De Aro Andrea Aparecida, Guerra Flávia Da Ré, Esquisatto Marcelo Augusto Marretto, Nakagaki Wilson Romero, Gomes Laurecir, Pimentel Edson Rosa
Department of Structural and Functional Biology, Institute of Biology, State University of Campinas-UNICAMP, Campinas, São Paulo, Brazil.
Microsc Res Tech. 2015 Jan;78(1):85-93. doi: 10.1002/jemt.22448. Epub 2014 Oct 18.
Dystrophin-deficient muscles have repeated cycles of necrosis and regeneration, being susceptible to injury induced by muscle contractions. Some studies have demonstrated that tendons are also affected in mdx mice, based especially on the changes in biomechanical properties arising from the respective linked muscles. However, most studies have focused only on alterations in the myotendinous junction. Thus, the purpose of this work was to study biochemical and morphological alterations in the Achilles tendons of 60-day-old mdx mice. Hydroxyproline quantification, showed higher collagen concentration in the mdx mice as compared with the control. No difference between the tendons of both groups was found in the noncollagenous proteins dosage, and in the amount of collagen type III detected in the western blotting analysis. The zymography for gelatinases detection showed higher amounts of metaloproteinase-2 (active isoform) and of metalloproteinase-9 (latent isoform) in the mdx mice. Measurements of birefringence, using polarization microscopy, showed higher molecular organization of the collagen fibers in the tendons of mdx mice in comparison to the control group, with presence of larger areas of crimp. Ponceau SS-stained tendon sections showed stronger staining of the extracellular matrix in the mdx groups. Toluidine blue-stained sections showed more intense basophilia in tendons of the control group. In morphometry, a higher number of inflammatory cells was detected in the epitendon of mdx group. In conclusion, the Achilles tendon of 60-day-old mdx mice presents higher collagen concentration and organization of the collagen fibers, enhanced metalloproteinase-2 activity, as well as prominent presence of inflammatory cells and lesser proteoglycans.
肌营养不良蛋白缺乏的肌肉会反复经历坏死和再生循环，容易受到肌肉收缩引起的损伤。一些研究表明，mdx小鼠的肌腱也会受到影响，特别是基于相关联肌肉的生物力学特性变化。然而，大多数研究仅关注肌-腱连接的改变。因此，本研究的目的是研究60日龄mdx小鼠跟腱的生化和形态学改变。羟脯氨酸定量分析显示，与对照组相比，mdx小鼠的胶原蛋白浓度更高。在非胶原蛋白剂量以及蛋白质印迹分析中检测到的III型胶原蛋白含量方面，两组肌腱之间未发现差异。明胶酶检测的酶谱分析显示，mdx小鼠中金属蛋白酶-2（活性异构体）和金属蛋白酶-9（潜伏异构体）的含量更高。使用偏振显微镜进行的双折射测量显示，与对照组相比，mdx小鼠肌腱中胶原纤维的分子排列更有序，出现更大面积的卷曲。丽春红S染色的肌腱切片显示mdx组细胞外基质染色更强。甲苯胺蓝染色切片显示对照组肌腱嗜碱性更强。在形态计量学方面，mdx组腱外膜中检测到更多的炎性细胞。总之，60日龄mdx小鼠的跟腱呈现出更高的胶原蛋白浓度和胶原纤维排列、增强的金属蛋白酶-2活性，以及炎性细胞的显著存在和较少的蛋白聚糖。
