Journal of Periodontal and Implant Science

Korean Academy of Periodontology (대한치주과학회)
권호 표지

Cellular study of replicative senescence in human periodontal ligament fibroblast using molecular biology

분자생물학을 이용하여 복제노화된 사람치주인대섬유모세포의 세포학적 연구

  • Kim, Byung-Ock (Dept. of Periodontology, College of Dentistry, Chosun University, Oral Biology Research Institute, Chosun University) ;
  • Cho, Il-Jun (Dept. of Periodontology, College of Dentistry, Chosun University) ;
  • Park, Joo-Cheol (Dept. of Oral Histology, College of Dentistry, Chosun University, Oral Biology Research Institute, Chosun University) ;
  • Kook, Joong-Ki (Dept. of Oral Biochemistry, College of Dentistry, Chosun University, Oral Biology Research Institute, Chosun University) ;
  • Kim, Heung-Joong (Dept. of Oral Anatomy, College of Dentistry, Chosun University, Oral Biology Research Institute, Chosun University) ;
  • Jang, Hyun-Seon (Dept. of Periodontology, College of Dentistry, Chosun University, Oral Biology Research Institute, Chosun University)
  • 김병옥 (조선대학교 치과대학 치주과학 교실, 조선대학교 치과대학 구강생물학연구소) ;
  • 조일준 (조선대학교 치과대학 치주과학 교실) ;
  • 박주철 (조선대학교 치과대학 구강조직학 교실, 조선대학교 치과대학 구강생물학연구소) ;
  • 국중기 (조선대학교 치과대학 구강생화학 교실, 조선대학교 치과대학 구강생물학연구소) ;
  • 김홍중 (조선대학교 치과대학 구강해부학 교실, 조선대학교 치과대학 구강생물학연구소) ;
  • 장현선 (조선대학교 치과대학 치주과학 교실, 조선대학교 치과대학 구강생물학연구소)
  • Published : 2005.09.30
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Abstract

Human periodontal ligament fibroblast(hPDLF) is very important to cure periodontal tissue because it can be diverged into various cells. This study examined the expression of MMP-1, TIMP-1, periodontal ligament specific PDLs22, Type I collagen, Fibronectin, TIMP-2, telomerase mRNA in a replicative senescence of hPDLF. The periodontal ligament tissue was obtained from periodontally healthy and non-carious human teeth extracted for orthodontic reasons at the Chosun University Hospital of Dentistry with the donors' informed consent. The hPDLF cells were cultured in a medium containing Dulbecco's modified Eagle medium(DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, Gibco BRL, USA) at 37C in humidified air with 5% $CO_2$. For the reverse transcription-polymerase chain reaction(RT-PCR) analysis, the total RNA of the 2, 4, 8, 16, 18, and 21 passage cells was extracted using a Trizol Reagent(Invitrogen, USA) in replicative hPDL cells. Two passage cells, i.e. young cells, served as the control, and ${\beta}-actin$ served as the internal control for RT-PCR The results of this study about cell morphology and gene expression according to aging of hPDLF using RT-PCR method are as follows: 1. The size of hPDLF was increased with aging and it was showed that the hPDLF was dying in the final passage. 2. PDLs22 mRNA was expressed in young hPDLF of the two, four, and six passage. 3. TIMP-1 mRNA was expressed in young hPDLF of the two and four passage. 4. There was a tendency that MMP-1 mRNA was weakly expressed over eighteen. 5. Type 1 collagen mRNA was expressed in almost all passages, but it was not expressed in the final passage. 6. Fibronectin mRNA was observed in all passages and it was weakly expressed in the final passage. 7. TIMP-2 and telomerase mRNA were not expressed in this study. Based on above results, it was observed that PDLs22, Type 1 collagen, Fibronectin, MMP-1. and TIMP-1 mRNA in hPDLF were expressed differently with aging. The study using the hPDLF that is collected from healthy patients and periodontitis patients needs in further study.

