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ABSTRACT Introduction: Skeletal muscle satellite cells are considered the unique source of stem cells for myogenic differentiation of adult skeletal muscle cells. Upon stimulation, the skeletal muscle satellite cell can be activated through specific signaling pathways, proliferate and differentiate into a muscle cell. An analysis of the effects of key signaling pathways could provide the basis for an in-depth study of skeletal muscle formation in athletes and muscle development. Objective: This paper analyzes the effects of key signaling pathways on skeletal muscle satellite cell proliferation and differentiation. Methods: We divided 32 athletes into four groups: control, stretching, experimental, and mixed groups. The control group received no training at all, the stretching group and the experimental group received stretching training on the right gastrocnemius. The mixed group also got weight climbing training in the stretching training, initial load 30% of the athlete's weight, increasing 25% each week until 100% of body weight, at the frequency of 3 times a week. After training, gene expression of live satellite cells was measured by intramuscular signaling. Results: The FGM level of the antagonistic group (3.56±0.21) was higher than in the control group (3.25±0.18). The gene expression of HGF mRNA was higher in the mixed group (2.16±0.24) followed by the antagonistic group (2.02±0.15), the stretching group (1.81±0.25), and the control group (1.03±0.06). Conclusion: Both stretching and antagonistic training can increase gene expression in signaling pathways. Antagonistic training significantly increased the expression of HGF, MGF, and mRNA. This activity can promote muscle bulking and skeletal muscle enlargements. Evidence Level II; Therapeutic Studies - Investigating the result.
RESUMO Introdução: As células satélites musculares esqueléticas são consideradas a única fonte de células-tronco para a diferenciação miogênica das células musculares esqueléticas adultas. Após a estimulação, a célula satélite muscular esquelética pode ser ativada através de vias de sinalização específicas, proliferar e diferenciar-se em célula muscular. Uma análise sobre os efeitos das principais vias de sinalização poderia estabelecer as bases para um estudo aprofundado da formação muscular esquelética nos atletas e do desenvolvimento muscular. Objetivo: Este artigo analisa os efeitos das principais vias de sinal na proliferação e diferenciação das células satélites musculares esqueléticas. Métodos: Dividimos 32 atletas em quatro grupos. Grupos controle, alongamento, experimental e grupo misto. O grupo controle não recebeu treinamento algum, o grupo de alongamento e o grupo experimental receberam treinamento de alongamento no gastrocnêmio direito. O grupo misto também obteve treinamento de escalada com peso no treino de alongamento, carga inicial de 30% do peso do atleta, aumentando 25% em cada semana até 100% do peso corporal. Na frequência de 3 vezes por semana. Após os treinos, a expressão genética das células satélites vivas foi medida por intermédio da sinalização proveniente de coleta intramuscular. Resultados: O nível de MGF do grupo antagônico (3.56±0.21) foi maior que no grupo controle (3.25±0.18). A expressão gênica do mRNA HGF foi maior no grupo misto (2.16±0.24) seguido pelo antagônico (2.02±0.15), o grupo de alongamento (1.81±0.25) e o grupo controle (1.03±0.06) Conclusão: Tanto o treinamento de alongamento quanto o treinamento antagônico podem aumentar a expressão genética nas vias de sinalização. O treinamento antagônico aumentou significativamente a expressão de HGF, MGF e mRNA. Essa atividade pode promover volume e hipertrofia muscular. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.
