GDF-5基因活化基质体内转染修复兔退变椎间盘的实验研究

The repairing effect of GDF-5 gene activated matrix transplantation in vivo on interdisc degeneration in rabbit

目的通过GDF5基因活化基质体内转染兔退变椎间盘,观察其修复效果。方法脂质体介导的pcDNA3.1(+)/GDF-5重组质粒混入Ⅰ型胶原支架,置入兔退变椎间盘;实验分为GAM组,普通支架组,脂质体对照组,2个月后RT-PCR和Western blot观察椎间盘髓核细胞GDF-5基因和蛋白表达的稳定性,间苯三酚法检测髓核蛋白多糖含量,流式细胞仪检测细胞凋亡率来观察修复效果。结果 RT-PCR结果显示术后2个月GDF-5基因稳定表达。GAM组蛋白多糖含量高于另外两组(<0.05),空白支架组和对照组之间没有差异(=0.601)。GAM组髓核细胞凋亡率明显低于另外两组(<0.01),空白支架组和对照组之间没有差异(=0.902)。结论利用GAM可以有效的进行GDF-5基因体内转染并表达,从而修复退变椎间盘。

Objective To study the repairing effect of GDF-5 gene activated matrix transplantation（GAM） in vivo on intervetebral disc degeneration（IDD） in rabbit. Methods The animals were divided into GAM group（ Ⅰ Collagen scaffold mixed with lipidosome mediated pcDNA3.1（＋）/GDF-5 recombinant plasmid was transplanted into the degenerative intervetebral disc）, SCAFFOLD group and lipidosome control group. The expression of GDF-5 gene and protein was observed by RT-PCR and Western blot methods respectively,the proteoglycans content and apoptosis rate in nucleus pulposus cells were determined by phloroglucin method and flowcytometer respectively after 2 months post-operation. Results RT-PCR and Western blot showed a stable expression of GDF-5 gene and protein in transfected disc 2 months after transplantation. Nucleus pulposus proteoglycan content was higher in GAM group than in other two groups（P〈 0.05）, with no difference between SCAFFOLD group and lipidosome control （P=0.601） .The apoptosis rate of nucleus pulposus cells was significant lower in GAM group than in other two groups （P〈0.01） , with no difference between SCAFFOLD group and lipidosome control （P=0.902） . Conclusion GDF-5 gene activated matrix ccould be effectively transfected and expressed to be involved in the repair of intervetebral disc degeneration.