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

点击翻译按钮获取中文摘要

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