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BACKGROUND: Clinical studies have confirmed that the injection of nerve growth factor into the nerve damage site can improve the motor function of skeletal muscle. OBJECTIVE: To observe the effect of nerve growth factor on the proliferation of primary skeletal muscle satellite cells in rats. METHODS: The original generation of rat skeletal muscle satellite cells were cultivated by the tissue block method, and the third generation of skeletal muscle satellite cells were divided into three groups. The control group was added with culture medium; the low concentration group and the high concentration group were added with culture medium containing 10, 20 U/mL nerve growth factors, respectively. At 2, 4 and 6 days after culture, the cell morphology was observed by inverted phase contrast microscope and hematoxylin-eosin staining, and the expression of α-actin was observed by immunohistochemistry. At 1, 2, 3, and 4 days after culture, CCK-8 assay was used to detect the cell proliferation activity. RESULTS AND CONCLUSION: (1) Inverted phase contrast microscope displayed that with the increase of culture time, the number of skeletal muscle satellite cells in each group increased gradually, and the cell morphology gradually changed from round to fusiform, spindle or polygonal, and gradually fused and arranged along the same direction, and the number of cells in high concentration group was more than that in low concentration group and control group (P 0.05). (4) The CCK-8 assay results showed that cell viability of low and high concentration groups was higher than that of control group (P=0.000). (5) Above results confirm that high and low concentrations of nerve growth factors could promote the proliferation of skeletal muscle satellite cells, but had no effect on the expression of α-actin in skeletal muscle satellite cells.
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OBJECTIVE: To investigate the effect of electroacupuncture (EA) on muscular atrophy and expression of microRNAs (Mir-1, Mir-133a, Mir-133b) and some proliferation-related factors of muscle satellite cells as histone deacetylase4 (HDAC4) and the paired box transcription factor Pax7 (Pax7) in skeletal muscle atrophy rats. METHODS: Twenty-four male SD rats were randomly and equally divided into sham operation, model and EA groups. The skeletal muscle atrophy model was established by transection of the right sciatic nerve. EA (2 Hz, 1 mA) was applied to the right "Zusanli"(ST36) and "Huantiao"(GB30) for 10 min, once a day, seven times a week for 2 weeks. The wet weight of bilateral gastrocnemius muscles was measured to calculate the ratio of weight between the affected gastrocnemius muscle and healthy gastrocnemius muscle. The cross-sectional area (CSA) of the gastrocnemius muscle on the affected side was measured after H.E. staining. The expression levels of Mir-1, Mir-133a, Mir-133b, HDAC4 mRNA and Pax7 mRNA in the gastrocnemius muscle tissue were detected using quantitative real time-PCR. RESULTS: Compared with the sham operation group, the ratio of wet weight and CSA of the gastrocnemius muscle, and the expression levels of Mir-1 and Mir-133a were significantly decreased in the model group (P<0.01, P<0.05), while the expression levels of HDAC4 mRNA and Mir-133b significantly up-regulated in the model group (P<0.05). Following EA intervention, the decreased levels of the ratio of wet weight and CSA of the gastrocnemius muscle were significantly suppressed (P<0.01), suggesting an inhibition of the skeletal muscle atrophy, and the expression levels of Pax7 and HDAC4 mRNAs were notably up-regulated (P<0.05), and those of Mir-1, Mir-133a and Mir-133b were significantly or further significantly down-regulated relevant to the model group (P<0.05). CONCLUSION: EA intervention can delay muscular atrophy in rats with denervated gastrocnemius muscle, which may be related with its function in up-regulating the expression of Pax7 and HDAC4 mRNAs and down-regulating the expression of Mir-1, Mir-133a and Mir-133b.
