Construction of eukaryotic expression plasmid human transforming growth factorβ3 and its transfection into precartilaginous stem cells

摘要

＜正＞Objective:To obtain seed cells for cartilage repair through constructing recombinant human transforming growth factorβ3 vector（hTGF-β3）and transfecting it into rat’s precartilaginous stem cells（PSCs）. Methods:Gene engineering technique was introduced to construct eukaryotic expression plasmid pcDNA3.1 （+）-hTGF-β3.PSCs of rats were isolated and purified with method of immunomagnetic microbeads.Then PSCs were cotransfected with plasmid hTGF-β3 and pcDNA3.1 （+）-enhanced green fluorescence protein（EGFP）by liner polyethyleneimine（PEI）.And 48 hours later the transient expression of EGFP was observed under a fluorescence microscope,and the expression of hTGF-β3 was detected with reverse transcription-polymerase chain reaction（RT- PCR）and enzyme linked immunosorbent assay（ELISA）. Results:The sequences of the recombinants were consistent with that from Genebank.Cotransfection of EGFP provided fast visual confirmation of successful transduction.The hTGF-β3 mRNA and protein expression could be detected by RT-PCR and ELISA. Conclusions:The recombinant plasmid is correctly constructed and successfully transfected into rat’s PSCs, which is an important step to treat epiphyseal injury or other osteo-cartilage diseases with transgenic therapy.