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

肌营养不良症肌肉质膜中水通道蛋白 4 耗竭的分子分析。

基本信息

批准号：
11670643
负责人：
WAKAYAMA Yoshihiro
金额：
$ 2.18万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670643/
关键词：
Duchenne dystrophy Fukuyama dystrophy aquaporin 4 protein genomic DNA mRNA depletion mechanism Duchenne筋ジストロフィー症福山型筋ジストロフィー骨格筋細胞膜 feeze fracture replica orthogonal arrays PCR RT-PCR

项目摘要

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

以前，我们的冻结电子显微镜研究表明，Duchenne肌肉营养不良症（DMD）的肌肉质膜肿瘤和福山先天性肌肉营养不良（FCMD）的儿童包含明显减少的正交阵列（OAS）。最近的投资显示，OAS的主要蛋白质成分是水通道蛋白家族的成员Aquaporin4（AQP4）。目前进行了投资，以分析蛋白质基因组DNA和mRNA水平的DMD和FCMD在肌肉质膜中AQP4耗竭的机制。根据6名患有FCMD 5名患有肌动症肌营养不良症患者的DMD儿童的6名男孩的知情同意，血液和肌肉样本被接受，1例患有肢体女性肌肉营养不良的患者1患有肌无力重为肌无力的患者和6例正常对照。在蛋白质水平下，使用兔抗AQP4和抗β-谱蛋白抗体以及单克隆抗…更多的肌营养不良蛋白抗体（Novocastra Dys-1）进行了免疫组织化学染色和免疫印迹分析。使用抗AQP4抗体的DMD和FCMD肌肉的免疫组织化学表明，DMD和FCMD肌肉由染色的肌纤维与呈阳性零星零散的肌纤维混合的负染色的肌纤维组成。使用抗肌营养不良蛋白抗体的DMD和FCMD肌肉的免疫染色分别为阴性和正面呈阳性。抗β-光谱抗体基本上染色的所有DMD和FCMD肌纤维。通过使用这三种抗体对正常和疾病控制肌肉的免疫染色显示，在所有肌纤维中，其细胞周围的阳性染色。免疫印迹分析表明，具有ANIT AQP4抗体的DMD和FCMD肌肉的染色强度显着降低：虽然DMD和FCMD肌肉的免疫染色反应与抗营养不良蛋白抗体均分别为阴性和阳性，并且与DMD和FCMD肌肉的阳性相似，并且与Anitib and procks and propprib and ant and proppriN和FCMD的propprin and proppriN and proppriN和aNIT型抗体相似。肌肉。 PCR在包括DMD和FCMD样品在内的所有血液样本中检测到AQP4分子的基因组DNA。此外，通过RT-PCR分析了DMD和FCMD肌肉的AQP4 mRNA，并在两种营养不良中明显降低。这项研究的结果表明，DMD和FCMD肌肉中AQP4的表达降低是由于这些肌肉中AQP4 mRNA的水平降低所致。较少的