Gene abnormality and ion-channel change in myotonic dystrophy

强直性肌营养不良的基因异常和离子通道变化

• 批准号: 06670671
• 负责人: YOSHII Fumihito
• 金额: $ 1.34万
• 依托单位: Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670671/
• 关键词: myotonic dystrophy; trinucleotide repeat; PCR method; myotonin protein kinase; immunohistochemical staining; ion-channel; patch clamp technique; 3.塩基反復配列; 筋緊張性ジストロフィー; CTGリピート; 細胞膜; DMキナーゼ; 免疫組織化学

The purposes of the study are to discover the relationship between the variety of clinical features in myotonic dystrophy (DM) and DM gene abnormality, and between DM gene abnormality and the changes in ion-channel currents of muscular cell membrane in DM patients.1. DNA analysis in myotonic dystrophyUsing the PCR method, we studied an expanded trinucleotide (CTG) repeat in a gene on 19 chromosome in patients with DM and healthy subjects. We found two bands on agarose gel in healthy subjects, but only one band appeared in patients with DM, probably due to enormous expansion of CTG repeat in the abnormal allele. For further evaluation we intend to use a specific probe for Southern blotting to study the exact repeat number of CTG in DM patients.2. Localization of myotonin protein kinase (DM-PK) in muscular cellsUsing rabbit anti-DM-PK polyclonal antibodies and indirect immunohistochemical staining method, we studied the localization of DM-PK in the muscular cells. Brownish reactive substances were observed along the inner side of the cell membrane in normal muscular cells, but no substances were observed in the muscular cells of the patients with DM.In the muscular cells of myositis, there were many reactive substances, especially in the cytoplasm of the regenerating muscular cells.3. Ion-channel currents of the muscular cell membraneIon-channel currents of the muscular cell membrane in patients with DM were measured by patch-clamp method. However, it failed to measure exactly the currents in mononuclear myoblasts or polynuclear muscular cells. We are now doing basic studies using muscular specimens of the animals.

该研究的目的是发现肌营养不良症（DM）和DM基因异常的临床特征之间的关系，DM基因异常与DM患者肌肉细胞膜的离子通道电流的变化之间的关系。1。 DNA分析PCR方法中，我们研究了DM和健康受试者患者的19个染色体中的基因中膨胀的三核苷酸（CTG）重复。我们在健康受试者中发现了两条琼脂糖凝胶的频带，但是在DM患者中只出现了一条条带，这可能是由于异常等位基因中CTG重复的巨大扩展所致。为了进一步评估，我们打算使用特定的探针来研究南部印迹，以研究DM患者中CTG的确切重复数量。2。肌醇蛋白激酶（DM-PK）在肌肉细胞中的定位，兔抗DM-PK多克隆抗体和间接免疫组织化学染色方法，我们研究了DM-PK在肌肉细胞中的定位。在正常肌肉细胞中沿细胞膜的内侧观察到棕褐色的反应性物质，但是在患有Dm的患者的肌肉细胞中未观察到任何物质，在肌炎的肌肉炎细胞中，有许多反应性物质，尤其是在再生肌肉细胞的细胞质中。3。 DM患者的肌肉细胞膜的肌肉细胞膜通道电流的离子通道电流通过贴剂钳方法测量。但是，它未能精确测量单核肌细胞或多核肌肉细胞中的电流。现在，我们正在使用动物的肌肉标本进行基础研究。