肝细胞生长因子在骨骼肌再生中的作用研究进展

骨骼肌损伤后的修复包括炎症反应期、修复期、组织重塑期三个阶段。而骨骼肌卫星细胞的激活、增殖与分化和骨骼肌伤后的修复有着密切的关系。骨骼肌损伤后,肝细胞生长因子(HGF)可以自分泌、旁分泌或内分泌的形式,调控肌卫星细胞功能,从而影响损伤骨骼肌的再生。其机制研究表明,HGF可能通过与其受体c-met结合,启动相关信号途径,参与骨骼肌卫星细胞激活、增殖、分化和迁移,从而影响骨骼肌再生进程。     

骨骼肌卫星细胞对肉品质的影响及其分化调控

骨骼肌卫星细胞是一种肌源性干细胞, 在骨骼肌的生长、发育及肌肉损伤修复中有着至关重要的作用。肌卫星细胞通过增殖、分化融合肌纤维形成新的肌核从而导致骨骼肌纤维的肥大以及骨骼肌纤维类型的相互转化, 进而影响肉品质的形成。文章从肌纤维的发育与肉品质形成、卫星细胞分化与肌纤维特征的相关性等方面, 对卫星细胞的Notch等经典遗传信号通路和miRNA等表观遗传调控及其对肉品质的影响进行了综述。     

肌卫星细胞在失重肌萎缩中的可塑性变化及机制

肌卫星细胞在骨骼肌生长发育和出生后骨骼肌损伤修复中起着重要的作用,但是有关肌萎缩中肌卫星细胞的可塑性变化、作用及其机制尚不清楚.本研究采用小鼠尾悬吊模拟失重效应诱导失重肌萎缩,动态分析了失重肌萎缩发生过程中不同类型肌纤维的肌卫星细胞数量和增殖、分化潜能可塑性的改变,发现在失重肌萎缩过程中,处于安静状态的肌卫星细胞显著增多、激活增殖的肌卫星细胞显著减少,而具有成肌分化潜能的肌卫星细胞有持续减少趋势.此外,在失重肌萎缩比目鱼肌单根肌纤维移出的体外培养中,证明了失重肌萎缩肌纤维肌卫星细胞可塑性降低的特征性变化.进一步,通过对比分析Smad3基因敲除及其同窝野生型小鼠,在失重肌萎缩中肌卫星细胞可塑性的差异性变化,揭示了Smad3在调控失重肌萎缩肌卫星细胞可塑性变化中的关键作用.     

蛋肉鸡骨骼肌卫星细胞的特性比较及分析

目的:比较蛋、肉鸡骨骼肌卫星细胞在增殖、分化速度及在细胞因子作用下细胞周期等方面所存在的特性差异,为人类肌肉疾病的研究和千细胞治疗提供一定的理论依据。方法:采用两步酶消化法体外原代培养获得7日龄蛋肉鸡骨骼肌卫星细胞,利用血球计数板进行细胞计数绘制出二者的细胞生长曲线;通过流式细胞仪检测经细胞因子bFGF和Myostatin处理后,蛋肉鸡骨骼肌卫星细胞的细胞周期变化情况。结果:体外相同的培养条件下,肉鸡肌卫星细胞的增殖、分化速度大于蛋鸡肌卫星细胞;且经相同剂量的同种细胞因子处理后,蛋鸡卫星细胞对于Myostatin的抑制作用十分敏感,而肉鸡则对bFGF的促进增殖的作用反应强烈。结论:肉鸡肌卫星细胞的增殖、分化速度大于蛋鸡肌卫星细胞,且二者对于同种细胞因子的敏感程度不同。     

肌卫星细胞研究进展

骨骼肌中的卫星细胞,长期以来就被认为是出生后骨骼肌生长、修复和维持的单能成肌干细胞。近年研究发现,卫星细胞与内皮细胞共同起源于胚胎血管祖细胞,且成年骨骼肌中存在多能干细胞,这些肌源多能干细胞在适当的微环境中具有多向分化潜能。这将为治疗包括帕金森病在内的多种临床退行性疾病提供自体干细胞的新来源。本文对肌卫星细胞的起源、增殖和成肌分化的分子调节机制,以及肌卫星细胞的多能干细胞潜能等方面的研究进展进行了综述。     