RESUMEN Introducción: Las células satélite del músculo esquelético se consideran la única fuente de células madre para la diferenciación miogénica de las células musculares esqueléticas adultas. Tras la estimulación, la célula satélite del músculo esquelético puede activarse a través de vías de señalización específicas, proliferar y diferenciarse en una célula muscular. Un análisis sobre los efectos de las vías de señalización clave podría sentar las bases para un estudio en profundidad de la formación del músculo esquelético en los atletas y del desarrollo muscular. Objetivo: Este trabajo examina los efectos de las vías de señalización clave en la proliferación y diferenciación de las células satélite del músculo esquelético. Métodos: Dividimos a 32 atletas en cuatro grupos. Grupos de control, de estiramiento, experimentales y mixtos. El grupo de control no recibió ningún entrenamiento, el grupo de estiramiento y el grupo experimental recibieron un entrenamiento de estiramiento en el gastrocnemio derecho. El grupo mixto también recibió entrenamiento de escalada con pesas en el entrenamiento de estiramiento, con una carga inicial del 30% del peso del atleta, aumentando un 25% cada semana hasta el 100% del peso corporal. Con una frecuencia de 3 veces por semana. Tras el entrenamiento, se midió la expresión génica de las células satélite vivas mediante la señalización de la recogida intramuscular. Resultados: El nivel de FGM del grupo antagonista (3,56±0,21) fue mayor que en el grupo de control (3,25±0,18). La expresión génica del ARNm del HGF fue mayor en el grupo mixto (2,16±0,24), seguido del grupo antagonista (2,02±0,15), el grupo de estiramiento (1,81±0,25) y el grupo de control (1,03±0,06) Conclusión: Tanto el entrenamiento de estiramiento como el antagonista pueden aumentar la expresión génica en las vías de señalización. El entrenamiento antagónico aumentó significativamente la expresión de HGF, MGF y mRNA. Esta actividad puede promover el aumento de volumen muscular y la hipertrofia. Nivel de evidencia II; Estudios terapéuticos - Investigación de resultados.
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Objective To investigate the effect of incremental load training on AMP-activated protein kinase (AMPK) phosphorylation in skeletal muscle satellite cells of aged mice. Methods Experimental mice were divided into 3 groups: young control group (YC group, n=12), old control group (OC group, n=12) and old training group (OT group, n=12). The mice in OT group received incremental load training, and CD45-/CD31-/Sca1-/VCAM (CD106) + cells were isolated by flow cytometry sorting. Desmin, Myod myogenic staining and myogenic differentiation culture were used for identification of muscle satellite cells, and the p-AMPK level of muscle satellite cells was detected by immunohistochemistry combined with Western blotting method. Results The expression levels of AMPK and p-AMPK in skeletal muscle satellite cells in YC group were significantly higher than those in OC group (P0.05), while p-AMPK expression level in OT group was significant higher than that in OC group (P<0.05). Conclusions Incremental load training can promote AMPK phosphorylation of skeletal muscle satellite cells in aged mice, and improve energy metabolism of skeletal muscle in aged mice.
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Objective:To study the effect of treadmill exercise and massage shortly after acute injury on expression of key growth factor in muscle satellite cells activation, insulin-like growth factor-1 (IGF-1), and mitogen-activated protein kinase (MAPK) / mitogen-activated protein kinase kinase (MEK) / extracellular regulated protein kinases (ERK)1/2 signaling pathways in muscle satellite cell in rats. Methods:A total of 40 SPF male adult Sprague-Dawley rats were randomly divided into groups A (n = 8), B (n = 8), C (n = 8), D (n = 8) and E (n = 8). Group A did not receive any treatment, while the other rats were contused the gastrocnemius muscle with self-made impactor. Group B received no intervention, groups C and D received massage and treadmill exercise shortly after injury, respectively, while group E received both treadmill exercise and massage shortly after injury. As the model was established, samples of gastrocnemius were obtained from all the rats 24 hours after injury, and observed under HE staining, detected the expression of MyoD1 and MyoG mRNA with reverse transcription polymerase chain reaction (RT-PCR), and expression of IGF-1, p-MAPK, p-MEK and p-ERKI/2 protein with Western blotting. Results:The expression of MyoD1 mRNA was more in groups C, D and E than in group B, which was the most in group C; less expression of MyoG mRNA, which was the most in group E (P < 0.05). The expression of p-MAPK, p-MEK and p-EPK1/2 was more in groups C, D and E than in group B, which was the most in group D (P < 0.05). The expression of IGF-1 increased in group C compared with that in group B (P < 0.05), and it decreased in group D (P < 0.05). Conclusion:Early intervention of treadmill exercise and massage may promote the activation of muscle satellite cells in different ways.
