OBJECTIVE Skeletal muscle undergoes rapid and profound atrophy in response to decreased mechanical loading,e.g.,limb immobilization and bed rest.Phosphatidylinositol 3 kinase(PI3K)/Akt signaling pathway is critical fo...OBJECTIVE Skeletal muscle undergoes rapid and profound atrophy in response to decreased mechanical loading,e.g.,limb immobilization and bed rest.Phosphatidylinositol 3 kinase(PI3K)/Akt signaling pathway is critical for regulating the balance between protein synthesis and degradation during disuse/inactivity-induced muscle atrophy.The present study aimed to investigate whether natural product Icaritin(ICT)required PI3K/Akt signaling to exert counteractive effect on skeletal muscle atrophy following mechanical unloading.METHODS Two oral dosages of ICT(80and 120mg·kg-1·d-1)were administrated daily to adult male rats with or without daily injection of PI3K/Akt signaling inhibitor wortmannin(15μg·kg-1·d-1)during 28-d hindlimb suspension(HS).Ex vivo muscle functional testing,histological and immunohistochemical analysis were performed to determine the changes of soleus muscle function,mean muscle fiber cross-sectional area(CSA)and fiber type distribution.Western blot and real-time PCR analysis were also performed to evaluate the protein or mRNA expression of the markers involved in PI3K/Akt signaling pathway.RESULTS After 28-d HS,soleus muscle underwent profound muscle atrophy(-52.7% muscle mass vs.pre-HS baseline).The high dose ICT treatment significantly attenuated the decreases in soleus muscle mass(+22.6% vs.HS),muscle fiber CSA(+52.8% vs.HS),as well as the muscle functional testing parameters during the unloading.Molecularly,the high dose ICT treatment significantly attenuated the decreases in protein synthesis markers at protein levels(phosphorylation of Akt and its downstream proteins)during the unloading,whereas the increases in protein degradation markers at mRNA(atrogin-1and MuRF-1)and protein(nuclear FOXO1 and FOXO3a)levels during the unloading were significantly attenuated by the high dose ICT treatment.The low dose ICT treatment moderately attenuated the above changes induced by the unloading.Mechanistically,Wortmannin could abolish the above effects of ICT.CONCLUSION ICT requires participation of PI3K/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading in a dose-dependent manner.展开更多
To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, th...To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.展开更多
Skeletal muscle accounts for 40%~45%of the body weight and is composed of a large number of parallel arrangement of muscle fiber bundles through attaching to the skeleton by the tendon.And robust skeletal muscles prov...Skeletal muscle accounts for 40%~45%of the body weight and is composed of a large number of parallel arrangement of muscle fiber bundles through attaching to the skeleton by the tendon.And robust skeletal muscles provide people the ability of daily activities such as walk,lift and so on.Muscle fibers are generated by multinucleated myotubes which form from the fusion of myoblasts with polarity development,and the synergistic effect between muscle fibers and spontaneous contraction provides support for the movement of the bodies and limbs.However,because of common clinical genetic defect,trauma,tumor,primary myopathy there are so many people cannot exercise like normal people.So it is an urgent task to treat these diseases.Traditional treatment methods are mainly through acupuncture,rehabilitation,drug treatment,etc.But these methods have long treatment times,poor efficacy,and high costs.Recently,the clinical researchers find that these diseases have similar pathological characteristics including structural damage,loss of skeletal muscle function,apoptosis and degradation of muscle fibers Therefore,there is academic and clinical value to study the repair and regeneration of skeletal muscle.Nowadays,tissue engineering provides a new idea for repairing damaged muscle tissue.Tissue engineering uses biological materials as a carrier to regulate the structural properties of materials.By applying biological/chemical/physical stimuli to mimic the microenvironment of the living body.And then construct a living tissue and transplant it into the body for wound