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目的通过改进肌肉组织蛋白的提取方法,利用微量组织进行Western blot(WB)方法诊断肌营养不良。方法比较不同的样品缓冲液和处理方式对鼠骨骼肌蛋白提取量的影响,采用筛选出的最佳方法对待检病例的微量肌肉组织进行蛋白提取和蛋白分析。结果将筛选出的样品缓冲液加入冰冻切片肌肉组织,通过振荡溶解,提取蛋白量多且损失小。从62例疑似肌营养不良病例的肌肉组织中提取足量蛋白,WB结果显示所检蛋白带清晰,部分病例存在蛋白带的异常。免疫组织化学染色显示Dys-R、Dys-C、Dys-N均重度表达异常的标本中,WB检测均未发现相应的目标蛋白带。免疫组织化学显示Dys轻度表达异常的标本中,WB检测显示目标蛋白带位置异常、带浅或正常。结论应用改进的蛋白提取方法可以节省组织标本用量,并进行蛋白分析诊断肌营养不良。临床高度疑诊疑似抗肌萎缩蛋白病的病例,免疫组织化学染色显示轻度缺失或正常,需要进一步行WB检测抗肌萎缩蛋白。
OBJECTIVE: To improve muscular dystrophy by improving the method of extracting protein from muscle tissue and using Western blot (WB) to detect muscular dystrophy. Methods The effects of different sample buffers and treatment methods on the amount of protein extracted from skeletal muscle of rats were compared. The optimal method for protein extraction and protein analysis was performed on the trace muscle tissues. Results The selected sample buffer was added to frozen sections of muscle tissue and dissolved by shaking to extract more protein and less loss. A sufficient amount of protein was extracted from the muscle tissue of 62 suspected cases of muscular dystrophy. The results of the WB showed that the detected protein band was clear, and in some cases, the protein band was abnormal. Immunohistochemical staining showed that there was no corresponding target protein band in the WB-detected specimens with abnormally expressed Dys-R, Dys-C and Dys-N. Immunohistochemistry showed mild abnormal expression of Dys specimens, WB test showed that the target protein band abnormal, with light or normal. Conclusion The improved protein extraction method can save the amount of tissue samples and carry out protein analysis to diagnose muscular dystrophy. Highly suspected cases of suspected dystrophinopathy, immunohistochemical staining showed mild deletion or normal, need further WB line detection of dystrophin.