骨骼肌组织来源的外泌体促进骨骼肌干细胞增殖并抑制其分化

骨骼肌干细胞(又称为肌卫星细胞)位于肌纤维膜与基底膜之间。肌纤维分泌各种细胞因子到骨骼肌干细胞微环境中,进而调节肌卫星细胞的功能。最近的研究发现,骨骼肌来源的外泌体以内分泌方式影响其他组织的功能,但是,骨骼肌组织分泌的外泌体是否以旁分泌的方式调控肌卫星细胞的功能,目前并不清楚。该研究发现,骨骼肌来源的外泌体能够显著促进肌卫星细胞增殖、抑制其分化,这为揭示外泌体介导的骨骼肌组织微环境调控肌卫星细胞功能提供了实验证据。     

鸡骨骼肌卫星细胞的分离培养、鉴定及生物学特性研究

采用I型胶原酶和胰蛋白酶二步消化,经体外培养获得鸡骨骼肌卫星细胞,并通过检测肌卫星细胞特异基因的表达进行鉴定。结果表明：二步消化法适用于鸡骨骼肌卫星细胞的分离和获取,此方法分离得到的鸡骨骼肌卫星细胞表达卫星细胞特异的标志基因desmin和Pax7,并具有良好的增殖和分化能力,为鸡骨骼肌细胞增殖、分化和再生机制的研究提供技术平台。      

微小RNA调控骨骼肌增殖、分化的研究进展

骨骼肌损伤、萎缩等疾病的防治一直是困扰临床医生的难题。这些骨骼肌疾病的康复离不开肌细胞增殖、分化的调节。微小RNA（microRNA）作为基因表达的调节因子,通过对骨骼肌细胞增殖、分化机制的开启、促进和抑制等方式,对骨骼肌发育过程中的初始肌细胞、成肌细胞以及生物体成熟以后的肌卫星细胞的增殖、分化均进行着精细调节。因此,研究microRNA调节骨骼肌增殖、分化的机制已成为科研工作者的当务之急,并具有广阔的应用前景。     

骨骼肌卫星细胞生物学特性及临床应用前景

骨骼肌卫星细胞是位于肌细胞膜和基膜之间的具有增殖和分化潜力的生肌干细胞,对于出生后骨骼肌的生长、再生修复和维持具有重要的意义.主要对卫星细胞的含量与分布、细胞标志、可塑性和不均一性等生物学特性做了综述,最后展望了卫星细胞移植在临床上的应用前景.     

肌卫星细胞激活和补给的分子调控与肌肉疾病

肌卫星细胞(muscle satellite cell,SC)作为生肌干细胞,参与司控生后骨骼肌的生长、修复和维持等重要过程.综述了NO-HGF,Myostatin,Notch等重要信号分子及卫星细胞自身的特殊微环境对SC激活和补给的分子调控机制,希冀将来可以从这两方面入手克服目前临床中肌卫星细胞移植治疗各种骨骼肌疾病的瓶颈.     

EGCG通过抑制wnt/β-catenin信号通路调节猪骨骼肌卫星细胞的增殖和分化

为了阐明Wnt/β-catenin信号通路在猪骨骼肌卫星细胞增殖分化中的作用,利用Wnt/β-catenin信号通路抑制剂(-)-表没食子儿茶素没食子酸酯(EGCG)处理猪骨骼肌卫星细胞,采用MTT、流式细胞术、免疫荧光和Western印迹等方法检测了细胞增殖和分化情况.结果显示,与对照组相比,EGCG以时间、浓度依赖方式抑制猪骨骼肌卫星细胞的增殖.流式细胞术检测细胞周期结果表明,与对照组相比,经EGCG处理后,猪骨骼肌卫星细胞的G1期细胞比例上升,而G2和S期细胞比例下降,这说明细胞被阻滞在G1期,细胞的增殖受到抑制.免疫荧光检测分化过程中MyHC的表达,与对照组相比,EGCG促进猪骨骼肌卫星细胞的分化,并降低增殖标志基因MyoD以及细胞周期蛋白D的表达量,而提高了分化标志基因MyoG和MyHC的表达量.在猪骨骼肌卫星细胞增殖分化过程中,EGCG降低β-联蛋白的表达量,且核内的β-联蛋白明显减少.结果表明,EGCG通过抑制Wnt/β-catenin信号通路抑制猪骨骼肌卫星细胞的增殖,促进其分化.     

Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.     

Skeletal muscle atrophy is associated with an increased expression of myostatin and impaired satellite cell function in the portacaval anastamosis rat

Proliferation and differentiation of satellite cells are critical in the regeneration of atrophied muscle following immobilization and aging. We hypothesized that impaired satellite cell function is responsible for the atrophy of skeletal muscle also seen in cirrhosis. Myostatin and insulin-like growth factor 1 (IGF1) have been identified to be positive and negative regulators, respectively, of satellite cell function. Using a rat model of cirrhosis [portacaval anastamosis (PCA)] and sham-operated controls, we examined the expression of myostatin, its receptor activinR2b, and its downstream messenger cyclin-dependent kinase inhibitor p21 (CDKI p21) as well as IGF1 and its receptor in the gastrocnemius muscle. Expression of PCNA, a marker of proliferation, and myogenic regulatory factors (myoD, myf5, and myogenin), markers of differentiation of satellite cells, were also measured. Real- time PCR for mRNA and Western blot assay for protein quantification were performed. PCA rats had lower body weight and gastrocnemius weight compared with sham animals (P < 0.05). PCNA and myogenic regulatory factors were lower in PCA rats (P < 0.05). Myostatin, activinR2b, and CDKI p21 were higher in the PCA animals (P < 0.05). The expression of IGF1 and its receptor was lower in liver and skeletal muscle of PCA animals (P < 0.05). These data suggest that skeletal muscle atrophy seen in the portacaval shunted rats is a consequence of impaired satellite cell proliferation and differentiation mediated, in part, by higher myostatin and lower IGF1 expression.     

Muscle cell culture as a tool in animal growth research

Muscle cell culture techniques have been used for several years in research on muscle growth and development. Several types of culture systems have been devised, including primary cultures from embryonic or postnatal muscle and myogenic cell lines. In addition, serum-free and serum-containing media have been developed to address specific muscle development questions. Many of these questions center around muscle cell differentiation and muscle cell physiology; and, more recently, muscle cell cultures have been used as bioassay tools for examining growth physiology in domestic animals. In our laboratory, skeletal muscle satellite cells have been studied in vitro to evaluate the effect of several protein hormones and growth factors on satellite cell proliferation and differentiation. Of the hormones examined, only the insulin-like growth factors/somatomedins and fibroblast growth factor have been shown to have a stimulatory effect on proliferation that could be physiologically significant. None of the major anterior pituitary hormones interacted directly with satellite cells to stimulate proliferation. With advances in serum-free medium formulations and cell separation techniques, more information can be obtained from experiments with muscle cell cultures. With appropriate design and interpretation, our knowledge of muscle growth in domestic animals will be expanded.     

巴什拜羊骨骼肌卫星细胞分离培养及诱导分化

为了获得巴什拜羊骨骼肌卫星细胞,本研究采集出生1日龄巴什拜羔羊后肢骨骼肌组织,采用两步酶消化法结合差速贴壁法分离纯化巴什拜羊骨骼肌卫星细胞,并对分离获得的骨骼肌卫星细胞进行了鉴定、传代培养及诱导分化等研究。结果表明,本研究采用的分离纯化方法可以高效获得巴什拜羊骨骼肌卫星细胞,RT-PCR检测结果表明骨骼肌卫星细胞标志性基因pax7、Myf5、MyoD、desmin和c-Met均呈阳性表达。获得的骨骼肌卫星细胞具有较强的增殖能力,连续传代12代,细胞的形态仍保持正常,且细胞的克隆形成率仍保持在50%以上,但是当细胞传代至第18代时,逐渐表现出较为明显的衰退。细胞的生长符合典型的"S"型生长曲线,且第2代和第8代细胞的生长曲线没有明显的差异,至第14代时细胞的增殖速度逐渐降低。采用低浓度马血清培养体系,可成功诱导巴什拜羊骨骼肌卫星细胞向肌管方向分化,诱导培养至第5天时,骨骼肌卫星细胞分化标志基因MyHC呈阳性表达。由此得出结论,本研究采用的骨骼肌卫星细胞分离纯化体系高效、可靠,可以满足较高纯度巴什拜羊骨骼肌卫星细胞的分离培养。     

Growth and sex effects on the expression of syndecan-4 and glypican-1 in turkey myogenic satellite cell populations

