Cloning, expression and functional analyses of human platelet-derived growth factor-B chain peptide for wound repair of cat corneal endothelial cells

• Published in: Chinese journal of traumatology 2009-02, Vol.12 (1), p.31-37
• Main Authors: LUO, Wen-juan, ZHAO, Gui-qiu, WANG, Chuan-fu, WANG, Li-mei, WANG, Xiao-ji
• Format: Article
• Language: English
• Subjects: Animals; Cats; Cell Proliferation - drug effects; Cells, Cultured; Cloning, Molecular; Corneal endothelium; Endothelium, Corneal - cytology; Endothelium, Corneal - drug effects; Gene expression; Humans; Immunohistochemistry; Phosphopyruvate Hydratase - analysis; Platelet derived growth factor; Proliferation; Protein Folding; Proto-Oncogene Proteins c-sis - chemistry; Proto-Oncogene Proteins c-sis - genetics; Proto-Oncogene Proteins c-sis - pharmacology; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - pharmacology; Wound Healing - drug effects
• ISSN: 1008-1275
• DOI: 10.3760/cma.j.issn.1008-1275.2009.01.006

Objective: To investigate the biological function of platelet-derived growth factor B (PDGF-B) on the survival and proliferation of cat corneal endothelial cells so as to provide bases for further studies of its role in wound repair and its clinical application.Methods: Total RNA was extracted from the placenta tissues of healthy pregnant women undergoing hysterotokotomy and PDGF eDNA was obtained with re-verse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-PDGF-B was constructed and expressed the recombinant PDGF-B in Escherichia coli (E.coli) BL21 (DE3). After purification and refolding on Ni2+-chelation affinity chromatography (NTA) column, it was used to culture cat corneal endothelial cells. Cell proliferation was tested by modified tertrazolium salt (MTT) and flow cytometer. And the morphologic change and the ultrastructure were ob-served under an inverted phase contrast microscope, a scan-ning electron microscope and a transmission electon microscope, respectively.Results: PDGF-B chain peptide (PDGF-BB) gene was successfully inserted into the prokaryotic expression vector, pET-28a(+). The purified recombined protein pET-PDGF-B showed a single band on sodium dodecyl sulfate polyacry-lamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the de-duced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal en-dothelial cells.Conclusions: The construction of recombinant prokary-otic expression vector pET-PDGF-B and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB protein. The expressed PDGF-BB promotes the prolif-eration of cultured cat corneal endothelial cells.