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Objective:To investigate the effects and mechanism of Tuina on denervation-induced atrophy. Methods:A total of 42 Sprague-Dawley rats were randomly divided into sham group (n = 6), model group (n = 18) and Tuina group (n = 18). The model group and Tuina group freed and excised right tibia nerve about one centimeter, while the sham group freed the right tibia nerve only. From the second day after operation, Tuina group accepted Tuina on the injured area, while the sham group and the model group were only fixed without any intervention. Six rats were sacrificed on the 14th, 21st and 28th day after operation in the model and Tuina groups, and the sham group was sacrificed on the 28th day after operation. The gastrocnemius muscles were measured wet weight ratio. The diameter and area of muscle cells were measured under HE staining. The expression of Pax7, MyoD, MyoG, microRNA-1, microRNA-133a and microRNA-206 in the gastrocnemius muscles were detected with reverse transcription real-time quantitative polymerase chain reaction. Results:Compared with the sham group, the wet weight ratio, the area of muscle cells (except the 14-day-Tuina group) and the diameter of muscle cells decreased at each time point in the model group and Tuina group (P < 0.05); compared with the model group, the wet weight ratio, muscle cell diameter and muscle cell area increased at each time point in Tuina group (P < 0.05). Compared with the sham group, the expression of Pax7 increased in the 14-day-model group (P < 0.05) and decreased in the 28-day-model group (P < 0.05), and it increased at each time point (except 28-day) in Tuina group (P < 0.05); compared with the model group, the expression of Pax7 increased at each time point in Tuina group (P < 0.05). Compared with the sham group, the expression of MyoD and MyoG increased at each time point in the model group and Tuina group (P < 0.05); compared with the model group, the expression of MyoD and MyoG increased at each time point (except 14-day) in Tuina group (P < 0.05). Compared with the sham group, the expression of microRNA-1 and microRNA-133a decreased, and microRNA-206 increased in the model group and Tuina group at 21-day (P < 0.05); compared with the model group, the expression of microRNA-1, microRNA-133a and microRNA-206 increased in Tuina group (P < 0.05). Conclusion:Tuina may activate the Pax7/MyoD/MyoG pathway by increasing the expression of muscle-specific microRNA, to promote the proliferation and differentiation of muscle satellite cells, and delay denervation-induced atrophy.
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Objective:To explore the effects and mechanism of electroacupuncture (EA) on expression of myostatin (MSTN), muscle-specific ring finger protein 1 (MuRF1/Trim63), F-box only protein 32 (Atrogin-1/ Fbxo32), myogenic differentiation antigen (Myod) and myogenin (Myog) in traumatic spinal cord injury (TSCI) rats. Methods:A total of 45 adult female Sprague-Dawley rats were randomly divided into sham operation group (n = 12) and operation group (n = 33). The TSCI model was established with the modified Allen's method. After modeling, there were 24 survival rats and they were randomly divided into model group (n = 12) and EA group (n = 12). EA group was electroacupunctured at Dazhui (DU 14), Mingmen (DU 4) and bilateral Zusanli (ST 36) for 10 minutes, once a day, six times a week for 28 days. Basso-Beattie-Bresnahan (BBB) score was tested before modeling, and three days, seven days, 14 days, 21 days and 28 days after modeling. The rats were measured their body mass before and 28 days after modeling. The ratio of gastrocnemius wet mass was calculated; the cross-sectional area (CSA) and fiber diameter were measured by HE staining; the expression of MSTN, Trim63, Fbxo32, Myod and Myog mRNA were tested with real-time quantitative polymerase chain reaction (qPCR). Results:Three days, seven days, 14 days, 21 days, and 28 days after modeling, the score of BBB was lower in the model group than in the sham operation group (P < 0.01); seven days, 14 days, 21 days, and 28 days after modeling, the score of BBB was higher in EA group than in the model group (P < 0.01). Compared with the sham operation group, the mass of rats, the gastrocnemius wet mass, the CSA and the diameter of the muscle fiber were smaller in the model group (P < 0.05), while the expression of MSTN, Trim63, Fbxo32, Myod and Myog mRNA were higher (P < 0.05). Compared with the model group, the mass of rats, the gastrocnemius wet mass, the CSA, the expression of Myod and Myog mRNA were higher (P < 0.05) in EA group, while the expression of MSTN, Trim63 and Fbxo32 mRNA were lower (P < 0.05). Conclusion:EA might delay the gastrocnemius atrophy in TSCI rats by down-regulating the expression of MSTN, Trim63, Fbxo32 mRNA and up-regulating the expression of Myod and Myog mRNA via controlling the differentiation of the muscle satellite cells and the degradation of protein in skeletal muscle cells.
