Construction and identification of recombinant human platelet-derived growth factor-B adenoviral vector and transfection into periodontal ligament stem cells
10.3321/j.issn:1002-0098.2008.10.003
- VernacularTitle:新型重组人血小板衍化生长因子B腺病毒载体的构建与转染牙周干细胞的研究
- Author:
Shu-Huan SHANG
1
;
Yu-Feng ZHANG
;
Bin SHI
;
Xiang-Rong CHENG
Author Information
1. 武汉大学口腔医学院
- Keywords:
Proto-oncogene proteins c-sis;
Adenoviruses,human;
Transfection;
Periodontalligament stem cells
- From:
Chinese Journal of Stomatology
2008;43(10):584-588
- CountryChina
- Language:Chinese
-
Abstract:
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 MTr eolorimetric 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 adenovirns 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.
