Construction of eukaryotic expression plasmid of bFGF gene in rats and its expression in tenocytes.
The bFGF plays an important role in embryonic development of tendons and ligaments and in the healing of injuried tendons and ligaments. The eukaryotic expression plasmid of rat basic fibroblast growth factor (bFGF) gene was constructed in order to further investigate the bFGF function in molecular regulatory mechanism in the repair of tendons and ligaments and to provide the foundation for the clinical application. The cDNA fragments of bFGF were cloned from the skin of rats by RT-PCR, and recombinated to the pMD18-T vector. The cDNA encoding bFGF was cloned from the pMD18-T vector by RT-PCR, digested with restriction enzyme EcoR I Pst I and bound to eukaryotic expression plasmid pIRES2-EGFP to construct eukaryotic expression plasmid pIRES2-EGFP-bFGF. The pIRES2-EGFP-bFGF was transfected into the tenocytes by lipid-mediated ransfection technique. MTT test was used to detect the biological activity of bFGF in supernatants after the transfection. The expression of type I and III collagen genes was detected by using RT-PCR. It was verified that the pIRES2-EGFP-bFGF was successfully constructed, and its transfection into tenocytes could significantly enhance the biological activity of bFGF, and increase the expression of type I and III collagen mRNA, suggesting that pIRES2-EGFP-mediated bFGF gene therapy was beneficial to the repair of tendons and ligaments.
Feng
Yong
Y
Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. fy981225@sina.com
Zheng
Dong
D
Yang
Shuhua
S
Li
Jing
J
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024061:Collagen Type III / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005562:Formazans / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013710:Tendons / Q000166:cytology / Q000378:metabolism*; D013778:Tetrazolium Salts / Q000378:metabolism; D014162:Transfection
D024042:Collagen Type I; D024061:Collagen Type III; D018076:DNA, Complementary; D005562:Formazans; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; C485184:enhanced green fluorescent protein; D016222:Fibroblast Growth Factor 2; D049452:Green Fluorescent Proteins; C079782:MTT formazan