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

我们以前的冷冻断口电子显微镜研究表明，杜氏肌营养不良症(DMD)和福山先天性肌营养不良症(FCMD)患儿的肌浆膜含有明显减少的正交阵(OA)。近年来的研究表明，OAS的主要蛋白成分是水通道蛋白家族成员水通道蛋白4(Aquaporin4，AQP4)。本研究从蛋白质、基因组DNA和mRNA水平分析了DMD和FCMD导致肌肉质膜AQP4缺失的机制。血液和肌肉样本来自6名男孩DMD，4名FCMD儿童，5名强直性肌营养不良症患者，1例四肢带状肌营养不良症患者，1名重症肌无力患者和6名正常对照。在蛋白质水平上，用兔抗AQP4、抗β-Spectrin抗体和单抗…进行免疫组织化学染色和免疫印迹分析较多的抗肌营养不良蛋白抗体(新星虫DYS-1)。用AQP4抗体对DMD和FCMD肌进行免疫组织化学染色，结果显示DMD和FCMD肌由染色阴性的肌纤维和阳性染色的散在肌纤维混合而成。抗dystrophin抗体对DMD和FCMD肌的免疫染色分别为阴性和阳性。抗β-SPECTIN抗体对DMD肌纤维和FCMD肌纤维基本染色。用这三种抗体对正常肌肉和疾病对照肌肉进行免疫染色，发现所有肌纤维的细胞周边都有阳性染色。免疫印迹分析显示，ANITAQP4抗体对DMD和FCMD肌肉的染色强度明显减弱，而抗Dystrophin抗体对DMD和FCMD肌肉的免疫染色反应分别为阴性和阳性，而ANITβ-SPECTIN抗体对DMD和FCMD肌肉的免疫染色强度与正常和疾病对照肌肉相似。用聚合酶链式反应检测DMD和FCMD血样中AQP4分子的基因组DNA。RT-PCR分析DMD和FCMD肌组织中AQP4基因的表达，发现DMD和FCMD肌营养不良患者AQP4基因表达均明显降低。本研究结果提示，AQP4在DMD和FCMD肌肉中的表达降低是由于这些肌肉中AQP4的表达水平降低所致。较少

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