repair.With the development of tissue engineering,we regulate the micro-structure of the cell matrix and applying mechanical stimulation to reconstruct a functional muscle microtissue in vitro.Such fabrication and mechanical loading strategy provide an easily adaptable platform to create functional muscle tissue constructs.And the mature muscle microtissues provide a useful tool for the clinical application to treat muscle injury.In this work,we regulate ECM microstructure and apply mechanical stimulation to construct three-dimensional muscle cell mechanical microenvironment.We first regulate the collagen fiber orientation by utilizing the characteristics of collagen gelation,which is a slow process and often need half an hour even more.Before gelation,we fabricate a PDMS mould with rectangle chamber and apply a strain along the long axis to a certain extent utilizing its elasticity.Then we pour collagen solution into the chamber and release the force a few minutes later and gelatinize.After gelation,we apply stretching on the end of the rectangle collagen hydrogel to promote further alignment.We adjust the strain loading time and the strain level to form different groups.Determining the optimal experimental conditions for myotube formation by counting the length and area ofthe myotube and immunofluorescence about relevant proteins.Later,we use western blot and RT-PCR to prove our result.It was found that the application of pre-strain and tensile stimulation can promote the alignment of collagen fibers.Results show that strain loading time and strain extent can regulate the morphology and function of myotubes.The muscle mechanical microenvironment we constructed promotes the formation and activity of polar multinuclear myotubes and builds mature muscle tissue,and determines the related molecular pathways and their transmission mechanisms during the mechanical conduction process.展开更多
The expression of Selenoprotein W(SelW)in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione(GSH)and the activity of glutathione peroxidase(GPx).SelW knock-dow...The expression of Selenoprotein W(SelW)in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione(GSH)and the activity of glutathione peroxidase(GPx).SelW knock-down was performed by RNA interference(RNAi)in cultured muscle cells and verified by Real-time PCR and Western blotting.In addition,cell viability,GSH content and GPx activity were assayed.The results showed that the mRNA level and protein expression of SelW were decreased successfully by 71.9%and 68.8%relative to control values,cell viability decreased by 21.5%,GSH increased by 29.76%,and GPx increased by 47.58%.WST assay showed that compared with blank control,the value of positive group dropped 21.5%;In GSH and GPx assay,compared with blank control the positive group increased29.76%and 47.58%separately.In conclusion,SelW knock-down by RNAi caused significant cytotoxity in skeletal muscle cells and led to compensatory increases in GSH content and GPx activity.These findings are consistent with the suggestions from bioinformatics indicating an antioxidative role for SelW in skeletal muscle cells.展开更多
Bone morphogenetic protein-2 （BMP-2） plays a key role in bone formation and maintenance. BMP-2 can stimulate longitudinal bone growth by increasing the growth plate chondrocyte proliferation, hypertrophy, and cartil...Bone morphogenetic protein-2 （BMP-2） plays a key role in bone formation and maintenance. BMP-2 can stimulate longitudinal bone growth by increasing the growth plate chondrocyte proliferation, hypertrophy, and cartilage matrix synthesis. The current study was designed to investigate the associations of the BMP-2 gene polymorphism with chicken skeletal traits. Northeast Agricultural University F2 resource population was used in this study. Body weight and body composition traits were measured in F2 population. Polymorphism between parental lines was detected by DNA sequencing, and PCR-fragment length polymorphism method was then developed to screen the population. The results showed that the BMP-2 gene polymorphism was associated with skeletal traits in F2 population. This research suggests that BMP-2 gene may be a candidate locus or linked to a major gene that affects skeletal traits in chickens.展开更多
