Journal of the korean academy of Pediatric Dentistry (대한소아치과학회지)

Korean Academy of Pediatric Dentistry (대한소아치과학회)
권호 표지

GENE EXPRESSION ANALYSIS OF THE DENTAL PULP IN HEALTHY AND CARIES TEETH

치아 우식증에 따른 치수내 유전자 발현 변화에 관한 분석

  • 오소희 (한림대학교 성심병원 소아치과) ;
  • 김종수 (단국대학교 치과병원 소아치과)
  • Received : 2010.04.02
  • Accepted : 2010.08.02
  • Published : 2010.08.31
Download PDF
⟨ Previous Next ⟩

Abstract

Deep caries may induce pulpitis and the pulpal tissue interacts with microbial invasion. The immune response to protect the pulpal tissue can be mediated by cellular signal molecules produced by the pulpal cells. The understanding of these processes is important to find future therapeutic method for the diseased pulp. The pulp tissue from sound teeth was set as control group (n=30) and the pulp tissue from decayed teeth was set as test group (n=30). Total RNA was extracted from the pulp of each group and it was used for cDNA microarray and reverse transcriptase-polymerase chain reaction(RT-PCR). The expression of TGF-${\beta}1$ was studied by immunohistochemistry. The results were as follows: 1. cDNA microarray analysis identified 520 genes with 6-fold or greater difference in expression level with 143 genes more abundant in health and 377 genes more abundant in disease. 2. The RT-PCR analysis was done for randomly selected 14 genes and the results supported the result of cDNA microarray assay. 3. TGF-${\beta}1$ was highly expressed in the carious pulp and it was found in odontoblast by immunohistochemistry. In conclusion, many cytokines were found to be significantly changed their expression in the diseased pulp(/M/>1.6).

치아 우식증은 범발성 질환이고 이에 대한 생체반응은 단순하지 않으며 질병 과정과 숙주의 활성 모두를 반영하는 복합적인 반응이다. 이러한 반응을 이해하기 위해서는 질병의 세포학적, 분자학적인 면을 이해하는 것이 필수적이다. 이에 본 연구는 임상적으로 건강한 치아와 우식이 진행된 치아로부터 얻어진 치수 안의 유전자 발현을 규명하고 우식 병소에서 일어나는 치유 및 재생에 관계되는 분자와 면역 세포들 사이의 분자 생화학적 상호작용을 규명하기 위해서 우식치아와 건전치아의 치수를 이용하여 cDNA 미세배열(microarray) 분석과 역전사효소 중합효소 연쇄반응 (RT-PCR) 분석, 그리고 면역화학염색법 (immunohistochemistry)을 시행하여 다음과 같은 결론을 얻었다. 1. cDNA 미세배열 분석 결과, 건전치아군인 대조군에서는 143개의 유전자가, 우식치아군인 실험군에서는 377개의 유전자가 1.6배이상 발현되었다. 2. 역전사효소 중합효소 연쇄반응 분석에서 14개의 유전자를 선택하였고 cDNA 미세배열 분석결과와 동일한 결과를 확인하였다. 3. TGF-${\beta}1$의 면역조직화학적 관찰 결과, 건전치에 비해 우식치의 상아모세포와 치수에서 특히 강하게 발현되었다.