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OBJECTIVE: To explore the effect of electroacupuncture (EA) serum on expression of myogenic differentiation antigen (Myod) and autophagy-related protein Beclin 1 in cultured muscle satellite cells of rats under starvation conditions. METHODS: The primary multifidus muscle satellite cells of one male SD rat were isolated and cultured to obtain the 3rd generation of cells. The EA serum was got from the rat received EA stimulation of bilateral "Weizhong" (BL40, 2 Hz/10 Hz, 1 mA, duration of 20 min, once daily for 7 days). The cell suspension (2×104/well) of the 3rd generation of cultured cells was transferred to each well of a 96-well plate in medium containing 10% fetal bovine serum (FBS). Twelve duplicate wells were set up for the blank control serum (without FBS), 10% FBS, 10% EA serum, 20% EA serum and 30% EA serum groups and incubated for 12 h and 24 h, respectively. Each well was supplemented with 10 µL CCK-8 reagent to be incubated for 1 h again for observing the state of cell proliferation. After culturing the primary muscle satellite cells in serum-free medium for 12 h, the cells were randomly divided into serum-free group, 10% fetal bovine serum group and optimal concentration electroacupuncture serum group, and serum of corresponding concentration was added respectively. The expression levels of Beclin 1 and cell-proliferation-related protein Myod were detected by Western blot. RESULTS: CCK-8 assay displayed that the proliferation levels were significantly higher at 12 h and 24 h after serum intervention in the 10% FBS, 10% EA serum, 20% EA serum and 30% EA serum groups than that in the blank control serum group (P0.05). As a result, 10% EA serum was selected as the optimal concentration for Western blot tests. No significant difference was found in the expression levels of Myod and Beclin 1 proteins among the serum-free, 10% FBS and 10% EA serum groups before intervention (P>0.05), and there was a marked up-regulation of Myod expression and an obvious down-regulation of Beclin 1 expression at 12 h in both the 10% EA serum and 10% FBS groups in comparison with their own pre-intervention (P0.05). CONCLUSION: EA serum can promote proliferation of cultured muscle satellite cells under starvation conditions, which is related to its functions in regulating expression of Beclin 1 and cell-proliferation-related protein Myod.
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Objective To explore any effect of calorie restriction on the proliferation of satellite cells in the skeletal muscles of elderly rats.Methods Twelve male C57BL rats aged 12 or 13 months were randomly divided in to an experimental group and a control group,each of 6.The control group was fed 75.09 kJ/d as normal,while the experimental group was provided with 45.05 kJ/d (60％ of normal).The intervention lasted for 15 weeks and each rat's weight was measured every week.After the intervention,limb muscle satellite cells were sorted by fluorescenceactivated cell sorting after digestion,and the cell cycle was analyzed.Western blotting was used to assess the expression of cyclin A,D1 and E.Results There was no significant difference in the average weight of the two groups before the experiment.After the 15 weeks the average weight of the experimental group had decreased significantly (to 19.5±0.4 g),and it was significantly lighter than that of the control group (31.9±0.5 g).The average percentage of the satellite cells in the G0/G1 phase had decreased significantly in the experimental group,but the percentage in the S phase had increased significantly.The expression of cyclin A and E was significantly greater in the experimental group compared with the control group,but the expression of cyclin D1 had decreased significantly.Conclusion Caloric restriction can delay the proliferation of satellite cells in the skeletal muscles of elderly mice.