Purpose： Fibronectin type III domain-containing protein 5 （FNDC5）, also known as irisin, is a myokine secreted from muscle in response to exercise and improves obesity and glucose homeostasis. However, the molecula...Purpose： Fibronectin type III domain-containing protein 5 （FNDC5）, also known as irisin, is a myokine secreted from muscle in response to exercise and improves obesity and glucose homeostasis. However, the molecular mecha- nisms that regulate FNDC5 expression and the functional significance of FNDC5 in skeletal muscle remain un- known. In this study, we explored the possible pathways that induce FNDC5 expression and delineated its metabol- ic effects on skeletal muscle. Methods： C2C12 myotubes were treated with various concentrations of Sp-cAMP, forskolin, and ionomycin respectively for various durations. FNDC5 and related metabolic genes＇ expressions were measured by quantitative real-time polymerase chain reaction （qRT-PCR）. Cyclic AMP responsive element-binding protein （CREB） phosphorylation was measured by Western blot. Oxidative phosphorylation was quantified by oxy- gen consumption rate （OCR） measurement using XF-96 analyzer （Seahorse Bioscience）. The statistical signifi- cance was calculated by one-way analysis of variance （ANOVA）. Data were considered significant when P 〈 0.05. Results： We found that cAMP and forskolin dose and time dependently increased FNDC5 expression in C2C12 myotubes. A synergistic effect of forskolin and ionomycin on FNDC5 expression was also found. CREB phosphoryl- ation was elevated in myotubes simultaneously upon these treatments. C2C12 myotubes over expressing CREB dis- plays increased FNDC5 expression as well, suggesting CREB was a regulator of FNDC5 expression. Functionally, irisin treatment enhanced mitochondrial biogenesis of C2C12 myotubes through increasing peroxisome proliferator- activated receptor gamma coactivator-1α （PGC-1α）, nuclear respiratory factor 1 （NRF1） and mitochondrial tran-scription factor A （TFAM） expressions, leading to increase myotube mitochondrial respirations and ATP produc- tion. Conclusions Our observation indicates that irisin is a metabolic modulator of skeletal muscle, whose expres- sion is controlled by cAMP pathway and intracellular level of calcium.展开更多
Myoblast-mediated gene transfer approach has agreat potential to be developed into a durable genetherapy method for various diseases. To increase theexpression level of human factor Ⅸ (hFⅨ) in musclecells, we constr...Myoblast-mediated gene transfer approach has agreat potential to be developed into a durable genetherapy method for various diseases. To increase theexpression level of human factor Ⅸ (hFⅨ) in musclecells, we constructed a series of hFⅨ expression vectorswith the retroviral vector frame and examined their hFⅨexpression in skeletal muscle cells. Firstly, we introducedhFⅨ minigene (hFⅨ ml or hFⅨ m2), which contains atruncated first intron (1. 4 kb or 0. 3 kb) of hFⅨ展开更多
Myoblast differentiation is an essential process during skeletal muscle development.C2C12 myoblast is a commonly used experimental model to study muscle cell differentiation in vitro.Dehydrogenase/reductase(SDR family...Myoblast differentiation is an essential process during skeletal muscle development.C2C12 myoblast is a commonly used experimental model to study muscle cell differentiation in vitro.Dehydrogenase/reductase(SDR family)member 3(DHRS3)is a highly conserved member in short-chain alcohol dehydrogenase/reductase superfamily and has been shown to be involved in the metabolism of retinol.Previous experimental results showed that the expression of DHRS3 increased significantly during the differentiation of myoblasts differentiation.However,the effect of DHRS3 on mouse muscle cell differentiation was unclear.The objective of current study was to determine if DHRS3 affected muscle cell differentiation,and if DHRS3 was involved in muscle regeneration.Protein expression was determined by western blot and immunofluorescence analysis.The activation and inhibition of DHRS3 increased and decreased C2C12 myoblast differentiation respectively,which indicated that DHRS3 could affect C2C12 myoblast differentiation.DHRS3 expression was significantly changed during muscle regeneration,with the regeneration of muscle injury,the expression of DHRS3 tended to increase first and then decrease.It suggested that DHRS3 might be involved in muscle regeneration.In summary,this study confirmed the involvement of DHRS3 in C2C12 myoblast differentiation and mouse skeletal muscle regeneration and provided a theoretical basis for further elucidating the molecular mechanism of muscle development.展开更多