Keywords

References

  1. Abiko Y : Passive immunization against dental caries and periodontal disease: development of recombinant and human monoclonal antibodies. Crit Rev Oral Biol Med, 11:140-158, 2000. https://doi.org/10.1177/10454411000110020101
  2. Smith AJ, Murray PE, Lumley PJ : Preserving the vital pulp in operative dentistry 1. A biological approach. Dent Update, 29:64-69, 2002.
  3. Smith AJ, Murray PE, Lumley PJ : Preserving the vital pulp in operative dentistry: 4. Factors influencing successful pulp capping. Dent Update, 29:225- 230, 2002.
  4. Smith AJ : Pulpal responses to caries and dental repair. Caries Res, 36:223-232, 2002. https://doi.org/10.1159/000063930
  5. Lockhart DJ, Winzele REA : Genomics, gene expression and DNA arrays. Nature, 405:827-836, 2000. https://doi.org/10.1038/35015701
  6. Brown PO, Botstein D : Exploring the new world of the genome with DNA microarrays. Nat Genet, 21:33-37, 1999. https://doi.org/10.1038/4462
  7. Debouck C, Goodfellow PN : DNA microarrays in drug discovery and development. Nat Genet, 21:48- 50; 1999. https://doi.org/10.1038/4475
  8. Paakkonen V, Ohlmeier S, Bergmann U, et al. : Analysis of gene and protein expression in healthy and carious tooth pulp with cDNA microarray and two-dimensional gel electrophoresis. Eur J Oral Sci, 113:369-379, 2005. https://doi.org/10.1111/j.1600-0722.2005.00237.x
  9. McLachlan JL, Smith AJ, Bujalska IJ, et al. : Gene expression profiling of pulpal tissue reveals the molecular complexity of dental caries. Biochim Biophys Acta. 25:1741:271-281, 2005.
  10. Lesot H, Begue-Kirn C, Kubler MD, et al. : Experimental induction of odontoblast differentiation and stimulation during reparative processes. Cell Mater, 3:211-217, 1993.
  11. Lendahl U, Zimmerman LB, McKay RDG : CNS stem cells express a new class of intermediate filament protein. Cell, 60: 585-595, 1990. https://doi.org/10.1016/0092-8674(90)90662-X
  12. About I, Maquin D, Lendahl U, et al. : Nestin expression in embryonic and adult human teeth under normal and pathological conditions. Am J Pathol, 157:287-295, 2000. https://doi.org/10.1016/S0002-9440(10)64539-7
  13. Imad A, Dominique LM, Urban L, et al. : Nestin Expression in Embryonic and Adult Human Teeth under Normal and Pathological Conditions. American J Patho, 157:1, 2000. https://doi.org/10.1016/S0002-9440(10)64508-7
  14. Tziafas D, Smith AJ, Lesot H : Designing new treatment strategies in vital pulp therapy. Dent, 28:77-92, 2000. https://doi.org/10.1016/S0300-5712(99)00047-0
  15. Bjorndal L, Darvann T : A light microscopic study of odontoblastic and non-odontoblastic cells involved in tertiary dentinogenesis in welldefined cavitated carious lesions. Caries Res, 33:50-60, 1999. https://doi.org/10.1159/000016495
  16. Melin M, Joffre-Romeas A, Farges JC, et al. : Effects of TGF-$\beta{1}$ on dental pulp cells in cultured human tooth slices. J Dent Res, 79:1689-1696, 2000. https://doi.org/10.1177/00220345000790090901
  17. Sloan AJ, Smith AJ : Stimulation of the dentinepulp complex of rat incisor teeth by transforming growth factorbeta isoforms 1-3 in vitro. Arch Oral Biol, 44:149-156, 1999. https://doi.org/10.1016/S0003-9969(98)00106-X
  18. McLachlan JL, Smith AJ, Sloan AJ : Cooper PR.Gene expression analysis in cells of the dentinepulp complex in ealthy and carious teeth. Arch Oral Biol, 48:273-283, 2003. https://doi.org/10.1016/S0003-9969(03)00003-7
  19. Peters K, Werner S, Chen G, et al. : Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development, 114:233-243, 1992.
