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

我们的冷冻断裂电镜研究表明，Duchenne型肌营养不良症（DMD）和福山型先天性肌营养不良症（FCMD）患儿的肌质膜含有明显减少的正交阵列（OAs）。近年来的研究表明，OAs的主要蛋白质成分是水通道蛋白4（AQP 4），它属于水通道蛋白家族。本研究从蛋白质、基因组DNA和mRNA水平分析DMD和FCMD对肌肉细胞质膜AQP 4的损伤机制。在知情同意的情况下采集6例DMD患儿、4例FCMD患儿、5例强直性肌营养不良症患儿、1例肢带型肌营养不良症患儿、1例重症肌无力患儿和6例正常对照者的血液和肌肉标本。在蛋白水平上，用兔抗AQP 4和抗β-血影蛋白抗体以及单克隆抗 ...更多信息 抗肌萎缩蛋白抗体（Novocastra DYS-1）。用抗AQP 4抗体对DMD和FCMD肌进行免疫组织化学染色，结果显示DMD和FCMD肌由阴性染色的肌纤维和阳性染色的散在肌纤维混合组成。DMD和FCMD肌肉抗Dystrophin抗体免疫组化分别为阴性和阳性。抗β-血影蛋白抗体基本上染色了所有DMD和FCMD肌纤维。用这三种抗体对正常和疾病对照肌肉的免疫染色显示，在所有肌纤维的细胞周边均呈阳性染色。免疫印迹分析显示，DMD和FCMD肌的抗AQP 4抗体染色强度明显减弱，抗Dystrophin抗体染色呈阴性，抗β-spectrin抗体染色呈阳性，与正常和疾病对照肌相似。采用PCR方法检测DMD和FCMD患者的AQP 4基因组DNA。此外，通过RT-PCR分析DMD和FCMD肌肉的AQP 4 mRNA，并且在两种营养不良中均显著降低。本研究结果提示，DMD和FCMD肌肉中AQP 4表达减少是由于这些肌肉中AQP 4 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