Keywords

References

  1. Polson AM, Caton J. Factors influencing periodontal repair and regeneration. J Periodontol 1982:53(10):617-625 https://doi.org/10.1902/jop.1982.53.10.617
  2. Kawanami M. Sugaya T, Gama H. et al. Periodontal healing after replantation of intentially rotated teeth with healthy and denuded root surfaces. Dental Traumatology 2001; 17(3): 127-133 https://doi.org/10.1034/j.1600-9657.2001.017003127.x
  3. Shimono M, Ishikawa T, Ishikawa H. et al. Regulatory mechanisms of periodontal regeneration. Microsc Res Tech 2003;60(5):491-502 https://doi.org/10.1002/jemt.10290
  4. Silva TA, Rosa AL, Lara VS. Dentin matrix proteins and soluble factors: intrinsic regulatory signals for healing and resoption of dental and periodontal tissues. Oral Diseases 2004; 10(2) :63-74 https://doi.org/10.1111/j.1601-0825.2004.00992.x
  5. Ohira T, Myokai F, Shiomi N, et al. Identification of genes diffentially regulated in rat alveolar bone wound healing by subtractive hybridization. J Dent Res 2004:83(7) :546-51 https://doi.org/10.1177/154405910408300707
  6. Sawa Y, Phillips A, Hollard J, et al. The in vitro life-span of human periodontal ligament fibroblasts. Tissue & Cell 2000; 32(2): 163-170 https://doi.org/10.1054/tice.2000.0100
  7. Nishimura F, Terranova VP, Braithwaite N, et al. Comparison of in vitro proliferative capacity of human periodontal ligament cells in juvenile and aged donors. Oral diseases 1997:3: 162-166 https://doi.org/10.1111/j.1601-0825.1997.tb00029.x
  8. West MD, Pereira-Smith OM, Smith JR. Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity. Exp Cell Res 1989: 184 (1): 138-147 https://doi.org/10.1016/0014-4827(89)90372-8
  9. Maruya Y, Sasano Y, Takahashi I, kagayama M, Mayanagi H. Expression of extracellular matrix molecules, MMPs and TIMPs in alveolar bone, cementum and periodontal ligaments during rat tooth eruption. J Electron Microscopy 2003:52(6) :593-604 https://doi.org/10.1093/jmicro/52.6.593
  10. Martin M, el Nabout R. Lafuma C, Crechet F, Remy J. Fibronectin and collagen gene expression during in vitro ageing of pig skin fibroblasts. Exp Cell Res 1990: 191 (1) : 8-13 https://doi.org/10.1016/0014-4827(90)90028-9
  11. Chung JH. Seo JY, Choi HR. et al. Modulation of skin collagen metabolism in aged and photo aged human skin in vivo. J Invest Dermatol 2001: 117 (5) : 1218-1224 https://doi.org/10.1046/j.0022-202x.2001.01544.x
  12. Park JC. Kim HJ. Jang HS. et al.. Isolation and characterization of cultured human periodontal ligament fibroblast-specific cDNAs. Biochem Biophys Res Commun 2001:282(5):1145-1153 https://doi.org/10.1006/bbrc.2001.4694
  13. 박영채, 양대승, 김재호, 등. Effects of Replicative Senescence on the Cell Cycle Regulation in Human Gingival Fibroblasts. 대한치주과학회지, 2001: 31 (1) : 135-148
  14. Hayflick L. and Moorhead PS. The serial cultivation of human diploid strains. Exp Cell Res 1961: 25: 585-621 https://doi.org/10.1016/0014-4827(61)90192-6
  15. Kang MK. Bibb C. Baluda MA. Rey O. Park NH. In vitro replication and differentiation of normal human oral keratinocytes. Exp Cell Res 2000; 258: 288-297 https://doi.org/10.1006/excr.2000.4943
  16. Dimri GP. Lee S. Basile G. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA. 1995; 92: 9363-9367 https://doi.org/10.1073/pnas.92.20.9363
  17. Cook H, Davies KJ, Harding KG, Thomas DW. Defective extracellular matrix reorganization by chronic wound fibroblasts is associated with alterations in TIMP-1. TIMP-2. and MMP-2 activity. J Invest Dermatol 2000; 115 (2): 225-233 https://doi.org/10.1046/j.1523-1747.2000.00044.x
  18. Hellio Le Graverand MP. Reno C. Hart DA. Gene expression in menisci from the knees of skeletally immature and mature female rabbits. J Orthop Res 1999;17(5):738-744 https://doi.org/10.1002/jor.1100170518
  19. Khorramizadeh MR, Tredget EE. Telasky C. Shen Q. Ghahary A. Aging differentially modulates the expression of collagen and collagenase in dermal fibroblasts. Mol Cell Biochem 1999; 194 (1-2) :99-108 https://doi.org/10.1023/A:1006909021352
  20. Paz MA, Gallop PM. Collagen synthesized and modified by aging fibroblasts in culture. In Vitro 1975; 11(5) ;302-312 https://doi.org/10.1007/BF02615641
  21. Abiko Y, Shimizu N, Yamaguchi M. Suzuki H, Takiguchi H, Effect of aging on functional changes of periodontal tissue cells. Ann Periodontol 1998;3(1): 350-369 https://doi.org/10.1902/annals.1998.3.1.350
  22. Caputi M, Baralle FE, Melo CA. Analysis of the linkage between fibronectin alternative spliced sites during ageing in rat tissues. Biochem Biophys Acta 1995;1263(1):53-59 https://doi.org/10.1016/0167-4781(95)00067-Q
  23. Rubio MA, Kim SH, Campisi J. Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells. J Biol Chem 2002; 277 (32) : 28609-18617 https://doi.org/10.1074/jbc.M203747200
  24. Shelton DN, Chang E, Whittier PS, Choi D, Funk WD. Microarray analysis of replicative senescence. Current biology 1999:9:939-945 https://doi.org/10.1016/S0960-9822(99)80420-5
  25. Coletta RD, Almeida OP, Reynolds MA, Sauk JJ. Alteration in expression of MMP-1 and MMP-2 but not TIMP-1 and TIMP-2 in hereditary gingival fibromatosis is mediated by TGF-$\beta$1 autocrine stimulation. J Periodontal Res 1999 ;34:457-463 https://doi.org/10.1111/j.1600-0765.1999.tb02281.x