Objective To research the relation between the time-dependent appearances of myofibroblasts during the repair of contused skeletal muscle in rat and wound age determination. Methods A total of 35 SD male rats were div...Objective To research the relation between the time-dependent appearances of myofibroblasts during the repair of contused skeletal muscle in rat and wound age determination. Methods A total of 35 SD male rats were divided into the control and six injured groups according to wound age as follows: 12 h, 1 d, 5 d, 7 d, 10 d and 14 d after injury. The appearances of myofibroblasts were detected by HE staining, immunohistochemistry and confocal laser scanning microscopy. Masson's trichrome staining was utilized to examine collagen accumulation in the contused areas. Results Immunohistochemical staining showed that α-SMA+myofibroblasts were initially observed at 5 d post-injury. The average ratio of myofibroblasts was highest at 14 d post-injury, with all samples, ratios more than 50%. In the other five groups, the average of α-SMA positive ratios were less than 50%. The collagen stained areas in the contused zones, concomitant with myofibroblast appearance, were increasingly augmented along with advances of posttraumatic interval. Conclusion The immunohistochemical detection of myofibroblasts can be applied to wound age determination. The myofibroblasts might be involved in collagen deposition during the repair of contused skeletal muscle in rat.展开更多
从生物能学观点看,冷环境下棕色脂肪解偶联蛋白1(uncoupling protein 1,UCP1)解偶联是机体产热和维持体温的重要机制,但成年人棕色脂肪含量极低,因而其产热的意义受到质疑。尽管研究证实运动促进脂肪棕色化,但其受环境温度、性别、训练...从生物能学观点看,冷环境下棕色脂肪解偶联蛋白1(uncoupling protein 1,UCP1)解偶联是机体产热和维持体温的重要机制,但成年人棕色脂肪含量极低,因而其产热的意义受到质疑。尽管研究证实运动促进脂肪棕色化,但其受环境温度、性别、训练水平等诸多因素影响较大,因此研究结果难以一致。研究从热力学角度探讨冷环境的运动适应以及能量转换过程,从分子层面揭示:运动时骨骼肌内的无效循环加快,通过Ca^(2+)跨膜转运、磷酸肌酸穿梭、α-磷酸甘油穿梭等路径,实现Ca^(2+)、磷酸肌酸、还原型辅酶Ⅰ(reduced nicotinamide adenine dinucleotide,NADH)跨膜转移,底物总量不发生变化,但伴随三磷酸腺苷(ATP)水解和产热。从热力学第一定律(ΔU=Q+W)来看,生物氧化释放的化学能(ΔU)是产热(Q)和肌肉对外做功,即肌肉收缩(W)之和。产热包括解偶联蛋白(uncoupling protein,UCP)解偶联产热和无效循环产热两部分,UCP1是脂肪棕色化和产热的重要标志。从热力学判断,冷环境下运动是否依赖脂肪产热以维持体温,在很大程度上还取决于无效循环的产热以及肌肉对外做功。因此,运动并非总能促进白色脂肪棕色化,甚至不能看作是运动促进健康的标志。展开更多
基金The project supported by National Natural Science Foundation of China(81201406)Direct Grant for Research,The Chinese University of Hong Kong(4054138)
文摘OBJECTIVE Skeletal muscle undergoes rapid and profound atrophy in response to decreased mechanical loading,e.g.,limb immobilization and bed rest.Phosphatidylinositol 3 kinase(PI3K)/Akt signaling pathway is critical for regulating the balance between protein synthesis and degradation during disuse/inactivity-induced muscle atrophy.The present study aimed to investigate whether natural product Icaritin(ICT)required PI3K/Akt signaling to exert counteractive effect on skeletal muscle atrophy following mechanical unloading.METHODS Two oral dosages of ICT(80and 120mg·kg-1·d-1)were administrated daily to adult male rats with or without daily injection of PI3K/Akt signaling inhibitor wortmannin(15μg·kg-1·d-1)during 28-d hindlimb suspension(HS).Ex vivo muscle functional testing,histological and immunohistochemical analysis were performed to determine the changes of soleus muscle function,mean muscle fiber cross-sectional area(CSA)and fiber type distribution.Western blot and real-time PCR analysis were also performed to evaluate the protein or mRNA expression of the markers involved in PI3K/Akt signaling pathway.RESULTS After 28-d HS,soleus muscle underwent profound muscle atrophy(-52.7% muscle mass vs.pre-HS baseline).The high dose ICT treatment significantly attenuated the decreases in soleus muscle mass(+22.6% vs.HS),muscle fiber CSA(+52.8% vs.HS),as well as the muscle functional testing parameters during the unloading.Molecularly,the high dose ICT treatment significantly attenuated the decreases in protein synthesis markers at protein levels(phosphorylation of Akt and its downstream proteins)during the unloading,whereas the increases in protein degradation markers at mRNA(atrogin-1and MuRF-1)and protein(nuclear FOXO1 and FOXO3a)levels during the unloading were significantly attenuated by the high dose ICT treatment.The low dose ICT treatment moderately attenuated the above changes induced by the unloading.Mechanistically,Wortmannin could abolish the above effects of ICT.CONCLUSION ICT requires participation of PI3K/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading in a dose-dependent manner.
