腺相关病毒介导的成骨蛋白-1和性别决定区Y框蛋白9双基因联合转染兔退变椎间盘的生物学效应

Biological effect of adeno-associated virus-mediated co-transfer with osteogenic protein-1 and sex determining region Y-box 9 encoding gene in rabbit degenerativie discs

目的 观察腺相关病毒（AAV）介导的成骨蛋白-1（OP-1）和性别决定区Y框蛋白9（SOX9）双基因体内转染对兔退变椎间盘的影响.方法 针刺损伤法制作兔退变椎间盘模型,于4周后,分别于造模椎间盘髓核内注射20μl双基因混合液（1∶1）、重组AAV（rAAV）-OP-1、rAAV-SOX9、rAAV-增强型绿色荧光蛋白（EGFP）、磷酸盐缓冲液（PBS）,于转染后9周行MRI观察椎间盘变化,逆转录-聚合酶链反应（RT-PCR）检测Ⅱ型胶原mRNA及蛋白多糖mRNA的变化.结果 MRI显示双基因组T2加权像信号强度明显恢复,与SOX9、OP-1组比差异有统计学意义（P＜0.05）；RT-PCR证实双基因组Ⅱ型胶原及蛋白多糖mRNA表达量均明显高于单一SOX9及OP-1组（P＜0.05）.结论 SOX9及OP-1双基因联合转染治疗退变椎间盘组织具有明显作用；双基因治疗椎间盘退变具有协同效用.

Objective To investigate the biological effect of the recombinant adeno-associated virus （rAAV）-mediated osteogenic protein-1 （OP-1） gene and sex determining region Y-box 9 （SOX9） gene transfer in rabbit degenerative intervertebral discs on MRI,collagen type Ⅱ and proteoglycan levels.Methods The lumbar disc degeneration model was established in the rabbits using percutaneous needle puncture.Twenty-five mature New Zealand white rabbits （13 males and 12 females,weighing 1.7-2.8 kg）were randomly divided into 5 groups：group A receiving 20 μl rAAV-OP-1 and rAAV-SOX9 （1 × 105 PFU）; group B receiving 20 μl rAAV-OP-1; group C receiving 20 μl rAAV-SOX9; group D receiving 20 μl rAAV-enhanced green fluorescent protein（EGFP） ; and group E receiving 20 μl PBS.Nine weeks later,the changes of the intervertebral generative discs were observed by MRI,and the changes in type Ⅱ collagen mRNA and proteoglycan mRNA were examined by using reverse transcriptase-polymerase chain reaction （RT-PCR）.Results The intervertebral generative discs recovered in a great measure on MRI,more significantly in double gene group than in single gene group （P ＜ 0.05）.The nucleus pulposus tissues from double gene group exhibited an increased expression of proteoglycan mRNA and Col2al mRNA as compared with single gene group （P ＜ 0.05）.Conclusion The rAAV-mediated OP-1 and SOX9 transfer is effective in the treatment of rabbit degenerative intervertebral discs with synergetic effects.