Rivero J L, Serrano A L, Barrey E, Valette J P, Jouglin M
Department of Anatomy, Faculty of Veterinary Science, University of Cordoba, Spain.
J Muscle Res Cell Motil. 1999 Feb;20(2):211-21. doi: 10.1023/a:1005461214800.
Combined methodologies of enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting, traditional myofibrillar ATPase (mATPase) histochemistry and immunocytochemistry of whole biopsied samples were used to study myosin heavy chain (MHC) isoforms in the equine gluteus medius muscle. The ELISA technique allowed the quantification of the three MHC isoforms known to be present in different horse muscles: slow (MHC-I) and two fast (termed MHC-IIA and MCH-IIX). The SDS-PAGE method resolved MHCs in three bands: MHC-I, MHC-IIX and MHC-IIA from the fastest to the slowest migrating band and a quantification by densitometry for each MHC isoform was also possible. The identity of these three MHCs was confirmed by immunoblots with specific monoclonal antibodies. Five fibre types were defined immunohistochemically according to their MHC content: I, I + IIA, IIA, the hybrid IIAX and IIX. When quantitative data obtained with the four different methodologies were combined and compared, they were consistent and, when considered together, showed significant correlation. Nevertheless, the percentage of MHC-IIA histochemically derived was underestimated, while that of MHC-IIX was overestimated in comparison with the immunocytochemical determination of these MHC isoforms. The percentage of MHC-I obtained by ELISA technique was underestimated. In short, these integrated methods for the analysis of MHCs at the protein level demonstrate that equine skeletal muscle does not express the MHC-IIB, so type II fibres have been misclassified in numerous previous studies based upon the vary traditional mATPase histochemistry. They also offer new prospects for muscle fibre typing in equine experimental studies and veterinary medicine.
采用酶联免疫吸附测定（ELISA）、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、免疫印迹、传统肌原纤维ATP酶（mATPase）组织化学以及全活检样本免疫细胞化学等联合方法，研究马臀中肌中的肌球蛋白重链（MHC）亚型。ELISA技术可对已知存在于不同马肌肉中的三种MHC亚型进行定量：慢肌型（MHC-I）和两种快肌型（分别称为MHC-IIA和MCH-IIX）。SDS-PAGE方法将MHC分离为三条带：从迁移最快到最慢的带依次为MHC-I、MHC-IIX和MHC-IIA，并且还可以通过光密度法对每种MHC亚型进行定量。用特异性单克隆抗体进行免疫印迹证实了这三种MHC的身份。根据其MHC含量，通过免疫组织化学定义了五种纤维类型：I型、I + IIA型、IIA型、杂交IIAX型和IIX型。当将用四种不同方法获得的定量数据进行合并和比较时，它们是一致的，并且综合考虑时显示出显著相关性。然而，与这些MHC亚型的免疫细胞化学测定相比，组织化学衍生的MHC-IIA的百分比被低估，而MHC-IIX的百分比被高估。ELISA技术获得的MHC-I的百分比被低估。简而言之，这些用于蛋白质水平MHC分析的综合方法表明，马骨骼肌不表达MHC-IIB，因此在许多先前基于传统mATPase组织化学的研究中，II型纤维被错误分类。它们还为马实验研究和兽医学中的肌纤维分型提供了新的前景。
