhoff AJ, Pena AS: CD14 and TLR4 gene polymorphisms in adult periodontitis. J Dent Res 2005, 84(11):1042-1046.
63.Na KS, Kim TH, Rahman P, Peddle L, Choi CB, Inman RD: Analysis of single nucleotide polymorphisms in Toll-like receptor 4 shows no association with ankylosing spondylitis in a Korean population. Rheumatol Int 2008, 28(7):627-630.
64.Umemura T, Katsuyama Y, Hamano H, Kitahara K, Takayama M, Arakura N, Kawa S, Tanaka E, Ota M: Association analysis of Toll-like receptor 4 polymorphisms with autoimmune pancreatitis. Hum Immunol 2009, 70(9):742-746.
65.Subramaniam K, Chen K, Joseph K, Raymond JR, Tholanikunnel BG: The 3''-untranslated region of the beta2-adrenergic receptor mRNA regulates receptor synthesis. J Biol Chem 2004, 279(26):27108-27115.
66.Conne B, Stutz A, Vassalli JD: The 3'' untranslated region of messenger RNA: A molecular ''hotspot'' for pathology? Nat Med 2000, 6(6):637-641.
67.Doi K, Noiri E, Nakao A, Fujita T, Kobayashi S, Tokunaga K: Functional polymorphisms in the vascular endothelial growth factor gene are associated with development of end-stage renal disease in males. J Am Soc Nephrol 2006, 17(3):823-830.
68.Mollen KP, Anand RJ, Tsung A, Prince JM, Levy RM, Billiar TR: Emerging paradigm: toll-like receptor 4-sentinel for the detection of tissue damage. Shock 2006, 26(5):430-437.
69.Gunsolley JC, Pandey JP, Quinn SM, Tew J, Schenkein HA: The effect of race, smoking and immunoglobulin allotypes on IgG subclass concentrations. J Periodontal Res 1997, 32(4):381-387.
70.Schenkein HA: Finding genetic risk factors for periodontal diseases: is the climb worth the view? Periodontol 2000 2002, 30:79-90.
71.Ordovas JM, Shen J: Gene-environment interactions and susceptibility to metabolic syndrome and other chronic diseases. J Periodontol 2008, 79(8 Suppl):1508-1513.
72.Palmer RM, Farkondeh N, Palmer PJ, Wilson RF: Astra Tech single-tooth implants: an audit of patient satisfaction and soft tissue form. J Clin Periodontol 2007, 34(7):633-638.
73.Zhang J, Sun X, Xiao L, Xie C, Xuan D, Luo G: Gene polymorphisms and periodontitis. Periodontol 2000 2011, 56(1):102-124.
74.Genes, drugs and race. Nat Genet 2001, 29(3):239-240.
75.Hsieh YY, Wan L, Chang CC, Tsai CH, Tsai FJ: STAT2*C related genotypes and allele but not TLR4 and CD40 gene polymorphisms are associated with higher susceptibility for asthma. Int J Biol Sci 2009, 5(1):74-81.
76.Shibuya E, Meguro A, Ota M, Kashiwagi K, Mabuchi F, Iijima H, Kawase K, Yamamoto T, Nakamura M, Negi A et al: Association of Toll-like receptor 4 gene polymorphisms with normal tension glaucoma. Invest Ophthalmol Vis Sci 2008, 49(10):4453-4457.
77.Song J, Kim DY, Kim CS, Kim HJ, Lee DH, Lee HM, Ko W, Lee G: The association between Toll-like receptor 4 (TLR4) polymorphisms and the risk of prostate cancer in Korean men. Cancer Genet Cytogenet 2009, 190(2):88-92.