Expression of osteoblast-specific genes in immortalized rat dental follicle cells

doi:10.3969/j.issn.2095-4344.2015.01.023

Abstract

Abstract:

BACKGROUND: During in vitro culture, dental follicle cells are easy to loss self-renewal capacity and become aging, and they are also very difficult to be purified and amplified in quantity, which limits the application of dental follicle cells in periodontal tissue engineering.
OBJECTIVE: To study the osteogenic effect of immortalized rat dental follicle cells.
METHODS: pSSR69-pAmpho plasmids containing SV40T-Ag were used to transfect 293 cells. Rat dental follicle cells were transfected with virus supernatant and screened by hygromycin to establish immortalized rat dental follicle cells (experimental group). Untransfected cells served as controls. RT-PCR was used to detect the osteogenic related factors (alkaline phosphatase, osteocalcin, bone morphogenetic protein 2, and Runx2) in the experimental group and control group.
RESULTS AND CONCLUSION: The results showed no statistical differences between the experimental group and the control group in the expressions of alkaline phosphatase, bone morphogenetic protein 2 and Runx2 (P > 0.05). The expression of osteocalcin was significantly higher in the experimental group than the control group (P < 0.05). These findings suggest that in the late osteogenesis differentiation, immortalized rat dental follicle cells may promote the secretion of osteocalcin and then make osteoblasts early entry into the bone calcification stage.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: Dental Sac, Cells, Gene Expression

CLC Number:

R394.2

Zheng Hong. Expression of osteoblast-specific genes in immortalized rat dental follicle cells[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(1): 130-134.

