Mouse growth and differentiation factor-5 protein and DNA therapy potentiates intervertebral disc cell aggregation and chondrogenic gene expression
Mouse growth and differentiation factor-5 protein and DNA therapy potentiates intervertebral disc cell aggregation and chondrogenic gene expression
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DOI：
10.1016/j.spinee.2007.05.012
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发表时间：
2008-03-01
期刊：
影响因子：
4.5
通讯作者：
Li, Xudong
中科院分区：
文献类型：
--
作者：
Cui, Min;Wan, Yuqing;Li, Xudong
背景情况：生长和分化因子-5（GDF-5）缺陷小鼠椎间盘（IVD）结构和细胞外基质异常。腺病毒介导的GDF-5对兔椎间盘细胞的生长有促进作用目的：本研究旨在探讨重组GDF-5蛋白和GDF-5 cDNA对体外培养的椎间盘细胞代谢的影响。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，并用于体外转染小鼠椎间盘细胞。治疗与GDF-5蛋白和cDNA进行了评估，通过测量细胞增殖，蛋白多糖的生产，和细胞外基质的基因expressions.RESULTS：生化分析显示，在培养的各种浓度的小鼠GDF-5（mGDF-5）蛋白的存在下，在小鼠IVD细胞的硫酸化糖胺聚糖（GAG）/DNA的比例升高。实时逆转录-聚合酶链反应（RT-PCR）表明，处理细胞与GDF-5蛋白增加的II型胶原和聚集蛋白聚糖基因的表达，以剂量依赖性的方式，但降低基质金属蛋白酶（MMP）-3基因的表达。免疫组织化学显示，与对照组相比，在培养物中用mGDF-5处理的小鼠IVD细胞的聚集增加。将小鼠GDF-5基因成功克隆到表达质粒载体中，并通过Western blot分析证实GDF-5蛋白的产生。II型胶原蛋白和聚集蛋白聚糖基因表达的细胞显着增加的细胞，核转染与GDF-5质粒转染的细胞与对照plasmid.CONCLUSIONS：这是第一次报告的小鼠GDF-5基因的克隆和使用的核转染方法转移DNA到IVD细胞。这些数据表明，重组蛋白和cDNA形式的GDF-5可以增加小鼠IVD细胞中细胞外基质蛋白基因的表达。未来需要尝试基因治疗，以治疗退行性椎间盘疾病与一种新的离体基因转移技术，以开发一种治疗，将缓解患者的条件与临床相关的轴向脊柱疼痛。(C)2008年爱思唯尔公司All rights reserved.
BACKGROUND CONTEXT: 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.PURPOSE: 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.STUDY DESIGN: The effects of recombinant GDF-5 protein and GDF-5 cDNA on mouse IVD cells will be evaluated in vitro.METHODS: 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.RESULTS: 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.CONCLUSIONS: 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. (C) 2008 Elsevier Inc. All rights reserved.