Experimental Study on BMSCs Transfected with Human Amelogenin Gene

Objective: This study was to construct the recombinant lentiviral vector of human amelogenin(Am) gene and infect dog's bone marrow stromal cells (BMSCs) with the recombinant lentivirus, in order to evaluate the feasibility of applying hAm gene-modified BMSCs as seeds for periodontal tissue engineering .

Methods: BMSCs were isolated from beagle dogs and expanded in vitro. The cDNA of hAm was linked into the vector plasmid. The recombinant plasmid FUAmW was identified by double endonuclease digestion and sequencing method. Recombinant lentivirus was prepared from 293T cells by polytheylenimine (PEI)-mediated transient cotransfection. The BMSCs and 293T Cells were infected by the generated lentivirus respectively. The infection efficiency was analysed by detection of green fluoresence protein (GFP) with fluorescent microscope and flow cytometer(FCM) in 72h. The expression of hAm gene was detected by reverse transcription polymerase chain reaction (RT-PCR).

Results: The sequence of the inserted fragment in recombinant plasmid was identical with the hAm sequence reported in Genebank. Green fluorescene was visible under fluorescent microscope, FCM assay showed that positive percentage was 67.38% and 31.75% in 293T and BMSCs respectively. hAm gene was expressed in infected groups by RT-PCR.

Conclusion: The recombinan lentiviral vector of hAm gene was constructed successfully and it could be transfected into cultured BMSCs. It may be used in the fields of periodontal tissue engineering as seeded cells .

Supported by National Natural Science Foundation of China. Grant No. 30672315)