Figures/Tables 2

References

[1] Hou LT, Liu CM, Chen YJ, et al.Characterization of dental follicle cells in developing mouse molar.Arch Oral Biol. 1999; 44(9):759-770.
[2] Owen TA, Aronow M, Shalhoub V,et al.Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix.J Cell Physiol. 1990;143(3):420-430.
[3] Handa K, Saito M, Tsunoda A,et al.Progenitor cells from dental follicle are able to form cementum matrix in vivo. Connect Tissue Res. 2002;43(2-3):406-408.
[4] Kémoun P, Laurencin-Dalicieux S, Rue J,et al.Localization of STRO-1, BMP-2/-3/-7, BMP receptors and phosphorylated Smad-1 during the formation of mouse periodontium.Tissue Cell. 2007;39(4):257-266.
[5] Zhao M, Xiao G, Berry JE,et al.Bone morphogenetic protein 2 induces dental follicle cells to differentiate toward a cementoblast/osteoblast phenotype.J Bone Miner Res. 2002; 17(8):1441-1451.
[6] Yao S, Norton J, Wise GE.Stability of cultured dental follicle cells.Cell Prolif. 2004;37(3):247-254.
[7] Avilion AA, Piatyszek MA, Gupta J, et al. Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues.Cancer Res. 1996;56(3):645-650.
[8] Ouyang H, Mou LJ, Luk C,et al.Immortal human pancreatic duct epithelial cell lines with near normal genotype and phenotype.Am J Pathol. 2000;157(5):1623-1631.
[9] Pollock K, Stroemer P, Patel S,et al.A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke.Exp Neurol. 2006;199(1): 143-155.
[10] Eldridge SR, Martens TW, Sattler CA,et al.Association of decreased intercellular communication with the immortal but not the tumorigenic phenotype in human mammary epithelial cells.Cancer Res. 1989;49(15):4326-4331.
[11] Kamata N, Fujimoto R, Tomonari M,et al.Immortalization of human dental papilla, dental pulp, periodontal ligament cells and gingival fibroblasts by telomerase reverse transcriptase.J Oral Pathol Med. 2004;33(7):417-423.
[12] 杨尊,刘婷,郑鸿,等.SV40Tag基因片段转染SD大鼠牙囊细胞后相关生物学特性初步研究[J].华西口腔医学杂志,2013,31(1): 4-7.
[13] Sharma GG, Gupta A, Wang H,et al.hTERT associates with human telomeres and enhances genomic stability and DNA repair.Oncogene. 2003;22(1):131-146.
[14] Bodnar AG, Ouellette M, Frolkis M,et al.Extension of life-span by introduction of telomerase into normal human cells. Science. 1998;279(5349):349-352.
[15] Kitagawa M, Tahara H, Kitagawa S,et al.Characterization of established cementoblast-like cell lines from human cementum-lining cells in vitro and in vivo.Bone. 2006;39(5): 1035-1042.
[16] Saito M, Handa K, Kiyono T,et al.Immortalization of cementoblast progenitor cells with Bmi-1 and TERT.J Bone Miner Res. 2005;20(1):50-57.
[17] Foddis R, De Rienzo A, Broccoli D,et al.SV40 infection induces telomerase activity in human mesothelial cells. Oncogene. 2002;21(9):1434-1442.
[18] Weinreb M, Shinar D, Rodan GA.Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization.J Bone Miner Res. 1990;5(8):831-842.
[19] Owen TA, Aronow M, Shalhoub V,et al.Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix.J Cell Physiol. 1990;143(3):420-430.
[20] Hauschka PV, Lian JB, Gallop PM.Direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue.Proc Natl Acad Sci U S A. 1975;72(10): 3925-3929.
[21] King GJ, Keeling SD, Wronski TJ.Histomorphometric study of alveolar bone turnover in orthodontic tooth movement.Bone. 1991;12(6):401-409.
[22] 关键,程宗生,王健平,等.成骨细胞中Runx2对机械离心力刺激的响应[J].华西口腔医学杂志,2010,28(1):38-40.
[23] Gersbach CA, Byers BA, Pavlath GK,et al.Runx2/Cbfa1 stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype.Exp Cell Res. 2004; 300(2):406-417.
[24] Boudreaux JM, Towler DA.Synergistic induction of osteocalcin gene expression: identification of a bipartite element conferring fibroblast growth factor 2 and cyclic AMP responsiveness in the rat osteocalcin promoter.J Biol Chem. 1996;271(13):7508-7515.
[25] Ducy P, Starbuck M, Priemel M,et al.A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.Genes Dev. 1999;13(8):1025-1036.
[26] Ducy P, Zhang R, Geoffroy V, et al.Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation.Cell. 1997;89(5):747-754.
[27] Jüttner KV, Perry MJ.High-dose estrogen-induced osteogenesis is decreased in aged RUNX2(+/-) mice.Bone. 2007;41(1):25-32.
[28] Morsczeck C.Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.Calcif Tissue Int. 2006;78(2):98-102.
[29] Morsczeck C, Schmalz G, Reichert TE,et al.Gene expression profiles of dental follicle cells before and after osteogenic differentiation in vitro.Clin Oral Investig. 2009;13(4):383-391.
[30] Pan K, Sun Q, Zhang J,et al.Multilineage differentiation of dental follicle cells and the roles of Runx2 over-expression in enhancing osteoblast/cementoblast-related gene expression in dental follicle cells.Cell Prolif. 2010;43(3):219-228.
[31] 郁卫东,秦书俭. 骨形态发生蛋白-2在骨形成过程中的作用机制[J].中国临床解剖学杂志,2000,18(1):82-83.
[32] Yamamoto N, Akiyama S, Katagiri T,et al.Smad1 and smad5 act downstream of intracellular signalings of BMP-2 that inhibits myogenic differentiation and induces osteoblast differentiation in C2C12 myoblasts.Biochem Biophys Res Commun. 1997;238(2):574-580.
[33] Noël D, Gazit D, Bouquet C,et al.Short-term BMP-2 expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells.Stem Cells. 2004; 22(1):74-85.
[34] Matsubara T, Kida K, Yamaguchi A,et al.BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation.J Biol Chem. 2008;283(43):29119-29125.
[35] Kémoun P, Laurencin-Dalicieux S, Rue J,et al.Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivatives (EMD) in vitro. Cell Tissue Res. 2007;329(2):283-294.