GDF-5体外转染BMSCs移植修复兔退变椎间盘的实验研究

The repairing effect of BMSCs transplantation transfected by GDF-5 gene on interdisc degeneration in rabbit

目的通过骨髓间充质干细胞(BMSCs)体外生长分化因子-5(GDF-5)基因转染修饰,然后移植入退变的椎间盘中,观察修复椎间盘退变的效果。方法脂质体介导的pcDNA3.1(+)/GDF-5重组质粒转染兔BMSCs,G418筛选稳定表达株;实验分为转染细胞组、BMSCs组、退变组,2个月后RT-PCR观察椎间盘髓核细胞GDF-5基因表达稳定性,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BMSCs转染GDF-5基因48h后以及移植手术后2个月时GDF-5基因表达稳定。转染细胞组和BMSCs组髓核蛋白多糖含量明显高于退变组(P<0.01),转染细胞组高于BMSCs组(P=0.05)。退变组髓核细胞凋亡率比其他两组显著增高(P<0.01),转染细胞组与BMSCs组之间没有差异(P=0.09)。结论移植BMSCs或者GDF-5基因转染的BMSCs对退变椎间盘有修复作用,后者的修复效果更好。

Objective To observe the repairing effect of bone mesenchymal stem cells （BMSCs） transplantation transfected by growth differentiation factor 5 （GDF-5） gene on intervetebral disc degeneration（IDD） in rabbit.Methods Lipidosome mediated pcDNA3.1 （＋）/GDF-5 recombinant plasmid was transfected into rabbit BMSCs,the lasting stock was screened by G418.The animals were divided into IDD group,BMSCs group and GDF-5＋ BMSCs group.The stability expression of GDF-5 mRNA was determined by RT-PCR,the proteoglycans content and the apoptosis rate were determind by phloroglucin method and flowcytometer respectively in nucleus pulposus cells after 2 months post-operation.Results RT-PCR showed a stable expression of GDF-5 gene in transfected BMSCs 48 hours post- transfection and 2 months post-operation.Nucleus pulposus proteoglycan content was significant lower in IDD group than in BMSCs group or GDF-5＋ BMSCs group （P〈0.01） with more higher in GDF-5＋ BMSCs group than in BMSCs group（P=0.05） The apoptosis rate of nucleus pulposus cells was obviously higher in IDD group than in BMSCs group or GDF-5＋ BMSCs group（P〈0.01）,but with no difference between GDF-5＋ BMSCs group and BMSCs group （P=0.05）.Conclusions GDF-5 plays an important role in repairing degenerated intervertebral discs.IDD can be better repaired by transplantation of BMSCs transfected by GDF-5.