Effect of human bone morphogenetic protein-7 gene transfection on proliferation of rabbit nucleus pulposus cells and synthesis of extracellular matrix / 中国组织工程研究

ABSTRACT

BACKGROUND: Previous studies demonstrated that recombinant human bone morphogenetic pretein-7 (hBMP-7) can effectively promote the synthesis of extracellular matrix, repair of damaged disc matrix, restore of degenerative disc height. It is hoped that BMP-7 can be used to control and reverse the intervertebral disc degeneration. However, because of the short half-life of recombinant protein and low biological activity, it is difficult to maintain BMP-7 high concentrations on degenerative disc. Gene therapy can prevent these defects effectively.OBJECTIVE: To study the effect of hBMP-7 gene transfeotion on biological activity of primary cultured rabbit nucleus pulposus cells in vitro, to determine the feasibility of hBMP-7 gene which will on gene therapy research of intervertebral disc degeneration,and to provide basis for further study.DESIGN, TIME AND SETTING: A randomized and controlled observation was performed at the Institute of Cardiothoracic Surgery of Changhai Hospital from December 2005 to September 2006.MATERIALS: Six healthy New Zealand white rabbits of either gender, averaging 4 weeks old and weighing 500 g, were used in this study, and Ad-hBMP7 was constructed by the Cardiothoracic Surgery Institute of Changhai Hospital.METHODS: After rabbits sacrifice under aseptic condition, the nucleus pulposus was got. After digested with Pronase, type Ⅱcollagenase and type Ⅱ DNAase (4 hours, 37 "(2), the cells were seeded in the Petri dishes and were kept in the incubator. After 7 days and then twice a week, the media were refreshed. The nucleus pulposus cells were infected by adenovirus integrated with hBMP7 gene. The cells which were transfected by adenovirus vector for Lac-Z gene and which were not transfected were adopted as control.MAIN OUTCOME MEASURES: The expression of hBMP-7 was determined by RTopCR and Western blot. The effect of hBMP-7 on cell proliferation was surveyed by MTT. Furthermore, the effect of hBMP-7 gene on synthesis of proteoglycan and type Ⅱcollagen was detected by modified dimethylmethylene blue method and ELISA, respectively.RESULTS: Gene sequencing and PCR showed that hBMP-7 gene was inserted correctly and no mutation happened. The pdmary cultured rabbit nucleus pulposus cells were identical with those reported in literature. After Ad-hBMP7 transduct into the nucleus pulposus cells, high level of hBMP-7 expression was observed and lasted over 3 weeks. Also hBMP-7 gene can promote cell proliferation and synthesis of proteoglycan and type II collagen with significant difference compared with control groups (P < 0.05).CONCLUSION: hBMP-7 gene mediated by adenovirus can be the target gene for the further study on gene therapy of intervertebral disc degeneration.