The adult skeletal muscle stem cells, satellite cells, are responsible for skeletal muscle growth and regeneration. Satellite cells represent a heterogeneous cell population that differentially express cell surface markers. The membrane-associated heparan sulfate proteoglycans, syndecan-4, and glypican-1, are differentially expressed by satellite cells during the proliferation and differentiation stages of satellite cells. However, how the population of syndecan-4- or glypican-1-positive satellite cells changes during proliferation and differentiation, and how sex and muscle growth potential affect the expression of these genes is unknown. Differences in the amount of satellite cells positive for syndecan-4 or glypican-1 would affect the process of proliferation and differentiation which would impact both muscle mass accretion and the regeneration of muscle. In the current study, the percentage of satellite cells positive for syndecan-4 or glypican-1 from male and female turkeys from a Randombred Control Line 2 and a line (F) selected for increased 16-week body weight were measured during proliferation and differentiation. Growth selection altered the population of syndecan-4- and glypican-1-positive satellite cells and there were sex differences in the percentage of syndecan-4- and glypican-1-positive satellite cells. This study provides new information on dynamic changes in syndecan-4- and glypican-1-positive satellite cells showing that they are differentially expressed during myogenesis and growth selection and sex affects their expression.     

Isolation,Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.     

Reduced troponin I phosphorylation and increased Ca2+-dependent ATP-consumption in triton X-skinned fiber preparations from Gαq overexpressor mice

Mechanical stress leads to satellite cell activation, which is an important event in the development, growth, and remodeling of postnatal skeletal muscle. Although there is a considerable knowledge on the events involved in skeletal muscle regeneration and development, the precise role of mechanical stress on activation of satellite cells remains unclear. Previously, satellite cells were isolated from adult bovine muscle and it was shown that the cells are multipotent, i.e., capable of proliferating and to differentiating into both myoblasts and adipocytes. This study investigated the cellular mechanisms by which cyclic mechanical stretching modulates the proliferation and differentiation of adult bovine satellite cells. The application of cyclic stretch induced the proliferation of satellite cells and inhibited their differentiation into myotubes. This response is believed to be closely related to the stretch-mediated changes in the expression of myogenic and cell cycle regulatory factors. Cyclic stretching increased the level of extracellular signal-regulated kinase (ERK) phosphorylation, whereas a specific ERK inhibitor (PD98058) blocked the stretch-mediated inhibition of myogenesis in a dose-dependent manner. Overall, this study demonstrates for the first time that cyclic mechanical stretch induces the proliferation of bovine satellite cells and suppresses their myogenic differentiation through the activation of ERK.     

ERK1/2 is required for myoblast proliferation but is dispensable for muscle gene expression and cell fusion

Skeletal muscle satellite cells, which are found between the muscle fiber and the basal lamina, remain quiescent and undifferentiated unless stimulated to remodel skeletal muscle or repair injured skeletal muscle tissue. Quiescent satellite cells express c-met and fibroblast growth factor receptors (FGFR) 1 and 4, suggesting these receptors are involved in maintaining the undifferentiated quiescent state or involved in satellite cell activation. Although the signaling pathways involved are poorly understood, the mitogen activated protein kinase (MAPK) cascade has been implicated in the regulation of skeletal muscle growth and differentiation by FGFs. In this study, we investigated if activation of the Raf-MKK1/2-ERK1/2 signaling cascade plays a role in FGF-dependent repression of differentiation and proliferation of MM14 cells, a skeletal muscle satellite cell line. Inactivation ofthe Raf-MKK1/2-ERK1/2 pathway in myoblasts through the overexpression of dominant negative mutants of Raf-1 blocks ERK1/2 activity and prevents myoblast proliferation. Additionally, inhibition of MKK1/2 by treatment with pharmacological inhibitors also blocks FGF-mediated stimulation of ERK1/2 and blocks the G1 to S phase transition of myoblasts. Unexpectedly, we found that inactivation of the Raf-ERK pathway does not activate a muscle reporter, nor does inactivation of this pathway promote myogenic differentiation. We conclude that FGF-stimulated ERK1/2 signaling is required during the G1 phase of the cell cycle for commitment of myoblasts to DNA synthesis but is not required for mitosis once cells have entered the S-phase. Moreover, ERK1/2 signaling is not required either to repress differentiation, to promote skeletal muscle gene expression, or to promote myoblast fusion.