基金Supported by the Ministry of Agricultural Nuarture of New Varieties Genetically Modified Organisms Significant Special Funding (2008ZX08007-002)
文摘To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.
文摘Skeletal muscle accounts for 40%~45%of the body weight and is composed of a large number of parallel arrangement of muscle fiber bundles through attaching to the skeleton by the tendon.And robust skeletal muscles provide people the ability of daily activities such as walk,lift and so on.Muscle fibers are generated by multinucleated myotubes which form from the fusion of myoblasts with polarity development,and the synergistic effect between muscle fibers and spontaneous contraction provides support for the movement of the bodies and limbs.However,because of common clinical genetic defect,trauma,tumor,primary myopathy there are so many people cannot exercise like normal people.So it is an urgent task to treat these diseases.Traditional treatment methods are mainly through acupuncture,rehabilitation,drug treatment,etc.But these methods have long treatment times,poor efficacy,and high costs.Recently,the clinical researchers find that these diseases have similar pathological characteristics including structural damage,loss of skeletal muscle function,apoptosis and degradation of muscle fibers Therefore,there is academic and clinical value to study the repair and regeneration of skeletal muscle.Nowadays,tissue engineering provides a new idea for repairing damaged muscle tissue.Tissue engineering uses biological materials as a carrier to regulate the structural properties of materials.By applying biological/chemical/physical stimuli to mimic the microenvironment of the living body.And then construct a living tissue and transplant it into the body for wound repair.With the development of tissue engineering,we regulate the micro-structure of the cell matrix and applying mechanical stimulation to reconstruct a functional muscle microtissue in vitro.Such fabrication and mechanical loading strategy provide an easily adaptable platform to create functional muscle tissue constructs.And the mature muscle microtissues provide a useful tool for the clinical application to treat muscle injury.In this work,we regulate ECM microstructure and apply mechanical stimulation to construct three-dimensional muscle cell mechanical microenvironment.We first regulate the collagen fiber orientation by utilizing the characteristics of collagen gelation,which is a slow process and often need half an hour even more.Before gelation,we fabricate a PDMS mould with rectangle chamber and apply a strain along the long axis to a certain extent utilizing its elasticity.Then we pour collagen solution into the chamber and release the force a few minutes later and gelatinize.After gelation,we apply stretching on the end of the rectangle collagen hydrogel to promote further alignment.We adjust the strain loading time and the strain level to form different groups.Determining the optimal experimental conditions for myotube formation by counting the length and area ofthe myotube and immunofluorescence about relevant proteins.Later,we use western blot and RT-PCR to prove our result.It was found that the application of pre-strain and tensile stimulation can promote the alignment of collagen fibers.Results show that strain loading time and strain extent can regulate the morphology and function of myotubes.The muscle mechanical microenvironment we constructed promotes the formation and activity of polar multinuclear myotubes and builds mature muscle tissue,and determines the related molecular pathways and their transmission mechanisms during the mechanical conduction process.
