Study On Skeletal Muscle Regeneration In Mdx Mice

Objective: Duchenne muscular dystrophy(DMD)is one of the highest incidence type of progressive muscular dystrophy with completely deficient of dystrophin. DMD is a X linked recessive lethal myopathy, characterized by progressive limb weakness and muscle atrophy, while the first symptom to the pelvic girdle muscle group weakness and gastrocnemius muscle pseudohypertrophy. After a lot of research, there are a variety of pathophysiological mechanisms involved in the formation and development of this diseases, such as oxidative stress、inflammation、apoptosis、 autophagy、intracellular calcium overload, etc. No matter what kind of change can cause degeneration and necrosis of muscle cells, how to repair the degeneration and necrosis, promoting muscle regeneration, restoration of muscle tissue function has become the focus of our study.At present, mdx mice is the classic models in studying muscular dystrophy. Like the DMD patients, cells membranes of mdx mice skeletal muscle did not express dystrophin protein. The histology of mdx mice is normal in the early, muscle cell degeneration and necrosis appeare in about 3 weeks, and showing symptoms of myasthenia gravis. With the development of the disease, muscle cell regeneration to occupy the dominant position, the disease tends to be stable, and weakness symptoms improved. While muscle cells regeneration of DMD patients is not obvious, weakness and becoming worse, and ultimately lose the ability to walk, even death. The pathogenesis of DMD and mdx mice both are dystrophin deficiency on muscle cell membrane, but the clinical manifestation of mdx mice is much lighter than the human. Regeneration well compensate for necrosis that may be the cause of mdx mice muscle mild weakness. So regeneration may play a decisive role in improving symptoms and delaying the progress of the disease.Regeneration of skeletal muscle is a necessary physiological process to maintain homeostasis, growth and development. The regeneration process involves: muscle cell degeneration and necrosis, activation of inflammation, inflammatory cytokine release, activation of stationary muscle satellite cells, and myoblast proliferation and differentiation. Muscle satellite cells are the main source of skeletal muscle growth and regeneration after the animal was born. When cells are stimulated by abnormal signals, they can proliferate and differentiate into new muscle fibers.Our study was designed to observe muscle histopathological differences between DMD patients and mdx mice, and the expression of regeneration associated gene in mdx mice at different stages, as well as Necdin and bex1 gene expression in mdx and C57/6 mice. To investigate the correlation between the two genes and the regeneration of mdx mice muscle cell, and finally, to provide a theoretical basis for the further discussion on the viability of muscle satellite cell activation factor treatment application for muscular dystrophy patients.Methods: The experiment was divided into two parts: Experiment 1,to observe muscle histopathological differences between DMD patients、 mdx mice and C57/6 mice; Experiment 2,to explore the expression of regeneration associated gene in mdx mice at different stages.Experiment 1: Select the DMD patients and normal persons, three cases each. All subjects were diagnosed by muscle biopsies with light microscopic and/or electronic microscopic examination. A signed informed consent was obtained in each subject. Open muscle biopsies of the bicipital or quadriceps muscles were performed on all subjects. Muscle biopsies were done for routine histological staining and immunohistochemical staining.C57BL/10 Sc Sn-Dmdmdx/JNju mice were used as the experimental animals and C57BL/6Sc Sn mice served as control group. There were four groups: 2weeks group, 4weeks group,8weeks group,12 weeks group. Each group included 3 mice. After 10% hydration aldehydes(350mg/kg body weight) intraperitoneal anesthesia was injected, extracted Bilateral quadriceps with routine histological staining.Experiment 2: The selection and grouping of experimental group and control group was the same with mentioned above. Each group included 3 mice. Muscle biopsies were used for study of gene expression profile and q RT-PCR detection. Gene chip technology were used to observe the expression of regeneration associated gene in mdx mice at different stages,and through the q RT-PCR to explore the expression of Necdin and bex1 gene in mdx and C57/6 mice.Data were analysed by spss 13.0.Results:1 The pathological characteristics of the muscle tissue in DMD patients.The histopathological changes of DMD patients in early symptoms and end-stage were observed through conventional staining. The pathological characteristics in early stages showed abnormal fibers varied in shape and size, rounded appearance, with muscle fibers degeneration, necrosis and regeneration, inflammatory cells infiltration. In contrast, Muscle tissue of end-stage in DMD patients presented connective tissue and adipose tissue hyperplasia obviously, remaining a small amount of muscle fiber only. Immunohistochemical staining showed dystrophin protein in muscle fiber membrane completely missing.2 The pathological characteristics of mdx mice muscle tissue at different stages.Through conventional staining,dynamic observation of different periods histopathological changes in mdx mice and control mice was performed.At 2weeks, mdx mice muscle tissue began to appear sporadic muscle fiber degeneration 、necrosis and inflammation cells infiltration, whereas, necrosis were hardly detectable in age-match control mice.At 4 weeks, numerous muscle fibers degeneration and swarms of muscle fiber necrosis arise, accompanied by more inflammatory cells infiltration, at the same time, scattered characterized by central nucleus, basophilic cytoplasm of regeneration fibers were Visible, On the contrary, control mice of 4weeks without the above pathological changes. At 8 weeks, pathological results as follows: Regeneration of muscle fiber clustered existence, necrotic fibers scattered distribution, conversely, Their normal control mice did not show obvious regeneration muscle fibers. At 12 weeks, The vast majority of muscle cells with multiple nucleus, and the nucleus to muscle membrane surrounding distribution, which indicated muscle cells mature gradually, however, the 12 weeks normal control mice did not demonstrat similar changes.3 The result of Gene chip(1)The result of cluster analysisCluster analysis of genes obtained from mdx mice in the 4 groups samples. Cluster thermograph acquired after Cluster analysis and each group of mdx mice samples clustered together. Among which gene expression was obvious difference between 2 weeks group and 12 weeks group in mdx mice.(2) The results of gene screeningComparison of Genetic testing results of mdx mice quadriceps muscle between 2 weeks and 12 weeks. p≦0.05,Fould-Change≧+2, Fould-Change≦﹣2 as the screening conditions. A total of differentially expressed genes were 2928, among them associated regeneration gene of muscle cells approximately have 368, 271 up-regulated and 97 decreased. Part of the difference genes is shown in Table 1. Necdin gene Fould-Change=10.1611,bex1 gene Fould-Change =-2.87293.4 The expression of Necdin and bex1 gene in skeletal muscle of mdx mice at different stages of the disease.q RT-PCR result showed that Necdin and bex1 exist in skeletal muscle cells of normal mice. Compared with control group, we found the gene levels of Necdin and bex1 were gradually enhanced in quadriceps of mdx mice at 4weeks group and 8weeks group, while decrease at 2 weeks group, the difference was statistically significant(p<0.05). In mdx mice, the gene levels of Necdin and bex1 were gradually enhanced in quadriceps at 8 weeks group compared with the rest of the groups, the difference was statistically significant(p<0.05).In C57/6 mice, the gene levels of Necdin and bex1 were gradually enhanced at 8 weeks group compared with other groups, the difference was statistically significant(p<0.05), and with the time prolonging the expression decrease gradually.Conclusions:1 With the observation of muscle histopathological of DMD patients and mdx mice, we can find regeneration of DMD patients muscle fibers is much less obviously than the mdx mice.2 A lot of gene participated in the regulation of mdx mice muscle regeneration. Necdin and bex1 gene existed in skeletal muscle cells of normal mice and had a high levels in the early stages of growth. Necdin and bex1 play a certain role in promoting the differentiation of mdx mice muscle cells, and may activate muscle satellite cells to promote muscle regeneration.