  20. Nakajima A, Shimizu S, Moriya H, et al. : Expression of fibroblast growth factor receptor-3 (FGFR3), signal transducer and activator of transcription- 1, and cyclin-dependent kinase inhibitor p21 during endochondral ossification: differential role of FGFR3 in skeletal development and fracture repair. Endocrinology, 144:4659-4668, 2003. https://doi.org/10.1210/en.2003-0158
  21. Mitsiadis TA, Henrique D, Thesleff I, et al. : Mouse Serrate-1(Jagged-1): Expression in the developing tooth is regulated by epithelial-mesenchymal interactions and fibroblast growth factor-4. Development, 124:1473-1483, 1997.
  22. Mitsiadis TA, Romeas A, Lendahl U, et al. : Notch2 protein distribution in human teeth under normal and pathological conditions. Exp Cell Res, 282:101- 109, 2003. https://doi.org/10.1016/S0014-4827(02)00012-5
  23. Baume, LJ : The biology of pulp and dentine. A historic, terminologic-taxonomic, histologic-biochemical, embryonic and clinical survey. Monogr Oral Sci, 8:210-220, 1980.
  24. Schroder, U : Effects of calcium hydroxide-containing pulp-capping agents on pulp cell migration, proliferation and differentiation. J Dent Res, 64:541- 548, 1985.
  25. Begue-Kirn C, Smith AJ, Loriot M, et al. : Comparative analysis of TGFbs, BMPs, IGFs, Msxs, fibronectin, osteonectin and bone sialoprotein gene expressions during normal and in vitro induced odontoblast differentiation. Int J Dev Biol, 38:405- 420, 1994.
  26. Nakashima K, Zhou X, Kunkel G, et al. : The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell, 108:17-29, 2002.
  27. Zhang X, Schwarz EM, Young DA, et al. : Cyclooxygenase -2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest, 109:1405-1415, 2002. https://doi.org/10.1172/JCI15681
  28. Camarda AJ, Butler WT, Finkelman RD, et al. : Immuno-cytochemical localization of gamma-carboxyglutamic acid-containing proteins (osteocalcin) in rat bone and dentin. Calcified Tissue International 40:349-355, 1987. https://doi.org/10.1007/BF02556698
  29. Owen TA, Bortell R, Yocum SA, et al. : Coordinate occupancy of AP-1 sites in the vitamin D responsive and CCAAT box elements by Fos-Jun in the osteocalcin gene: model for phenotype suppression of transcription. Proc Nat Acad Sci USA, 87:9990-9994, 1990. https://doi.org/10.1073/pnas.87.24.9990
  30. Hirata M, Yamaza T, Mei YF, et al. : Expression of osteocalcin and Jun D in the early period during reactionary dentin formation after tooth preparation in rat molars. Cell tissue Res 319:455-465, 2005. https://doi.org/10.1007/s00441-004-1035-y
  31. Yoshiba N, Yoshiba K, Nakamura H, et al. : Immunohistochemical localization of HLA-DR-positive cells in unerupted and erupted normal and carious human teeth. J Dent Res, 75:1585-1589, 1996. https://doi.org/10.1177/00220345960750081001
  32. Kamal AMM, Okiji T, Kawashima N, et al : Defense responses of dentin/pulp complex to experimentallyinduced caries in rat molars: An immunohistochemical study on kinetics of pulpal 1a antigen -expressing cells and macrophages. J Endod, 23:115-120, 1997. https://doi.org/10.1016/S0099-2399(97)80257-9
  33. Ohshima H, Sato O, Kawahara I, et al. : Responses of immunocompetent cells to cavity preparation in rat molars: an immunohistochemical study using OX6-monoclonal antibody. Connect Tissue Res, 32:303-311, 1995. https://doi.org/10.3109/03008209509013738
  34. Bergenholtz G, Nagaoka S, Jontell M : Class II antigen expressing cells in experimentally induced pulpitis. Int Endod J, 24:8, 1991.
  35. Ohshima H, Sato O, Kawahara, et al. : Responses of immunocompetent cells to cavity preparation in rat molars: an immunohistochemical study using OX6-monoclonal antibody. Connect Tissue Res, 32:303-311, 1995. https://doi.org/10.3109/03008209509013738
