Effects of adeno-associated virus-2 mediated human BMP-7 gene transfection on the phenotype of nucleus pulposus cells

English Keywords:Gene transfection  Bone morphogenetic protein-7  Recombinant adeno-associated virus type-2  Nucleus pulposus cell

English Abstract:

  【Abstract】 Objective：To investigate the effects of recombinant adeno-associated virus-2 expressing human bone morphogenetic protein-7（rAAV2-hBMP7） on the phenotype of canine nucleus pulposus（NP）cells.Method：The NP cells were derived from lumbar 4-5 intervertebral disc of 1 year old beagle dog.The 2nd passage NP cells were cultured and transfected by rAAV2-hBMP7 at multiplicities of infection of 1×105 genomes per cell（experiment group），while the control group cells by recombinant adeno-associated virus-2 expressing enhanced green fluorescent protein（rAAV2-EGFP）.NP cells were assessed semi-qualitatively for BMP7 expression by RT-PCR at 4，7 and 14 days after transfection.BMP7 protein expressed by NP cells were assayed by Western blot at 7 and 14 days post-transfection.Aggrecan，type Ⅰ and type Ⅱ collagen secreted by NP cells were qualitatively assayed at 4，7 and 14 days after transfection in the transfection and control groups by dimethylmethylene blue（DMMB） and ELISA method.Result：The NP cell transfected by rAAV2-hBMP7 expressed human BMP7 mRNA and BMP7 protein.Their expression level reached peak at the 7th days.However，these proteins could not be detected in the control group at any time points.In experiment group，the accumulation of proteoglycans mRNA and protein at 7 and 14 days after transfection were remarkably higher than that of 4 days after transfection.Moreover，the accumulation of proteoglycans mRNA and protein at 14 days was higher than that of 7 days（P<0.05）.However，the accumulation of proteoglycans mRNA and protein showed no significant difference at any time points in the control group（P>0.05）.The accumulation of proteoglycans mRNA and protein in the experiment group was not different from that of the control group at 4 days，but higher than that of control group at 7 and 14 days（P<0.05）.The accumulation of type Ⅰ collagen mRNA and protein in each groups showed no difference at any time points.Moreover，there was no difference at each time point in both groups（P<0.05）.In the experiment group，the accumulation of Ⅱ collagen mRNA and protein at 7 and 14 days were remarkably higher than that at 4 days.Moreover，the accumulation of Ⅱ collagen mRNA and protein at 14 days post-transfection was higher than that at 7 days（P<0.05）.However，the accumulation of Ⅱ collagen mRNA and protein showed no significant difference at any time points in the control group（P>0.05）.The accumulation of Ⅱ collagen mRNA and protein in experiment group was not different from that of the control group at 4 days，but higher than that of the control group at 7 and 14 days（P<0.05）.Conclusion：The NP cells transfected by the rAAV2-hBMP7 can overexpress hBMP7.The rAAV2-hBMP7 is capable of promoting the expression of proteoglycans and type Ⅱ collagen of NP cells.