Construction of eukaryotic expression plasmid human transforming growth factorβ3 and its transfectio

来源 :Chinese Journal of Traumatology | 被引量 : 0次 | 上传用户:xinmo2009
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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. 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 (RT-PCR) and enzyme linked immunosorbent assay (ELISA). sequences of the recombinants were consistent with that from Genebank. Small transfection 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.
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