Constructing adeno-associated virus-TGFbeta3 and comparing its biological effect on proteoglycan synthesis in dedifferentiated nucleus pulpous cells with adenovirus-TGFbeta1.
OBJECTIVE
To construct adeno-associated virus (AAV) expression system for transforming growth factor beta3 (TGFbeta3 ) and detect its biological effect on proteoglycan synthesis of the earlier and later dedifferentiated rabbit lumbar disc nucleus pulpous (NP) cells, which was compared with that of adenovirus (AV) expression system for TGFbeta1.


METHODS
TGFbeta3 gene was obtained using PCR. Its upstream contained restriction enzyme site Kpn I, and its downstream contained restriction enzyme site Sal I. Using the restriction enzyme sites of PCR product of TGFbeta3 and the corresponding multiple cloning site (MCS) in plasmid AAV, TGFbeta3 was subcloned into AAV. The recombinant plasmid AAV-TGFbeta3 was transfected into H293 cells with Lipofectamine 2000, and the expression of TGFbeta3 gene was detected using immunofluorescent analysis. After AAV-TGFbeta3 virus particle with infectious activity was packaged, TGFbeta3 expression in NP cells was detected by immunoblotting, and its biological effect on proteoglycan synthesis was detected by antonopulos method and compared with that of AV-TGFbeta1 in the earlier and later dedifferentiated NP cells.


RESULTS
For the earlier dedifferentiated NP cells, AAV-TGFbeta3 slowly and stably enhanced proteoglycan synthesis, but AV-TGFbeta1 rapidly and transiently enhanced its synthesis. For the later dedifferentiated NP cells, AAV-TGFbeta3 stably enhanced proteoglycan synthesis, but AV-TGFbeta1 inhibited its synthesis.


CONCLUSIONS
AAV expression system can mediate TGFbeta3 gene to be expressed stably, and AAV-TGFbeta3 can enhance proteoglycan synthesis of the earlier and later dedifferentiated NP cells.
Sai
Jia-Ming
JM
Department of Orthopaedic Surgery, the Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003. sjm5689523@126.com
Hu
You-Gu
YG
Wang
De-Chun
DC
D000818:Animals; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology*; D010920:Placenta / Q000166:cytology*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011509:Proteoglycans / Q000096:biosynthesis*; D011817:Rabbits; D053773:Transforming Growth Factor beta1 / Q000235:genetics*; D053782:Transforming Growth Factor beta3 / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis
D004274:DNA, Recombinant; D011509:Proteoglycans; D053773:Transforming Growth Factor beta1; D053782:Transforming Growth Factor beta3; D014764:Viral Proteins