CONSTRUCTING AAV-TGFβ_1 AND COMPARISING ITS BIOLOGICAL EFFECTS ON PROTEOGLYCAN SYNTHESIS OF NUCLEUS PULPOUS CELLS WITH AV-TGFβ_1

摘要

Objective To construct adeno-associated virus express system for TGFβ_1(AAV-TGFβ_1) andrncompare its biological effects on proteoglycan synthesis of the rabbit lumbar disc nucleus pulpous (NP) cells with adenovirus (Ad) express system for TGFβ_1 (AV-TGFβ_1). Methods TGFB, gene was obtained by polymerase chain reactions (PCR). The upstream of TGFβ_1 contained restriction enzyme site of EcoR I , and the restriction enzyme site of Sal I was at the downstream of TGFβ_1. Using the multiple cloning sites (MCS) in plasmid AAV and the corresponding contained restriction enzyme site in PCR product of TGFB,, TGFB, gene was subcloned into AAV. The recombinant plasmid AAV-TGFβ_1 was detected by restriction enzyme digestion and DNA sequencing. Then, AAV-TGFβ_1 virus was packaged and TGFβ_1 expression mediated by AAV was detected by immunofluence analysis in H293 cells. AAV transfection rate to NP cells was evaluated with AAV-PEGF. After NP cells were respectively trans-fected by AAV-TGFβ_1 virus and AV-TGFβ_1 virus, proteoglycan synthesis was detected and compared by using Anto-nopulos methods. Results DNA sequencing revealed that the PCR-amplified TGFβ_1 gene was consistent with NCBI Gene Bank. The recombinant plasmid was proved to be constructed successfully by restriction enzyme digestion. AAV could be transfected into NP cells and mediate an efficient expression of TGFβ_1 protein. AV-TGFβ_1 virus could quickly enhance the proteoglycan synthesis of the NP cells, but its biological effect was transient. AAV-TGFβ_1 virus could enhance stably proteoglycan synthesis. Conclusion AAV-TGFβ_1 virus was successful constructed and enhanced stably proteoglycan synthesis of NP cells.