使用自动化毛细管 Western 分析系统检测人骨骼肌中的 dystrophin Dp71。
Detection of Dystrophin Dp71 in Human Skeletal Muscle Using an Automated Capillary Western Assay System.
机构信息
Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd., Edogawa, Tokyo 134-8634, Japan.
Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Nishi, Kobe 651-2180, Japan.
出版信息
Int J Mol Sci. 2018 May 23;19(6):1546. doi: 10.3390/ijms19061546.
BACKGROUND
Dystrophin Dp71 is one of the isoforms produced by the gene which is mutated in patients with Duchenne muscular dystrophy (DMD). Although Dp71 is expressed ubiquitously, it has not been detected in normal skeletal muscle. This study was performed to assess the expression of Dp71 in human skeletal muscle.
METHODS
Human skeletal muscle RNA and tissues were obtained commercially. Mouse skeletal muscle was obtained from normal and DMD mice. Dp71 mRNA and protein were determined by reverse-transcription PCR and an automated capillary Western assay system, the Simple Western, respectively. Dp71 was over-expressed or suppressed using a plasmid expressing Dp71 or antisense oligonucleotide, respectively.
RESULTS
Full-length Dp71 cDNA was PCR amplified as a single product from human skeletal muscle RNA. A ca. 70 kDa protein peak detected by the Simple Western was determined as Dp71 by over-expressing Dp71 in HEK293 cells, or suppressing Dp71 expression with antisense oligonucleotide in rhabdomyosarcoma cells. The Simple Western assay detected Dp71 in the skeletal muscles of both normal and DMD mice. In human skeletal muscle, Dp71 was also detected. The ratio of Dp71 to vinculin of human skeletal muscle samples varied widely, indicating various levels of Dp71 expression.
CONCLUSIONS
Dp71 protein was detected in human skeletal muscle using a highly sensitive capillary Western blotting system.
背景
Dp71 是肌营养不良蛋白基因发生突变的杜氏肌营养不良症（DMD）患者产生的一种异构体。虽然 Dp71 广泛表达，但在正常骨骼肌中未检测到。本研究旨在评估 Dp71 在人骨骼肌中的表达。
方法
商业获得人骨骼肌 RNA 和组织。从小鼠的正常和 DMD 肌肉中获得小鼠骨骼肌。通过逆转录 PCR 和自动化毛细管 Western 检测系统（Simple Western）分别确定 Dp71 mRNA 和蛋白。使用表达 Dp71 的质粒或反义寡核苷酸分别过表达或抑制 Dp71。
结果
从人骨骼肌 RNA 中 PCR 扩增出全长 Dp71 cDNA。通过在 HEK293 细胞中转染 Dp71 或在横纹肌肉瘤细胞中用反义寡核苷酸抑制 Dp71 表达，在 Simple Western 中检测到约 70 kDa 的蛋白峰被确定为 Dp71。Simple Western 检测到正常和 DMD 小鼠的骨骼肌中均存在 Dp71。在人骨骼肌中也检测到了 Dp71。人骨骼肌样本中 Dp71 与波形蛋白的比值差异很大，表明 Dp71 的表达水平存在差异。
结论
使用高度敏感的毛细管 Western 印迹系统在人骨骼肌中检测到 Dp71 蛋白。
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