[Experimental study on human periodontal ligament cells transfected with human amelogenin gene].
OBJECTIVE
To construct the recombinant lentiviral vector of human amelogenin gene, infect human periodontal ligament cells with the recombinant lentivirus, and evaluate the feasibility of applying modified PDLCs as seeds for a further periodontal reconstruction.


METHODS
The mature peptide of hAm cDNA was cloned and linked into the vector plasmid, the recombinant plasmid FUAmW was confirmed by double enzyme digestion and sequence analysis. Recombinant lentivirus was prepared from 293T cells by polytheylenimine (PEI)-mediated transient cotransfection. The hPDLCs and 293T cells were infected with the generated lentivirus. The infection efficiency was analysed by detection of green fluorescence protein (GFP) with fluorescent microscope and flow cytometer 72 hours later. The expression of hAm gene was detected by reverse transcription polymerase chain reaction (RT-PCR).


RESULTS
The sequence of inserted fragment in recombinant plasmid was identical to the hAm sequence reported in Genebank. Green fluorescence was visible under fluorescent microscope, FCM assay showed that positive percentage was 69.46% and 33.99% in 293T and hPDLCs, respectively. The targeted gene was obtained in the experimental groups by RT-PCR.


CONCLUSIONS
The recombinan lentiviral vector of hAm gene is constructed successfully and it could be transfected into cultured hPDLCs. hAm gene and seed cells may be used for further study in the fields periodontal tissue engineering. Supported by National Natural Science Foundation of China (Grant No. 30672315).
Yu
Guang
G
Department of Periodontology, College of Stomatology, Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200011, China.
Shu
Rong
R
Sun
Ying
Y
Cheng
Lan
L
Song
Zhong-Chen
ZC
Zhang
Xiu-Li
XL
D053523:Amelogenin / Q000235:genetics*; D002460:Cell Line; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D016086:Lentivirus; D010513:Periodontal Ligament; D014162:Transfection
D053523:Amelogenin; D049452:Green Fluorescent Proteins