Cui Min, Wan Yuqing, Anderson D Greg, Shen Francis H, Leo Brian M, Laurencin Cato T, Balian Gary, Li Xudong
Department of Orthopaedic Surgery, University of Virginia, Hospital Drive, Cobb Hall, P.O. Box 800374, Charlottesville, VA 22908, USA.
Spine J. 2008 Mar-Apr;8(2):287-95. doi: 10.1016/j.spinee.2007.05.012. Epub 2007 Jul 6.
Growth and differentiation factor-5 (GDF-5)-deficient mice showed abnormalities in intervertebral disc (IVD) structure and extracellular matrix. Adenovirus-mediated GDF-5 delivery can promote the growth of rabbit disc cells.
The aim of the present study was to investigate the effect of recombinant GDF-5 protein and GDF-5 complementary DNA (cDNA) on the metabolism of IVD cells.
The effects of recombinant GDF-5 protein and GDF-5 cDNA on mouse IVD cells will be evaluated in vitro.
Mouse disc cells in vitro were treated with recombinant GDF-5 protein. Mouse GDF-5 cDNA was cloned into an expression vector and was used to transfect mouse disc cells in vitro. Therapy with GDF-5 protein and cDNA was assessed by measuring cell proliferation, proteoglycan production, and extracellular matrix gene expression.
Biochemical assays revealed an elevated sulfated glycosaminoglycan (GAG)/DNA ratio in mouse IVD cells that were cultured in the presence of various concentrations of mouse GDF-5(mGDF-5) protein. Real-time reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that treating the cells with GDF-5 protein increased the expression of the collagen Type II and aggrecan genes in a dose-dependent manner but decreased matrix metalloproteinase (MMP)-3 gene expression. Immunohistochemistry showed an increase in the aggregation of mouse IVD cells that were treated with mGDF-5 in culture compared with the control group. The mouse GDF-5 gene was successfully cloned into an expression plasmid vector, and GDF-5 protein production was confirmed by Western blot analysis. Type II collagen and aggrecan gene expression by the cells increased significantly in the cells that were transfected by nucleofection with the GDF-5 plasmid compared with cells that were transfected with a control plasmid.
This is the first report of the cloning of the mouse GDF-5 gene and use of the nucleofection method to transfer DNA into IVD cells. The data suggest that both recombinant protein and the cDNA forms of GDF-5 can increase the expression of genes for extracellular matrix proteins in mouse IVD cells. Future attempts at gene therapy to treat degenerative disc disease with a novel ex vivo gene transfer technique are needed to develop a therapy that would alleviate the condition of patients with clinically relevant axial spine pain.
生长分化因子5（GDF - 5）缺陷小鼠的椎间盘（IVD）结构和细胞外基质出现异常。腺病毒介导的GDF - 5递送可促进兔椎间盘细胞生长。
本研究旨在探讨重组GDF - 5蛋白和GDF - 5互补DNA（cDNA）对IVD细胞代谢的影响。
将在体外评估重组GDF - 5蛋白和GDF - 5 cDNA对小鼠IVD细胞的作用。
用重组GDF - 5蛋白处理体外培养的小鼠椎间盘细胞。将小鼠GDF - 5 cDNA克隆到表达载体中，用于体外转染小鼠椎间盘细胞。通过测量细胞增殖、蛋白聚糖产生和细胞外基质基因表达来评估GDF - 5蛋白和cDNA的治疗效果。
生化分析显示，在不同浓度的小鼠GDF - 5（mGDF - 5）蛋白存在下培养的小鼠IVD细胞中，硫酸化糖胺聚糖（GAG）/DNA比值升高。实时逆转录 - 聚合酶链反应（RT - PCR）表明，用GDF - 5蛋白处理细胞以剂量依赖方式增加了II型胶原蛋白和聚集蛋白聚糖基因的表达，但降低了基质金属蛋白酶（MMP）-3基因的表达。免疫组织化学显示，与对照组相比，在培养中用mGDF - 5处理的小鼠IVD细胞聚集增加。小鼠GDF - 5基因成功克隆到表达质粒载体中，通过蛋白质印迹分析证实了GDF - 5蛋白的产生。与用对照质粒转染的细胞相比，用GDF - 5质粒进行核转染的细胞中II型胶原蛋白和聚集蛋白聚糖基因表达显著增加。
这是关于小鼠GDF - 5基因克隆以及使用核转染方法将DNA导入IVD细胞的首次报道。数据表明，重组蛋白和cDNA形式的GDF - 5均可增加小鼠IVD细胞中细胞外基质蛋白基因的表达。未来需要尝试用新型体外基因转移技术进行基因治疗以治疗椎间盘退变疾病，从而开发出一种能够缓解具有临床相关轴向脊柱疼痛患者病情的疗法。