基金supported by China Postdoctoral Science Foundation(LRB06-262)Funds of Northeast Forestry University
文摘The expression of Selenoprotein W(SelW)in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione(GSH)and the activity of glutathione peroxidase(GPx).SelW knock-down was performed by RNA interference(RNAi)in cultured muscle cells and verified by Real-time PCR and Western blotting.In addition,cell viability,GSH content and GPx activity were assayed.The results showed that the mRNA level and protein expression of SelW were decreased successfully by 71.9%and 68.8%relative to control values,cell viability decreased by 21.5%,GSH increased by 29.76%,and GPx increased by 47.58%.WST assay showed that compared with blank control,the value of positive group dropped 21.5%;In GSH and GPx assay,compared with blank control the positive group increased29.76%and 47.58%separately.In conclusion,SelW knock-down by RNAi caused significant cytotoxity in skeletal muscle cells and led to compensatory increases in GSH content and GPx activity.These findings are consistent with the suggestions from bioinformatics indicating an antioxidative role for SelW in skeletal muscle cells.
基金Supported by National "973" Project of China (2006CB102105)National "863" Project of China (2006AA10A120)Earmarked Fund for Modern Agro-industry Technology Research System (nycytx-42-G1-07)
文摘Bone morphogenetic protein-2 （BMP-2） plays a key role in bone formation and maintenance. BMP-2 can stimulate longitudinal bone growth by increasing the growth plate chondrocyte proliferation, hypertrophy, and cartilage matrix synthesis. The current study was designed to investigate the associations of the BMP-2 gene polymorphism with chicken skeletal traits. Northeast Agricultural University F2 resource population was used in this study. Body weight and body composition traits were measured in F2 population. Polymorphism between parental lines was detected by DNA sequencing, and PCR-fragment length polymorphism method was then developed to screen the population. The results showed that the BMP-2 gene polymorphism was associated with skeletal traits in F2 population. This research suggests that BMP-2 gene may be a candidate locus or linked to a major gene that affects skeletal traits in chickens.
文摘Purpose： Fibronectin type III domain-containing protein 5 （FNDC5）, also known as irisin, is a myokine secreted from muscle in response to exercise and improves obesity and glucose homeostasis. However, the molecular mecha- nisms that regulate FNDC5 expression and the functional significance of FNDC5 in skeletal muscle remain un- known. In this study, we explored the possible pathways that induce FNDC5 expression and delineated its metabol- ic effects on skeletal muscle. Methods： C2C12 myotubes were treated with various concentrations of Sp-cAMP, forskolin, and ionomycin respectively for various durations. FNDC5 and related metabolic genes＇ expressions were measured by quantitative real-time polymerase chain reaction （qRT-PCR）. Cyclic AMP responsive element-binding protein （CREB） phosphorylation was measured by Western blot. Oxidative phosphorylation was quantified by oxy- gen consumption rate （OCR） measurement using XF-96 analyzer （Seahorse Bioscience）. The statistical signifi- cance was calculated by one-way analysis of variance （ANOVA）. Data were considered significant when P 〈 0.05. Results： We found that cAMP and forskolin dose and time dependently increased FNDC5 expression in C2C12 myotubes. A synergistic effect of forskolin and ionomycin on FNDC5 expression was also found. CREB phosphoryl- ation was elevated in myotubes simultaneously upon these treatments. C2C12 myotubes over expressing CREB dis- plays increased FNDC5 expression as well, suggesting CREB was a regulator of FNDC5 expression. Functionally, irisin treatment enhanced mitochondrial biogenesis of C2C12 myotubes through increasing peroxisome proliferator- activated receptor gamma coactivator-1α （PGC-1α）, nuclear respiratory factor 1 （NRF1） and mitochondrial tran-scription factor A （TFAM） expressions, leading to increase myotube mitochondrial respirations and ATP produc- tion. Conclusions Our observation indicates that irisin is a metabolic modulator of skeletal muscle, whose expres- sion is controlled by cAMP pathway and intracellular level of calcium.