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The robust capacity of skeletal muscle stem cells (SkMSCs, or satellite cells) to regenerate into new muscles in vivo has offered promising therapeutic options for the treatment of degenerative muscle diseases. However, the practical use of SkMSCs to treat muscle diseases is limited, owing to their inability to expand in vitro under defined cultivation conditions without loss of engraftment efficiency. To develop an optimal cultivation condition for SkMSCs, we investigated the behavior of SkMSCs on synthetic maltose-binding protein (MBP)-fibroblast growth factor 2 (FGF2)-immobilized matrix in vitro. We found that the chemically well-defined, xeno-free MBP-FGF2-immobilized matrix effectively supports SkMSC growth without reducing their differentiation potential in vitro. Our data highlights the possible application of the MBP-FGF2 matrix for SkMSC expansion in vitro.
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In Vitro Techniques , Maltose-Binding Proteins , Muscle, Skeletal , Muscles , Stem CellsABSTRACT
Hypoxic condition of skeletal muscle is caused not only by hypoxia exposure but also by exercise and disease etc. It is thought that clarifying a mechanism of response to hypoxia in vivo is useful for developing better training methods and treatment strategies. However, at present, research dealing with the effects of hypoxic exposure on skeletal muscle have not shown consistent results. Hypoxic exposure results in angiogenesis or muscle atrophy as morphological changes in skeletal muscle. Applications of hypoxic exposure include intermittent hypoxic exposure and hypoxic training, both of which may lead to angiogenesis in a mechanism different from normal hypoxic exposure. In this review, we present some findings on the effects of hypoxia exposure on skeletal muscle and discuss whether satellite cells are involved in promoting angiogenesis by hypoxia.
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Objective To investigate the cell-fusion modes and contractive characteristics of myotubes formed by muscle satellite cells in vitro. Methods The muscle satellite cells in quadriceps femoris of six 2-day-old SD rats were isolated, cultured and identified by immunofluorescence; the cell-fusion modes and contractive characteristics of myotubes were observed and recorded under microscope. Results The positve cells expressing marker Pax7 were obtained by immunofluorescence, and the purity was 90.3%. Myotubes were fused by several cells, cell and myotube or myotube and myotube. There were myotubes of spontaneous contraction on the conditions of no acetylcholine, no electric stimulation or no contiguous cells. Conclusion The myotubes of spontaneous contraction are fused by muscle satellite cells through three cell-fusion modes in vitro.
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@#Objective To observe effects and mechanism of electroacupuncture (EA) on denervated skeletal muscle atrophy. Methods Forty-nine male Sprague-Dawley rats were randomly divided into normal group (group A, n=7), natural recovery group (group B, n=21) and EA group (group C, n=21). The groups B and C, established the model of denervated skeletal muscle atrophy by transecting the sciatic nerve of rats, were divided into subgroups of 7 days, 14 days, 21 days postoperation, seven in each subgroup. Electroacupuncture was given to the group C at Zusanli (ST36) and Chengshan (BL57) once a day since 24 hours after modeling. The muscle wet weight ratio of the affected gastrocnemius was determined. Cross-sectional area and fiber diameter of the gastrocnemius were measured with HE staining. The expression of insulin-like growth factor-1 (IGF-1), Myostatin and proliferating cell nuclear antigen (PCNA) protein and gene in the gastrocnemius were detected with Western blotting and RT-PCR. Results The wet weight ratio, cross-sectional area and fiber diameter were less in the groups B and C than in the group A (P<0.001), and they were more in the group C than in the group B (P<0.001). Compared with the group B, the protein and gene of IGF-1, PCNA increased in the group C (P<0.05), while the Myostatin decreased (P<0.05). Conclusion Electroacupuncture can increase the expression of IGF-1 and decrease the expression of Myostatin, to promote the proliferation of satellite cell, which may relate with the prevention of denervated skeletal muscle atrophy.
