[Construction and identification of recombinant human platelet-derived growth factor-B adenoviral vector and transfection into periodontal ligament stem cells].
OBJECTIVE
To construct a recombinant human platelet-derived growth factor-B (PDGF-B) adenoviral vector and to transfect it into human periodontal ligament stem cells (PDLSC).


METHODS
The recombinant plasmid pAd-PDGF-B was constructed by homologous recombination and confirmed by restriction endonucleases digestion. Recombinant adenovirus was packaged in HEK293 cells. PDLSC were transfected with recombinant adenovirus and PDGF-B expression was confirmed. Expression of collagen type I gene was determined by quantitative analysis of the products of RT-PCR. The cell proliferation was determined with MTT colorimetric assay.


RESULTS
The recombinant plasmid pAd-PDGF-B was confirmed by restriction endonucleases digestion. EGFP expression was observed on the third day after transfecting, and the expression of PDGF-B was detected. Immunohistochemical methods revealed that PDGF-B was expressed in PDLSC. Levels of expression of collagen type I gene were increased significantly by transfer of the exogenous PDGF-B gene to PDLSC. At the same time, findings indicated that Ad-PDGF-B stimulated PDLSC proliferation. MTT assay indicated the absorbance of PDLSC by stimulating with Ad-PDGF-B was (0.68 +/- 0.02), P < 0.01.


CONCLUSIONS
Using the AdEasy system, the human PDGF-B recombinant adenovirus can be rapidly obtained. These results indicate that recombinant adenoviruses encoding PDGF-B transgenes could modulate proliferative activity of PDLSC, enhance the high expression of collagen type I and lay the foundation for periodontal tissue regeneration and dental implant gene therapy.
Shang
Shu-huan
SH
Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School of Stomatology, Wuhan University, Wuhan 430079, China.
Zhang
Yu-feng
YF
Shi
Bin
B
Cheng
Xiang-rong
XR
D000256:Adenoviridae / Q000235:genetics; D002478:Cells, Cultured; D005822:Genetic Vectors; D006801:Humans; D010513:Periodontal Ligament / Q000166:cytology*; D020574:Proto-Oncogene Proteins c-sis / Q000235:genetics*; D013234:Stem Cells; D014162:Transfection
D020574:Proto-Oncogene Proteins c-sis