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Objective To construct human acidic fibroblast growth factor (aFGF) recombinant eu karyotic expression vector and transfect it into muscle satellite cells(MSCs) of rat,in purpose of further study the method to set up cell bank.Methods The aFGF gene was cloned from human total RNA which was obtained from human skeletal muscle tissue by RT-PCR method.Human interleukin 2 (IL-2) signal peptide sequence (SPS) was obtained by direct chemosynthesis method.Then aFGF and SPS were fused to obtain SPS-aFGF.Finally,directional cloning SPS-aFGF into pEGFP-N1,the recombinant (pEGFP-N1-SPS-aFGF) was obtained.The recombinant was confirmed by endonuclease digestion and DNA sequencing.MSCs were purified by difference-speed adherence method and were ideontified by immunofluorescence assay.The correct cells were divided into 3 groups:Experimental group (aFGF +N 1),control group (N 1),blank group (blank).All the groups were transfected by Lipofectamine 2000TM Reagent,and pEGFP-N1-SPS-aFGF,pEGFP-N1 were respectively added in experimental group and control group while blank group was added none plasmid.Fluorescence microscope was employed to detect transfection efficiency tendency along with time changes.The expression of target gene was detected by fluorescent quantitation PCR and Western blot.Results (1) The sequencing of pEGFP-N1-SPS-aFGF was completely correct and the outcome of endonuclease was equal to actual ban s-ize.(2)The expression of GFP in transfected cells were observed by fluorescencemicroscope and transfection efficiency reached the peak at 72 h.(3)Real-time fluorescent quantitation PCR proved strong aFGF mRNA expression in transfected cells (the average relative expression of experimental group was 1464.95)with aFGF gene,while it was detected a little in the other groups (the average relative expression of control group was 1.016 and blank group was 1.000) (P ＜ 0.05).Western blot also proved strong expression in Experimental group then the other two groups.Conclusion aFGF eukaryotic expression vector was successfully constructed and transfected into MSCs.This study may be expected to obtain some specific functions cells.
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Objective To probe the transplantation of skeletal muscle satellite cells of new-born rats for repairing skeletal muscle contusions. Methods Thirty-six Sprague-Dawley adult rats were separated at random into experimental and control groups (including a normal saline control group and a normal control group). Contusions were created on the left hind legs of rats in the experimental group. The cells were cultured and antibodies were identified through immunocytochemical staining and fluorescence labeling. In the experimental group, 0.2 ml of a muscle satellite cell suspension was slowly injected into the medial and lateral heads of the right calf gastrocnemius. The normal saline control group was injected with an equal volume of normal saline solution at the same sites. On the 5th, 10th and 15th day after the operation, EMG wave-amplitude and wave-width were recorded. The recovery rate of the muscle's wet weight and the gray value of a muscle fiber transverse section were determined.Two-factors analysis of variance was performed on the data. Results Before transplantation the muscle satellite cells were bright red after staining and their nuclei were blue after fluorescence labeling. After the operation the number of cells fluorescing and the strength of the fluorescence gradually decreased. The transplanted cells formed myotubes, melted into the muscle tissue and gradually took part in the restoration. The stained muscle satellite cell nuclei were free in intercellular space and melted into the basement membrane gradually. The outlines of the muscle tissue became progressively clearer. EMGs showed the fibrillation potentials as well as positive sharp waves reduced, but they too tended to become similar to those from the normal side. Restoration of EMG wave-amplitude and wave-width in the experimental group was better than in the saline control group and close to those in the normalcontrol group. The gray value of the muscle fiber transverse section area under HE staining was significantly lower at the 5th day post transplantation in both the experimental group and the saline control group than in the normal control group. At the 10th and 15th days post transplantation, the average gray value in the experimental group had increased significantly more than in the saline control group, and it was close to the normal control group average. In the experimental group the recovery rate of muscle wet weight was close to 1, but in the saline control group it was less than 1. Conclusions Transplanting skeletal muscle satellite cells of new-born rats can produce evident effects in renovating injured muscles.
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Objective To evaluate the effects of pulsed ultrasound at different intensities on the healing of standardized contusions in an animal model. Methods Forty-eight 3-month-old, male Sprague-Dawley rats were given experimental contusions of the right gastrocnemius muscle before being divided into four groups randomly ( n =12 in each group): (1) a muscle injury control group (muscle injury without treatment); (2) a muscle injury and pulsed ultrasound (0.25 W/cm2 ) group; (3) a muscle injury and pulsed ultrasound ( 0.5W/cm2 ) group; and (4)a muscle injury and pulsed ultrasound ( 0.75 W/cm2 ) group. Pulsed ultrasound treatment ( frequency 3 mega Hz)was started 24 hours post injury and delivered 5 min daily for 14 days on the injured right hindlimb. At days 4, 7 and 14 after injury, muscle samples were analyzed through hematoxylin-eosin staining and immunohistochemistry for the detection of muscle satellite cells and desmin. Results The average optical density (IOD) of desmin-positive mononucleated cells had increased significantly at days 4, 7 and 14 post injury in the treatment groups compared to the control group, but with no statistically significant difference among the 3 ultrasound treatment groups. Conclusions The pulsed ultrasound treatment played a beneficial role in skeletal muscle regeneration after contusion. There was no significant dose-dependent effect over the intensity range of 0.25-0.75 W/cm2.