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Objective To investigate the effect of Panax notoginsenosides monomers ginsenoside Rg 1 in inhibiting hepatic fibro-sis .Methods The rat model of hepatic fibrosis was established by using 50% Ccl4 ,total 35 d .The different doses of Rg1was ad-ministered by hypodermical injection .At the end of the treatment ,the pathological changes of hepatic tissue were observed by light and transmission electron microscope .The stereological method was adopted to measure the volume density (Vvm) ,area density (Svm) ,specific surface(Qm) and surface number density (Nam) of liver cell mitochondria in various groups .Results The stereo-logical data of liver cell mitochondria showed that the statistical differences existed among various groups .Vvm in the Panax Notog-insenosides ,low dose Rg1 and isotonic saline groups were significantly increased compared with the normal control group with sta-tistical difference(P0 .05);Vvm in the high ,middle and low dose Rg1 ,Panax No-toginsenosides and colchicine groups showed the decreasing trend compared with the isotonic saline group without statistical differ-ence(P>0 .05) .Svm in the low dose Rg1 ,Panax Notoginsenosides ,colchicine and isotonic saline groups were significantly increased compared with the normal group with statistical difference (P0 .05) .Conclu-sion Rg1 has antifibrosis effects of Panax notoginsenosides ,even exceeds Panax notoginsenosides in some aspects ,and the above-mentioned effect is positively correlated with dose .Rg1 is an ideal drug for preventing and treating liver fibrosis .
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We investigate the effects of training and detraining on the satellite cell activation in thoroughbred horse muscles after an exhaustive exercise. Six horses were subjected to conventional training for 18 weeks and detraining for 6 weeks. Before training (Pre), after 10-week and 18-week training (10Tr and 18Tr), and after 6-week detraining (DTr), an incremental exercise test (IET) was performed on inclined treadmill to measure VO<sub>2</sub>max and the velocity at a plasma lactate of 4 mmol/l (VLA4). Biopsy samples from gluteus medius muscle was obtained before and at 1 minute (1min), 3 hour (3hr), 6 hour (6hr) and 1 day after each IET. Number of muscle satellite cell were counted in type identified muscle fibers by immuno-histochemical stain images. The levels of mRNA expressions were determined using real time RT-PCR system. The number of satellite cells in 10Tr was significantly higher in type IIa fibers (0.31±0.10) than Pre (0.15±0.06). As compared to each value before IET, IL-6 mRNA expression (fold change) increased remarkably at 6hr after IET in 10Tr (x 2290.2) and 18Tr (x 2304.2), but not in both Pre (x 260.0) and DTr (x 853.3). IGF-I and Myogenin mRNA expressions were significantly increased at 1 day after IET in 18Tr (x 6.6 and x 3.3), but not in both Pre and DTr. These results suggested that the increased reactivity of satellite cells by training for 18 weeks is almost disappeared after detraining for 6 week, as well as VO<sub>2</sub>max and VLA4.
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@#Skeletal muscle has high regenerative capability. It is able to regenerate completely after acute traumatic damage, while the repairing process often accompanied by fibrosis in the chronic degenerative conditions such as muscular dystrophy and repeated muscle fiber damage. Through in-depth study on the mechanisms of skeletal muscle fibrosis in the past decade, it has been found that a variety of cells and regulatory molecules involved in the process, especially muscle satellite cells-derived myofibroblasts, transforming growth factor β (TGF-β) and other fibrosis promoting growth factors. This review focused on the cellular and molecular mechanisms of the skeletal muscle fibrosis and relevant antagonistic strategies.
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Skeletal muscle has high regenerative capability. It is able to regenerate completely after acute traumatic damage, while the repairing process often accompanied by fibrosis in the chronic degenerative conditions such as muscular dystrophy and repeated muscle fi-ber damage. Through in-depth study on the mechanisms of skeletal muscle fibrosis in the past decade, it has been found that a variety of cells and regulatory molecules involved in the process, especially muscle satellite cells-derived myofibroblasts, transforming growth factor β(TGF-β) and other fibrosis promoting growth factors. This review focused on the cellular and molecular mechanisms of the skeletal muscle fibrosis and relevant antagonistic strategies.
