Wakayama Yoshihiro, Jimi Takahiro, Inoue Masahiko, Kojima Hiroko, Murahashi Makoto, Kumagai Toshiyuki, Yamashita Sumimasa, Hara Hajime, Shibuya Seiji
Department of Neurology, Showa University Fujigaoka Hospital, 1-30 Fujigaoka, Aoba-ku, Yokohama 227-8501, Japan.
Arch Neurol. 2002 Mar;59(3):431-7. doi: 10.1001/archneur.59.3.431.
In Duchenne muscular dystrophy (DMD), previous freeze-fracture electron microscopic studies demonstrated that muscle plasma membrane contained markedly decreased numbers of orthogonal arrays. Recent investigations showed that orthogonal arrays were composed of aquaporin 4 (AQP4) molecules, a member of the water channel protein family.
To study whether the immunostainability of anti-AQP4 antibody is reduced in muscles of patients with DMD and whether, if it is reduced, the problem is at the genomic DNA, messenger RNA (mRNA), or posttranscriptional level.
We analyzed the muscle and blood samples from 6 boys with DMD, 6 normal control subjects, and 12 patients with neuromuscular diseases at the protein, genomic DNA, and mRNA levels. At the protein level, immunohistochemical staining and immunoblot analysis were performed. At the genomic DNA and mRNA levels, the polymerase chain reaction and reverse transcription polymerase chain reaction, respectively, were used to screen for mutations in the AQP4 gene.
At the protein level, immunohistochemical staining of our originally generated rabbit anti-AQP4 antibody in DMD muscles was markedly reduced. Most of the DMD myofibers showed negative staining with sporadic partially positive fibers at their myofiber surface, whereas the control muscles displayed continuous myofiber surface staining. Immunoblot analysis showed that the content of AQP4 in DMD muscles was remarkably decreased. Amplification of leukocyte genomic DNA by polymerase chain reaction showed that the patients with DMD had genomic DNA of the AQP4 molecule. Quantitative reverse transcription polymerase chain reaction demonstrated that DMD skeletal muscles contained markedly decreased AQP4 mRNA compared with controls.
The reduction in AQP4 in DMD muscles results from decreased levels of AQP4 mRNA in DMD myofibers.
在杜氏肌营养不良症（DMD）中，以往的冷冻断裂电子显微镜研究表明，肌肉质膜中正交排列的数量显著减少。最近的研究表明，正交排列由水通道蛋白家族成员水通道蛋白4（AQP4）分子组成。
研究DMD患者肌肉中抗AQP4抗体的免疫染色性是否降低，以及如果降低，问题是出在基因组DNA、信使核糖核酸（mRNA）还是转录后水平。
我们在蛋白质、基因组DNA和mRNA水平上分析了6名DMD男孩、6名正常对照受试者以及12名神经肌肉疾病患者的肌肉和血液样本。在蛋白质水平上，进行了免疫组织化学染色和免疫印迹分析。在基因组DNA和mRNA水平上，分别使用聚合酶链反应和逆转录聚合酶链反应来筛查AQP4基因中的突变。
在蛋白质水平上，我们最初制备的兔抗AQP4抗体在DMD肌肉中的免疫组织化学染色明显减少。大多数DMD肌纤维呈阴性染色，其肌纤维表面有散在的部分阳性纤维，而对照肌肉则显示肌纤维表面连续染色。免疫印迹分析表明，DMD肌肉中AQP4的含量显著降低。通过聚合酶链反应扩增白细胞基因组DNA表明，DMD患者具有AQP4分子的基因组DNA。定量逆转录聚合酶链反应表明，与对照组相比，DMD骨骼肌中AQP4 mRNA明显减少。
DMD肌肉中AQP4的减少是由于DMD肌纤维中AQP4 mRNA水平降低所致。
