Establishment of Down's syndrome periodontal ligament cells by transfection with SV40T-Ag and hTERT

Abstract

Down's syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down's syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down's syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (< 40), while STPDL and STPDLDS cells continued to grow with more than 80 population doublings. Primary cultured periodontal ligament cells obtained from the patient showed a chromosome pattern characteristic of Down's syndrome with trisomy 21, whereas STPDLDS samples showed a large number of abnormal chromosomes in those results. Gene expression analysis revealed that expression of DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down's syndrome patients.

Keywords: DSCR-1; Down’s syndrome; Periodontal ligament cells; SV40T-Ag; hTERT.

Fig. 1

A Representative images of pPDL (a), STPDL (b) and STPDLDS (c) obtained with an optical microscope. Immunohistochemical staining for SV40T-Ag was positive in STPDL (e) and STPDLDS (f), but not pPDL (d). B Levels of hTERT protein expression determined by ELISA. Each experiment was performed at least three times and values are shown as the mean ± SD. **P < 0.01, Students, t test

Fig. 2

Population doubling (PD) of (●) pPDL, (◇) STPDL, and (×) STPDLDS. The x axis indicates incubation days and the y axis PD number

Fig. 3

Expressions of RUNX2, Osterix, ALP, OPN, OCN, Periostin, EGFR, ColXII, α-SMA, and GAPDH in pPDL, STPDL and STPDLDS, as shown by semi-quantitative reverse transcription PCR analysis results

Fig. 4

Expression of DSCR1 in STPDLDS was higher than that in STPDL. Total cellular RNA was extracted and mRNA levels of DSCR1 and GAPDH were examined using quantitative real time PCR analysis. Results are shown as the mean ± SD of 3 samples. *P < 0.05 student’s t test