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Skeletal muscle fiber has a great ability to hypertrophy during growth and in response to exercise stimuli, and atrophy during aging and in response to disuse. Because the muscle fiber is a multinucleated cell, the region of cytoplasm governed by a single myonucleus (myonuclear domain; MND) is a very important factor for understanding muscle plasticity. Although the MND size varies with fiber type, metabolic property and species, it was considered that the size was maintained constantly during muscle adaptation. Recently, however, there have been many studies demonstrating the variability of the MND size. In some of these studies, it is hypothesized that muscle hypertrophy is achieved by increase in protein synthesis rate until the ‘ceiling’ of MND size and subsequent addition of myonuclei by satellite cell activation. On the other hand, during muscle atrophy, the myonuclei seems to be long lasting as ‘muscle memory’ storing information about previous size to prepare for recovery of muscle fiber. Understanding the variability of MND size more deeply would provide fundamental insights into the mechanism of skeletal muscle plasticity.
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Bex1 protein is upregulated in regenerating muscle and interacts with calmodulin, a Ca2+-binding protein involved in cell cycle regulation. Following cardiotoxin-induced injury the regenerating muscle of Bex1 knock-out mice exhibits prolonged cell proliferation and delayed cell differentiation compared to wild-type mice. To gain insight into this process, we compared the regenerating myogenic morphologies of Bex1 knock-out and wild-type mice at several time points. Bex1-positive cells were identified by double immunofluorescence staining. These studies demonstrated that a population of cells that are Bex1-positive after injury are c-Met/basal lamina-positive and Mac-1-negative indicating that they are derived from at least a subset of myogenic progenitor/satellite cells but not invading immune cells. In addition, in regenerating muscle, Bex1 co-localizes with calmodulin in the cytoplasm of the late myoblast or early myotube stage of myogenesis. These results suggest that Bex1 participates in muscle regeneration through the regulation of satellite cell proliferation and differentiation by its interaction with calmodulin. Current studies of Bex1 may provide a new molecular tool for the identification of activated satellite cell and open the way to new or improved therapeutic regimens against progressive muscular atrophy.
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Animals , Mice , Calmodulin , Cell Cycle , Cell Differentiation , Cell Proliferation , Cytoplasm , Fluorescent Antibody Technique , Mice, Knockout , Muscle Development , Muscle Fibers, Skeletal , Muscles , Muscular Atrophy, Spinal , Myoblasts , Regeneration , Satellite Cells, Skeletal MuscleABSTRACT
Bex1 protein is upregulated in regenerating muscle and interacts with calmodulin, a Ca2+-binding protein involved in cell cycle regulation. Following cardiotoxin-induced injury the regenerating muscle of Bex1 knock-out mice exhibits prolonged cell proliferation and delayed cell differentiation compared to wild-type mice. To gain insight into this process, we compared the regenerating myogenic morphologies of Bex1 knock-out and wild-type mice at several time points. Bex1-positive cells were identified by double immunofluorescence staining. These studies demonstrated that a population of cells that are Bex1-positive after injury are c-Met/basal lamina-positive and Mac-1-negative indicating that they are derived from at least a subset of myogenic progenitor/satellite cells but not invading immune cells. In addition, in regenerating muscle, Bex1 co-localizes with calmodulin in the cytoplasm of the late myoblast or early myotube stage of myogenesis. These results suggest that Bex1 participates in muscle regeneration through the regulation of satellite cell proliferation and differentiation by its interaction with calmodulin. Current studies of Bex1 may provide a new molecular tool for the identification of activated satellite cell and open the way to new or improved therapeutic regimens against progressive muscular atrophy.
Subject(s)
Animals , Mice , Calmodulin , Cell Cycle , Cell Differentiation , Cell Proliferation , Cytoplasm , Fluorescent Antibody Technique , Mice, Knockout , Muscle Development , Muscle Fibers, Skeletal , Muscles , Muscular Atrophy, Spinal , Myoblasts , Regeneration , Satellite Cells, Skeletal MuscleABSTRACT
Objective: To study the mechanism of Tuina in the treatment of skeletal muscle injury. Methods: Rabbits were heavily beaten at gastrocnemius muscle to make acute contusion model and then treated respectively by early Tuina and routine Tuina. The number of satellite cells of skeletal muscles was observed. Results: The number of the satellite cells continued to grow in both groups, and it began to increase significantly 3-5 days after Tuina treatment. Early Tuina treatment produces larger number of satellite cells than routine Tuina treatment.Conclusion: Early Tuina treatment is helpful to the marked recovery of skeletal muscles by increasing the number of satellite cell.