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Objective To investigate the regenerating effect of muscle satellite cells autografted onto mus-cles injured by chronic compartment syndrome (CCS). Methods Twenty-four adult rabbits were randomly divided into an experimental group (model-transplantation), a non-graft group (model) and a control group (transplanta-tion). The CCS model was established in the experimental and non-graft groups. Transplantation was done in the ex-perimental and control groups. Satellite cells from the experimental and control group rabbits were isolated and then proliferated in vitro. The specific protein was identified by immnochemistry before engrafting. Then the 4',6-diamidi-no-2-phenylindole-tagged satellite cells were transplanted back to the original soleus muscles. After transplantation,the proliferation and differentiation of the satellite cells in vivo was observed and morphological changes at the site of the injury were compared by HE staining. Results At the 28th day after grafting, the satellite cells from the com-pressed soleus muscles in the experimental group had increased significantly, whereas those in the control group had remained the same. In both the graft group and the non-graft group, HE staining showed a large cluster of myofibers and interstitial fibers necrosed in the compressed muscle, while the skeletal muscle fibers and interstitial fibers were in-tact in the control group. At the 28th day after engrafting, the engrafted muscle showed much repair; but the non-en-grafted muscle demonstrated dominant fibrosis. The morphology of the skeletal muscle in the control group was normal.Conclusions Autografting of muscle satellite cells after a small amount of expansion in vitro could improve their regen-eration efficiency for repairing a large cluster of damaged myofibers caused by chronic compartment syndrome.
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AIM: To observe the effects of H_2O_2 on apoptosis of skeletal muscle satellite cells (SMSC)and mitochondrial membrane potential (MMP), and protective effect of erythropoietin(EPO). METHODS: SMSC in vitro were divided into three groups: H_2O_2 group, H_2O_2+EPO group and control. Apoptosis rate and the means were obverted by monofluorescence flow cytometry. The morphological change of apoptosis cells were observed under fluorescence microscopy after Hoechst 33258 staining. RESULTS: The cells in H_2O_2 group show the highest apoptosis rate (22.13±1.79)%. In H_2O_2+EPO group, apoptosis rate were (16.47±2.53)%, (4.97±0.55)% and (2.93±0.47)% according to the EPO treated levels (10, 20 or 40 kU/L), respectively. MMP level in H_2O_2 group was the lowest 9.70±0.09. MMP levels in H_2O_2+EPO group were 12.67±0.32, 27.90±0.66, 44.53±0.93, respectively according to the EPO treated levels (10, 20 or 40 kU/L). In control group, apoptosis rate was 1.93±0.57 and MMP was 51.37±0.64. In H_2O_2 group and H_2O_2+ low dosage EPO group, Hoechst 33258 staining showed obvious apoptosis. CONCLUSION: EPO inhibits the apoptosis induced by H_2O_2 and stabilizes the MMP, which is related to the dosage of EPO.
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Objective To investigate the whole differentiating process of myogenic satellite cells in vitro,and the expression of N-ACh receptors.Methods Skeletal muscle satellite cells were isolated by trypsin digestion in vitro and purified by preferential attachment method.The cells were observed morphologically and identified by ?-sarcomeric actin,desmin and skeletal myosin antibodies.Contraction of myotubes was observed after adding ACh in the culture medium, N-AChRs in myotube were also be identified by bungarotoxin.Results Cultured satellite cells in vitro differentiated into myoblasts and fused to form myotubes later.These cells were positive by ?-sarcomeric actin, skeletal myosin and desmin antibodies' staining.Adding ACh directly in the medium stimulated the contraction of myotubes.No obvious expression of ACh receptors was observed on the surface of satellite cells.Cluster of receptors aggregated on myotubes from neonatal rats' satellite cells cultured in differentiation media.Conclusion The myotubes differentiated from myogenic satellite cells have the function of contraction.N-ACh receptors were synthesized gradually during the process of differentiation.