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Elucidation of molecular mechanism of utrophin expression in dystrophic skeletal muscle and its application to molecular therapy

营养不良性骨骼肌中utropin表达的分子机制的阐明及其在分子治疗中的应用

基本信息

批准号：
11470153
负责人：
TAKEDA Shin'ichi
金额：
$ 9.54万
依托单位：
National Institute of Neuroscience, NCNP
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470153/
关键词：
utrophin dystrophin mdx mice DMD adenovirus vector stabilization of mRNA cytokines IL-6 免疫応答炎症反応免疫反応

项目摘要

Duchenne muscular dystrophy (DMD) is an X-linked lethal disorder caused by a defect in the DMD gene, which encodes the cytoskeletal protein dystrophin. Utrophin is an autosomal homologue of the DMD gene product dystrophin, and augmented expression of endogenous utrophin is expected to provide an alternative therapeutic approach for DMD. We previously reported that immune response against a (β-galactosidase-expressing adenovirus vector, AxCALacZ, resulted in accumulation of endogenous utrophin expression on the extrasynaptic sarcolemma in dystrophin-deficient mdx mice (Hum Gene Ther 11 : 669-680, 2000). In order to determine which cytokine is involved in the regulation of utrophin expression, we directly injected several cytokines separately into neonatal mdx muscles and tested whether the expression of utrophin is increased on the sarcolemma or not. Importantly, among the cytokines tested, solely IL-6 successfully increased expression of utrophin. Moreover, increase of utrophin mRNA was detected from rIL-6-injected mdx muscles by the quantitative real-time RT-PCR study. Further, IL-6 expression was elevated in AxCALacZ-infected mdx muscle in the early stage, and anti-IL-6R antibody treatment blocked enhanced utrophin expression in AxCALacZinfected mdx muscle. We should point out, however, that over-expression of utrophin due to recombinant IL-6 treatment lasted only a week. In addition, expression of utrophin was not evident in normal C57BL/10 neonatal muscles injected with IL-6 (Hum Gene Ther 13 : 509-518, 2002). Taken together, these results suggest that IL6 can induce over-expression of utrophin on the extrasynaptic sarcolemma but requires pre-existing factors in neonatal mdx muscle to fully regulate utrophin expression.

杜氏肌营养不良症（DMD）是由DMD基因缺陷引起的X连锁致死性疾病，DMD基因编码细胞骨架蛋白肌营养不良蛋白。肌营养不良蛋白是DMD基因产物肌营养不良蛋白的常染色体同源物，并且预期内源性肌营养不良蛋白的增强表达为DMD提供替代的治疗方法。我们先前报道了针对表达β-半乳糖苷酶的腺病毒载体AxCALacZ的免疫应答导致在肌营养不良蛋白缺陷型mdx小鼠中突触外肌膜上内源性肌营养不良蛋白表达的积累（Genetics Ther 11：669-680，2000）。为了确定哪种细胞因子参与utrophin表达的调节，我们将几种细胞因子分别直接注射到新生mdx肌肉中，并测试肌膜上utrophin的表达是否增加。重要的是，在测试的细胞因子中，仅IL-6成功地增加了utrophin的表达。此外，通过定量实时RT-PCR研究，从rIL-6注射mdx的肌肉中检测到utrophin mRNA的增加。此外，在AxCALacZ感染的mdx肌肉中，IL-6表达在早期阶段升高，并且抗IL-6 R抗体治疗阻断了AxCALacZ感染的mdx肌肉中utrophin表达的增强。然而，我们应该指出的是，由于重组IL-6治疗引起的utrophin的过度表达仅持续了一周。此外，在用IL-6注射的正常C57 BL/10新生儿肌肉中utrophin的表达不明显（Escherogene Ther 13：509-518，2002）。两者合计，这些结果表明，IL 6可以诱导过度表达的utrophin的突触外肌膜，但需要预先存在的因素，在新生儿mdx肌肉，以充分调节utrophin的表达。