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

Duchenne肌营养不良症(DMD)是一种X连锁的致死性疾病，由编码细胞骨架蛋白dystrophin的基因突变引起。最近的研究表明，骨骼肌中的nNOS通过钝化α-肾上腺素能受体激活引起的血管收缩反应，在运动骨骼肌血流调节中发挥关键作用。我们推测，dystrophin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，我们获得了在平滑肌α-肌动蛋白启动子的转录控制下表达14kb全长人dystrophin基因的转基因小鼠。然后将这些小鼠与MDX小鼠杂交，产生了三个独立的SMTg/MDX系，它们只在SMC中含有dystrophin基因。半定量RT-PCR分析和免疫组织化学染色检测到转基因基因的表达模式，表明转基因基因在SMC中有特异性表达。我们分析了SMTg/mdx小鼠的组织学特征，如中心核、纤维大小变异和CK浓度，并与C57BL/10对照组和mdx小鼠进行了比较。此外，我们还成功地通过RGD修饰的腺病毒将LacZ基因表达到血管平滑肌细胞(VSMC)中，用于针对DMD血管肌肉的基因治疗。我们还建立了两步转移法将基因定向稳定地转移到肌肉细胞中的方法。我们表明，检测不同于成纤维细胞的肌营养不良蛋白在肌管中的表达有助于DMD的基因诊断

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