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Objective To find the features of cryo-injured infarcted model and the therapeutic effects of skeletal satellites cells(SCs) on myocardial infarction(MI).Methods SCs were implanted into the center of cryo-injured myocardium.Immunohistochemistry,confocal immunofluorescence double-labelling staining and transmission electron microscopy were used to study the state of SCs.Results After cryo-injured frontal wall of the left ventricle(LV) for approximately(15 s) by aluminium rod with(5 mm) in diameter,it made 43% weight of LV ischemia and 17% weight of LV infarcted.At 2nd week,injured area occured as transmural infarct.After transplantation,the implanted cells survived in large numbers.The transplanted cells proliferated remarkably at early phase and differentiated into many multinuclear myotubes after 2 weeks.Some of them developed into muscle fibers with better sarcomeres,but the gap junction was not formed between implanted cells and cardiac myocytes.Conclusions Cryo-injured infarcted model is a proper model to study myocardial infarction.Skelatal satellite cell transplantation will repair morphological structure partly of myocardial infarction area.
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This review explores novel possibilities of skeletal muscle hypertrophy mechanism based on previous studies. Hypertrophy and/or regeneration of skeletal muscles are caused by activation of satellite cells, induced by mechanisms such as growth factors and cytokines. Many unsolved problems, however, yet remain concerning signaling pathways, activated by such substances, and regulation of transcriptional factors and cell cycles.In recent years, possible involvement of not only satellite cells but also tissue-specific stem cells in skeletal muscle hypertrophy has been reported from studies made on stem cell transplantation in muscle regeneration.It has been elucidated that differentiation plasticity of tissue-specific stem cells contributes to hypertrophy and/or regeneration of skeletal muscles. Moreover, satellite cells have come to be regarded as one kind of tissue-specific stem cells, since they have been known to exhibit diversity, by their differentiating into not only skeletal muscle cells but also other tissue cells, as well as self renewal capacity.This review also summarizes findings on signaling pathways based on ligand receptor characterization. Among the various signaling pathways, focus was especially placed on the possibilities of Wnt signaling pathway and Notch signaling pathway on skeletal muscle hypertrophy, indicating that Wnt expression in adult muscle stem cells leads to regulation of skeletal muscle hypertrophy.It has also become evident that Notch signaling pathway is associated with the activation of satellites cells, and that functional decline of such signaling pathway causes a decline in the activation of satellite cells. Such functional decline of Notch signaling pathway has also been revealed as the reason for the decline of regenerative capacity of skeletal muscles due to advancing age. Further discussion on the involvement of Notch signaling pathway in skeletal muscle hypertrophy is made based on these results. As indicated above, the roles of ligands and/or receptors of canonical growth factors, signaling pathways, and transcriptional factors contributing to skeletal muscle hypertrophy were reexamined in this review, after which the contribution of tissue-specific cells to skeletal muscle hypertrophy was discussed based on the results of muscle regeneration studies. Furthermore, focus was centered on Wnt and Notch signaling pathways, both of which are attracting renewed attention, and study was made on the possibility of the involvement of these pathways in skeletal muscle hypertrophy process.
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Satellite cells, as the major stem cells, are responsible for postnatal skeletal muscle growth, regeneration and maintenance. As a result, satellite cells have great potential as therapeutic agents. Activation of satellite cells in vivo is a key link in the muscle regeneration processes. And their replenishment is necessary to keep the capacity of skeletal muscle to regenerate after recurrence of muscle damage. Cellular and molecular regulation of activation and replenishment for satellite cells issues were reviewed. Hoping through the points of nitric oxide-hepatocyte growth factor (NO-HGF), myostatin and Notch signaling and the niche of satellite cells to overcome the recent obstruction in cell therapy in clinic myopathy, such as Duchenne muscle destrophy.
