Cloning, expression and functional analyses of human platelet-derived growth factor-B chain peptide for wound repair of cat corneal endothelial cells.
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 cDNA was obtained with reverse 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 observed under an inverted phase contrast microscope, a scanning 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 polyacrylamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the deduced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal endothelial cells.


CONCLUSIONS
The construction of recombinant prokaryotic 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 proliferation of cultured cat corneal endothelial cells.
Luo
Wen-Juan
WJ
Department of Ophthalmology, Affiliated Hospital, Medical College, Qingdao University, Qingdao 266003, China.
Zhao
Gui-Qiu
GQ
Wang
Chuan-Fu
CF
Wang
Li-Mei
LM
Wang
Xiao-Ji
XJ
D000818:Animals; D002415:Cats; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D007150:Immunohistochemistry; D010751:Phosphopyruvate Hydratase / Q000032:analysis; D017510:Protein Folding; D020574:Proto-Oncogene Proteins c-sis / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D014945:Wound Healing / Q000187:drug effects*
D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D010751:Phosphopyruvate Hydratase