文摘Myoblast-mediated gene transfer approach has agreat potential to be developed into a durable genetherapy method for various diseases. To increase theexpression level of human factor Ⅸ (hFⅨ) in musclecells, we constructed a series of hFⅨ expression vectorswith the retroviral vector frame and examined their hFⅨexpression in skeletal muscle cells. Firstly, we introducedhFⅨ minigene (hFⅨ ml or hFⅨ m2), which contains atruncated first intron (1. 4 kb or 0. 3 kb) of hFⅨ
基金Supported by the Natural Science Foundation of Heilongjiang Province(C2017025)。
文摘Myoblast differentiation is an essential process during skeletal muscle development.C2C12 myoblast is a commonly used experimental model to study muscle cell differentiation in vitro.Dehydrogenase/reductase(SDR family)member 3(DHRS3)is a highly conserved member in short-chain alcohol dehydrogenase/reductase superfamily and has been shown to be involved in the metabolism of retinol.Previous experimental results showed that the expression of DHRS3 increased significantly during the differentiation of myoblasts differentiation.However,the effect of DHRS3 on mouse muscle cell differentiation was unclear.The objective of current study was to determine if DHRS3 affected muscle cell differentiation,and if DHRS3 was involved in muscle regeneration.Protein expression was determined by western blot and immunofluorescence analysis.The activation and inhibition of DHRS3 increased and decreased C2C12 myoblast differentiation respectively,which indicated that DHRS3 could affect C2C12 myoblast differentiation.DHRS3 expression was significantly changed during muscle regeneration,with the regeneration of muscle injury,the expression of DHRS3 tended to increase first and then decrease.It suggested that DHRS3 might be involved in muscle regeneration.In summary,this study confirmed the involvement of DHRS3 in C2C12 myoblast differentiation and mouse skeletal muscle regeneration and provided a theoretical basis for further elucidating the molecular mechanism of muscle development.
基金This study was financially supported in part bythe research funds from the National Natural Science Foundation of China （81202383）, the Doctoral Program of Higher Education of China （20122104110025） and Program for Young Innovative Research Team in China University of Political Science and Law.
文摘Objective To research the relation between the time-dependent appearances of myofibroblasts during the repair of contused skeletal muscle in rat and wound age determination. Methods A total of 35 SD male rats were divided into the control and six injured groups according to wound age as follows: 12 h, 1 d, 5 d, 7 d, 10 d and 14 d after injury. The appearances of myofibroblasts were detected by HE staining, immunohistochemistry and confocal laser scanning microscopy. Masson's trichrome staining was utilized to examine collagen accumulation in the contused areas. Results Immunohistochemical staining showed that α-SMA+myofibroblasts were initially observed at 5 d post-injury. The average ratio of myofibroblasts was highest at 14 d post-injury, with all samples, ratios more than 50%. In the other five groups, the average of α-SMA positive ratios were less than 50%. The collagen stained areas in the contused zones, concomitant with myofibroblast appearance, were increasingly augmented along with advances of posttraumatic interval. Conclusion The immunohistochemical detection of myofibroblasts can be applied to wound age determination. The myofibroblasts might be involved in collagen deposition during the repair of contused skeletal muscle in rat.