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Objective To establish a method of isolation and purification of rat skeletal muscle satellite cells,and observe the characteristics of proliferation and myotube cell formation of skeletal muscle satellite cells cultured in vitro. MethodsPurified skeletal muscle satellite cells were obtained by improved two-step enzymatic digestion method and pre-plating technique.Immunohistochemical staining was employed to identify the skeletal muscle satellite cells cultured in vitro.The growth of skeletal muscle satellite cells cultured in vitro was examined by MTT assay.The differentiation of skeletal muscle satellite cells was observed by inverted microscopy. Results The skeletal muscle satellite cells with higher purity were obtained and confirmed by the high expression of desmin.When cultured in vitro,the latent phase of skeletal muscle satellite cells was the first to the second day,and the platform phase was the fifth to the sixth day.Myotube cells gradually formed when cell confluence was more than 60% to 70% or differential medium with lower fetal bovine serum was used.Conclusion The combination of improved two-step enzymatic digestion method and pre-plating technique serve as an easy and practical way to obtain skeletal muscle satellite cells with higher purity.Skeletal muscle satellite cells can form myotube cells with contraction characteristics without any special induction.
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Objective To investigate the effect of auto-skeletal muscle satellite cell implantation into ischemic myocardium on cardiac function and the mechanisms.Methods Approximately 10 7 to 10 9 muscle satellite cells(SCs)were cultivated in vitro.The left anterior descending(LAD)artery was ligated in Wistar rats to create myocardial infarction(MI).Some rats only underwent sham operation served as control.Two weeks after MI,autologous SCs,serum-free culture medium and sodium chloride injection were injected into ischemic myocardium of implantation rats(n=15),control rats(n=15)and myocardium around LAD of sham operation rats(SO,n=15),respectively.Four weeks after injection,hemodynamic parameters and cardiac function in all groups were evaluated by polygraph system,capillary density in the ischemic myocardium was demonstrated by immunohistochemical method,serum VEGF concentration was examined by ELISA,and the differentiated myofibers from SCs in the infarcted site were observed by pathologic examination and immunohistochemical method.Results Four weeks after injection,the SCs had progressively differentiated into striated muscle fibers in the myocardial infarction site,and immunohistochemical analysis confirmed their skeletal muscle origin.Compared with the SO rats,systolic blood pressure(SBP),diastolic blood pressure(DBP),mean ar-tery pressure(MAP),left ventricular systolic pressure(LVSP)and dp/dtmaxwere markedly decreased(P
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Objective Satellite cells(SCs) transplantation was tried to improve cardiac function in this experiment.Methods A myocaridium infarction was created in 23 male Suzhong pigs by ligating the diagonal ramus,and the alive pigs were divided into the experimental group and the control group.SCs were extracted,purified,cultured by modified Dorfman method,and were transplanted into myocardium by intramyocardial injection.By pathomorphological research,we observed the therapeutic outcomes of heart failure.Results ⑴10 pigs were alive(5 in the control group,and the other 5 in the experimental group).The successful rate of making mould was 43 5%.⑵After cultured in vitro for 3 days,SCs were spindle-shaped,and sticked to walls.SCs growth were stasis and each other fusing into form myotube when we delayed to divede Petri dishs or reduced the concentration of Fetus Bovine Serum(FBS) in the culture medium.⑶In the experimental group,neonate skeletal muscle cells were observed in the infarction regions,which were multi-nuclei near edges.These cells aligned in an accordance direction.Intercalated disks were not found between them.The capillary density in experimental group was much higher than the control group.Conclusions Autograft of SCS can be alive in acceptor,and improve the heart function by altering cardiac compliance and configuration,or by stimulating the blood capillary proliferation.SCs don't form intercalated disks with remnant myocardial cells,so synchro-contraction will not occur between them,but malign arrhythmia are not found.