Gene expression profiles using microarrays in mdx and mdx/MMP-2-/- mice at 1 and 3 months of age

Matrix metalloprotease (MMP) -2 has been reported to be up-regulated in skeletal muscle in the lethal X-linked muscle disorder Duchenne muscular dystrophy (DMD), which is caused by loss of dystrophin. However, the role of MMP-2 in dystrophin-deficient muscle is not well known. The aim of this study was to verify the role of MMP-2 in dystrophin-deficient muscle by using mdx mice with genetic ablation of MMP-2 (mdx/MMP-2-/-). Gene expression profiles were analyzed in the skeletal muscle of mdx and mdx/MMP-2-/- mice at 1 and 3 months of age. Tibialis anterior muscle was isolated from four groups of mice (mdx and mdx/MMP-2-/- mice at 1 and 3 months of age). Total RNA was purified and prepared for hybridization to Affymetrix Mouse Genome 430 2.0 arrays (Affymetrix Inc., Santa Clara, CA, USA) using Affymetrix reagents and protocols. The mRNA levels of differentially expressed genes from gene chip analysis were confirmed by quantitative real-time PCR assay.已有研究表明，在由抗肌萎缩蛋白（dystrophin）缺失引发的致死性X连锁肌肉疾病——杜氏肌营养不良症（Duchenne muscular dystrophy, DMD）的骨骼肌组织中，基质金属蛋白酶（Matrix metalloprotease, MMP）-2的表达水平呈上调状态。然而，MMP-2在抗肌萎缩蛋白缺失型肌肉组织中的具体作用仍未明确。 本研究旨在通过构建MMP-2基因敲除的mdx小鼠（mdx/MMP-2-/-），验证MMP-2在抗肌萎缩蛋白缺失型肌肉组织中的作用。研究者分别对1月龄与3月龄的mdx小鼠（mdx mice）及mdx/MMP-2-/-小鼠的骨骼肌组织进行基因表达谱分析。 本次实验共设置四组小鼠：1月龄mdx小鼠、1月龄mdx/MMP-2-/-小鼠、3月龄mdx小鼠及3月龄mdx/MMP-2-/-小鼠，研究者从各组小鼠体内分离胫骨前肌组织。随后提取各组组织的总RNA并纯化，使用Affymetrix公司的试剂与实验流程，将其制备为可与Affymetrix小鼠基因组430 2.0基因芯片（Affymetrix Inc., 美国加利福尼亚州圣克拉拉市）进行杂交的样本。最后通过实时荧光定量PCR（quantitative real-time PCR）检测，验证了基因芯片分析中筛选出的差异表达基因的mRNA表达水平。