Abstract:

AIM： To investigate the expression of human bone morphogenetic protein （hBMP）-2 gene transfected to rabbit adipose-derived.stem cell （ADSCs） by adenoviral vector and analyze the cells＇ osteoblastic differentiation potential. METHODS： This experiment was performed at the Central Laboratory of Medical College of Wuhan University and Laboratory of Department of Pathology, Medical College of Three Gorges University from December 2005 to September 2006. （1）Two 4-month old healthy New Zealand flap-eared rabbits were used and the plasmid vectors of pAd-BMP-2 and pAd-Lac Z were offered by Dr Oliver from Center of Molecule and Orthopaedics of Harvard medical school and Dr Li respectively. The E.coli DH5a and 293 cells were preserved by this lab. （2）After the rabbits were anesthetized, double inguina fat pad was excised and outer envelope, connective tissue and small vessels were excluded, sheared and centrifugated and the ADSCs were cultured in vitro. When constructed the pAd-hBMP-2 plasmid vector and recombinated the virus, the 3-passage ADSCs well grown were harvested, cultured in 60 mm culture dish at 5x105. Ad-hBMP-2 virus was transfected for 10-20 MOI. Two groups were the hBMP-2 transfected group and the Control group with Lac Z gene. The cells were cultured on complete osteoblastic medium. （3）The morphology and localized regions of mineralization of the cells were observed under light inverted microscope. Growth curve was drawn by MTT and proliferation timing was calculated. The expression of alkaline phosphatase （ALP）, hBMP-2, Collagen Ⅰ and Osteocalcin were measured by the cytochemisty, reverse transcription polymerase chain reaction （RT-PCR）, immunohistochemistry and Western blot, respectively. RESULTS： （1）The hBMP-2 transfected ADSCs became the polygon form and its growth curve double time showed shorter than the control group. （2）The cytochemisty of ALP of the hBMP-2 transfected group showed significant higher than the control group after transfected for 7 days, 10 days and 14 days（P 〈 0.01 ）. Localized regions of mineralization showed inaurate became more and large. （3）The expressions of hBMP-2, Collagen Ⅰ and Osteocalcin were significantly higher than the control group at days 7 and 14 after RT-PCR, Western blot and immunohistochemiscal method. CONCLUSION： The ADSCs transfected by the adenoviral vector rjnediated hBMP-2 gene have stronger potential to differentiate into osteoblasts. The hBMP-2 gene has showed high and long time expression on the cells.[著者文摘]