Functional analysis of dystrophin in vascular smooth muscle cells and gene therapy to the smooth muscle in Duchenne muscular dystrophy

杜氏肌营养不良症（DMD）是一种由编码细胞骨架蛋白营养不良蛋白的基因突变引起的x连锁致命疾病。最近的研究表明，骨骼肌中的nNOS通过减弱α -血管收缩剂对α -肾上腺素能受体激活的反应，在运动骨骼肌血流调节中起关键作用。我们假设肌营养不良蛋白缺乏也会导致血管平滑肌细胞（VSMCs）中nNOS的减少，导致血管功能障碍并加剧肌肉病理。因此，我们在平滑肌α -肌动蛋白启动子的转录控制下，产生了表达14 Kb全长人肌营养不良蛋白cDNA的转基因小鼠。然后将这些小鼠与mdx小鼠杂交，得到三个独立的SMTg/mdx系，这些系仅在SMCs中含有肌营养不良蛋白基因。半定量RT-PCR分析和免疫组化染色检测其表达模式，表明转基因在SMCs中的特异性表达。与C57BL/10对照小鼠和mdx小鼠相比，我们正在分析SMTg/mdx小鼠的组织学特征，如中心成核、纤维大小变异性和CK浓度。此外，我们还成功利用rgd修饰腺病毒利用肌肉特异性启动子在血管平滑肌细胞（VSMC）中表达LacZ基因，用于针对DMD血管肌的基因治疗。我们还开发了通过两步转移将基因定向和稳定地传递到肌肉细胞的方法。我们发现分析肌管中与成纤维细胞分化的肌营养不良蛋白的表达对DMD的遗传诊断是有用的

Duchenne muscular dystrophy (DMD) is an X-linked fatal disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin. Recent studies indicate that nNOS in skeletal muscle plays a key role in the regulation of the blood flow within exercising skeletal muscle by blunting the vasoconstrictor response to alpha-vasoconstrictor response to alpha-adrenergic receptor activation. We hypothesized that dystrophin deficiency also causes the reduction of nNOS in vascular smooth muscle cells (VSMCs), leads to vascular dysfunction and exacerbates muscle pathology. We therefore generated transgenic mice expressing 14 Kb full-length human dystrophin cDNA under the transcriptional control of the smooth muscle alpha-actin promoter. These mice were then crossed with mdx mice, resulting in three independent SMTg/mdx lines which harbor the dystrophin gene only in SMCs. The expression pattern was detectable by semi-quantitative RT-PCR analysis and immunohistotochemical staining, which showed the specific expression of transgene in SMCs. We are analyzing histological characteristics of SMTg/mdx mices such as central nucleation, fiber size variability, and CK concentrations as compared to C57BL/10 control mice and mdx mice.In addision, we have succeeded that LacZ gene was expressed in Vascular smooth muscle cell (VSMC) by RGD-modification adenovirus using muscle specific promoter for gene therapy targeting to vascular muscle of DMD.We also developed the methods of targeted and stable gene deliverly into muscle cells by a two-step transfer.We showed that the analysis of dystrophin expression in myotubes which were differenciated from fibloblasts is useful for genetic diagnosis of DMD