  36. Dinarello, CA : Biologic basis for interleukin-1 in disease. Blood, 87:2095-2147, 1996.
  37. Thomas H, Klaus P : The intriguing biology of the tumour necrosis factor/tumour necrosis factor receptor superfamily: players, rules and the games. Immunol, 115:1-20. 2005. https://doi.org/10.1111/j.1365-2567.2005.02143.x
  38. Nathan C : Points of control in inflammation. Nature, 420: 846-852, 2002 https://doi.org/10.1038/nature01320
  39. McLachlan JL, Sloan AJ, Smith AJ, et al : S100 and cytokine expression in caries Infect Immun, 72:4102-4108, Jul 2004. https://doi.org/10.1128/IAI.72.7.4102-4108.2004
  40. DeLarco JE, Todaro GJ : Growth factors from murine sarcoma virus transformed cells. Proc natn Acad Sci USA, 75:4001-4005,1978. https://doi.org/10.1073/pnas.75.8.4001
  41. Partanen AM, Ekblom P, Thesleff I : Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth. Dev Biol, 111:84-94, Sep 1985. https://doi.org/10.1016/0012-1606(85)90437-3
  42. Twardzik DR : Differential expression of transforming growth factor-alpha during prenatal development of the mouse. Cancer Res, 45:5413-5416, 1985.
  43. Wilcox JN, Derynck R : Developmental expression of transforming growth factors alpha and beta in mouse fetus. Molec Cell Biol, 8: 3415-3422, 1988.
  44. Kronmiller JE, Upholt WB, Kollare J : Effects of retinol on the temporal expression of transforming growth factor-a mRNA in the embryonic mouse mandible. Archs Oral Biol, 38:185-188, 1993. https://doi.org/10.1016/0003-9969(93)90205-Z
  45. Wang Y, Kowalski PE, Thalmann I, et al. : Otoconin- 90, the mammalian otoconial matrix protein, contains two domains of homology to secretory phospholipase A2. Proc Natl Acad, 95:15345-15350, 1998. https://doi.org/10.1073/pnas.95.26.15345
  46. Diaz BL, Arm JP : Phospholipase A2, Prostaglandins, Leukot. Essent Fat Acids, 69:87-97, 2003. https://doi.org/10.1016/S0952-3278(03)00069-3
  47. Kujubu DA, Fletcher BS, Varnum BC, Lim RW, et al. : TIS10, a phorbol ester tumor promoterinducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem, 266:12866-12872, 1991.
  48. Sancho D, Gomez M, Viedma F, Esplugues E, Gordon-Alonso M, Garcia-Lopez MA, de la Fuente H, Martinez-A C, Lauzurica P, Sanchez-Madrid F.CD69 downregulates autoimmune reactivity through active transforming growth factor-beta production in collagen-induced arthritis. J Clin Invest, 112:872-882, 2003. https://doi.org/10.1172/JCI19112
  49. Buchaille ML, Couble H, Magloire, et al. : Expression of the Small Leucine-Rich Proteoglycan Osteoadherin/ Osteomodulin in Human Dental Pulp and Developing Rat Teeth R. Bone, 27:265-270, 2000. https://doi.org/10.1016/S8756-3282(00)00310-0
  50. Oldberg A, Franze NA, Heinegard D : The primary structure of a cell-binding bone sialoprotein. J Biol Chem, 263:19430-19432, 1988.
  51. ChenJ, Sasaguri K, Sodek J, et al. : Enamel epithelium expresses bone sialoprotein (BSP). European Journal of Oral Sciences, 106: 331-336, 1998.
  52. Garcia1 JMQ, Martins MD, Jaeger RG, et al. : immunolocalization of bone extracellular matrix proteins( type I collagen, osteonectin and bone sialoprotein) in human dental pulp and cultured pulp cells. International Endodontic Journal, 36:404-410, 2003. https://doi.org/10.1046/j.1365-2591.2003.00669.x
  53. Mark TJ, Saleh M, Susannah LH : Inactivation of the Murine Pyruvate Dehydrogenase (Pdha1) Gene and Its Effect on Early Embryonic Development. Mol Gen and Met, 74:293-302, 2001. https://doi.org/10.1006/mgme.2001.3249
  54. O'Regan AW, Nau GJ, Chupp GL, et al : Osteopontin (Eta-1) in cell-mediated immunity: teaching an old dog new tricks. Immunol Today, 21:475-478, 2000. https://doi.org/10.1016/S0167-5699(00)01715-1