Objective: To obtain seed cells for cartilage repair through constructing recombinant human transforming growth factor β3 vector (hTGF-β3) and transfecting it into rat's precartilaginous stem cells (PSCs).Method...Objective: To obtain seed cells for cartilage repair through constructing recombinant human transforming growth factor β3 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 (RTPCR) 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.展开更多
基金the National Natural Science Foundation of China(No.30571873).
文摘Objective: To obtain seed cells for cartilage repair through constructing recombinant human transforming growth factor β3 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 (RTPCR) 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.
