Gene abnormality and ion-channel change in myotonic dystrophy

本研究的目的是探讨强直性肌营养不良（DM）临床表现的多样性与DM基因异常的关系，以及DM基因异常与DM患者肌细胞膜离子通道电流变化的关系.强直性肌营养不良症的DNA分析使用PCR方法，我们研究了DM患者和健康受试者19号染色体上基因中的扩展三核苷酸（CTG）重复序列。我们在琼脂糖凝胶上发现健康人有两条带，而糖尿病患者只有一条带，可能是由于异常等位基因中CTG重复序列的大量扩增。为了进一步的研究，我们打算用特异性的探针进行Southern杂交，以研究DM患者CTG的确切重复数.肌紧张素蛋白激酶（DM-PK）在肌细胞中的定位应用兔抗DM-PK多克隆抗体和间接免疫组织化学染色方法，研究了DM-PK在肌细胞中的定位。正常肌细胞胞膜内侧有沿着分布的棕色反应物质，而DM患者肌细胞内无反应物质;肌炎肌细胞内有大量反应物质，尤其是再生肌细胞胞浆内.肌细胞膜离子通道电流用膜片钳法测定糖尿病患者肌细胞膜离子通道电流。然而，它未能准确测量单核成肌细胞或多核肌细胞的电流。我们现在正在用动物的肌肉标本做基础研究。

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.