Molecular analysis of the depletion of aquaporin 4 in the muscle plasma membrane of muscular dystrophies.

我们先前的冷冻断裂电镜研究表明，男孩杜氏肌营养不良症（DMD）和儿童福山先天性肌营养不良症（FCMD）的肌质膜含有明显减少的正交阵列（OAs）数量。最近的研究表明，OAs的主要蛋白质成分是水通道蛋白4 (aquaporin4, AQP4)，它是水通道蛋白家族的成员。本研究旨在从蛋白质基因组DNA和mRNA水平分析DMD和ffcmd肌质膜中AQP4缺失的机制。在知情同意的情况下，采集了6例DMD男童患者、4例FCMD儿童患者、5例肌强直性营养不良患者、1例肢带性肌肉营养不良患者、1例重症肌无力患者和6例正常对照者的血液和肌肉样本。在蛋白水平上采用兔抗AQP4、抗β-谱蛋白抗体和抗…More抗肌营养不良蛋白单克隆抗体（Novocastra dys1）进行免疫组化染色和免疫印迹分析。用抗AQP4抗体对DMD和FCMD肌肉进行免疫组化，结果显示DMD和FCMD肌肉是由阴性染色的肌纤维与阳性染色的散发性肌纤维混合而成。抗肌营养不良蛋白抗体对DMD和FCMD肌肉的免疫染色分别为阴性和阳性。抗β-谱蛋白抗体对DMD和FCMD肌纤维基本全部染色。用这三种抗体对正常肌和疾病对照肌进行免疫染色，所有肌纤维的细胞外周均呈阳性染色。免疫印迹分析显示，抗AQP4抗体对DMD和FCMD肌肉的染色强度明显降低，抗抗肌营养不良蛋白抗体对DMD和FCMD肌肉的免疫染色反应分别为阴性和阳性，抗β-spectrin抗体对DMD和FCMD肌肉的免疫染色反应与正常和疾病对照肌相似。PCR检测包括DMD和FCMD在内的所有血样中AQP4分子的基因组DNA。通过RT-PCR分析DMD和FCMD肌肉中AQP4 mRNA的表达，两种营养不良患者的AQP4 mRNA表达均明显降低。本研究结果提示，DMD和FCMD肌肉中AQP4表达降低是由于这些肌肉中AQP4 mRNA水平降低所致。少

Previous our freeze fracture electron microscopic studies demonstrated that the muscle plasma membrance of boys with Duchenne muscular dystrophy (DMD) and children with Fukuyama congenital muscular dystrophy (FCMD) contained the markedly reduced number of orthogonal arrays (OAs). Recent investigations revealed that the major protein component of OAs was aquaporin4 (AQP4) which was a member of water channel protein family. Present investigations were undertaken to analyze the mechanism of the depletion of AQP4 in the muscle plasma membranes with DMD and FCMD at protein genomic DNA and mRNA levels. The blood and muscle samples were taken under informed consent from 6 boys with DMD 4 children with FCMD 5 patients with myotonic dystrophy 1 patient with limb-girdle muscular dystrophy 1 patient with myasthenia gravis and 6 normal controls. At protein level immunohistochemical staining and immunoblot analyses were done using rabbit anti AQP4 and anti β-spectrin antibodies and monoclonal anti … More dystrophin antibody (Novocastra DYS-1). The immunohistochemistry of DMD and FCMD muscles using anti AQP4 antibody showed that the DMD and FCMD muscles were composed of the negatively stained myofibers mixed with the positively stained sporadic myofibers. The immunostainability of DMD and FCMD muscles using anti dystrophin antibody was negative and positive respectively. The anti β-spectrin antibody stained basically all of the DMD and FCMD myofibers. The immunostaining of normal and disease control muscles by using these three antibodies revealed the positive staining at their cell periphery in all myofibers. Immunoblot analyses showed that the staining intensity of DMD and FCMD muscles with anit AQP4 antibody was markedly decreased : while the immunostaining reactions of DMD and FCMD muscles with anti dystrophin antibody were negative and positive, respectively and those of DMD and FCMD muscles with anit β-spectrin antibody were positive similar to those of normal and disease control muscles. The genomic DNA of AQP4 molecule was detected by PCR in all blood samples including DMD and FCMD samples. Further the AQP4 mRNA of DMD and FCMD muscles was analyzed by RT-PCR and was markedly decreased in both dystrophies. The results of this study suggested that the reduced expression of AQP4 in DMD and FCMD muscles was due to the decreased level of AQP4 mRNA in these muscles. Less