Lack of galectin-1 results in defects in myoblast fusion and muscle regeneration.

Galectin-1 has been implicated in the development of skeletal muscle, being maximally expressed at the time of myofiber formation. Furthermore, in the presence of exogenous galectin-1, mononuclear myoblasts show increased fusion in vitro. In the current study, we have used the galectin-1 null mouse to elucidate the role of galectin-1 in skeletal muscle development and regeneration. Myoblasts derived from the galectin-1 mutant showed a reduced ability to fuse in vitro. In galectin-1 null mutants, there was evidence of a delay in muscle fiber development at the neonatal stage and muscle fiber diameter was reduced when compared with wild-type at the adult stage. Muscle regeneration was also compromised in the galectin-1 mutant with the process being delayed and a reduced fiber size being maintained. These results, therefore, show a definitive role for galectin-1 in fusion of myoblasts both in vitro, in vivo, and in regeneration after recovery from induced injury.     

miR-431 promotes differentiation and regeneration of old skeletal muscle by targeting Smad4

The myogenic capacity of myoblasts decreases in skeletal muscle with age. In addition to environmental factors, intrinsic factors are important for maintaining the regenerative potential of muscle progenitor cells, but their identities are largely unknown. Here, comparative analysis of microRNA (miRNA) expression profiles in young and old myoblasts uncovered miR-431 as a novel miRNA showing markedly reduced abundance in aged myoblasts. Importantly, elevating miR-431 improved the myogenic capacity of old myoblasts, while inhibiting endogenous miR-431 lowered myogenesis. Bioinformatic and biochemical analyses revealed that miR-431 directly interacted with the 3′ untranslated region (UTR) of Smad4 mRNA, which encodes one of the downstream effectors of TGF-β signaling. In keeping with the low levels of miR-431 in old myoblasts, SMAD4 levels increased in this myoblast population. Interestingly, in an in vivo model of muscle regeneration following cardiotoxin injury, ectopic miR-431 injection greatly improved muscle regeneration and reduced SMAD4 levels. Consistent with the finding that the mouse miR-431 seed sequence in the Smad4 3′ UTR is conserved in the human SMAD4 3′ UTR, inhibition of miR-431 also repressed the myogenic capacity of human skeletal myoblasts. Taken together, our results suggest that the age-associated miR-431 plays a key role in maintaining the myogenic ability of skeletal muscle with age.     

Transcriptome analysis of gene expression changes upon enzymatic dissociation in skeletal myoblasts

Single-cell RNA-sequencing analysis is one of the most effective tools for understanding specific cellular states. The use of single cells or pooled cells in RNA-seq analysis requires the isolation of cells from a tissue or culture. Although trypsin or more recently cold-active protease (CAP) has been used for cell dissociation, the extent to which the gene expression changes are suppressed has not been clarified. To this end, we conducted detailed profiling of the enzyme-dependent gene expression changes in mouse skeletal muscle progenitor cells, focusing on the enzyme treatment time, amount and temperature. We found that the genes whose expression was changed by the enzyme treatment could be classified in a time-dependent manner and that there were genes whose expression was changed independently of the enzyme treatment time, amount and temperature. This study will be useful as reference data for genes that should be excluded or considered for RNA-seq analysis using enzyme isolation methods.     

长链非编码RNA在成肌分化中的作用

人类基因组90％可以发生转录,但98％的转录产物为不具有蛋白编码能力的非编码 RNA （ noncoding RNA, ncRNA）。长链非编码RNA （ long noncoding RNA, lncRNA）是指转录本超过200 nt的非编码RNA,曾一度被认为是转录的“噪音”,不具有任何生物学功能。然而,近年报道lncRNA广泛参与成肌分化,可在RNA水平通过多种方式调控成肌分化进程,是成肌分化的重要调节因子。     

CNTF 联合RA 诱导成肌细胞类神经分化及与REST 的相关性

目的 探讨睫状神经营养因子（CNTF）联合视黄酸（RA）诱导成肌细胞类神经分化的方法以 及该诱导过程与神经元限制性沉默因子（REST）相关性。方法 以CNTF 诱导C2C12 去分化,再以RA 诱导 C2C12 类神经分化。通过细胞形态学、细胞免疫荧光、流式细胞术、Western blot 等技术对去分化及类神经 分化的细胞进行鉴定及相关分析,比较细胞类神经分化前后REST 表达变化。结果 诱导后细胞聚集生长,呈 典型的神经球样。细胞免疫荧光染色显示神经球能够被神经特异性标志物NSE、Sox1、GFAP 标记,神经球 离散为单个细胞接种后生长状态良好,神经干细胞标志物（nestin）呈阳性表达。Western blot 结果显示CNTF 去分化诱导后成肌细胞分化相关蛋白Myogenin、P21 表达降低,类神经分化后NSE、Sox1、GFAP 表达增多, 且成肌细胞特异性标志物Desmin 及REST 表达下降。流式细胞仪分析表面标志物NSE 显示诱导组与对照组 比较细胞有差异,且诱导组特异性阳性细胞率达84% 左右。结论 CNTF 联合RA 可诱导成肌细胞类神经分化, 且该诱导过程可能与REST 表达降低相关。     

成年大鼠骨骼肌成肌细胞的培养和鉴定

目的探索成年大鼠骨骼肌成肌细胞的高浓度培养方法。方法以成年同种系Wistar大鼠为研究对象,采用两步消化法获取大鼠骨骼肌卫星细胞,进行体外培养,对获得的细胞进行形态学研究,以免疫组织化学方法进行鉴定。结果细胞增殖旺盛,分化良好,可融合成肌管。免疫细胞化学染色显示,骨骼肌卫星细胞呈弱阳性,肌管呈强阳性。结论两步消化法体外培养的骨骼肌卫星细胞具有良好的增殖与分化能力,用此种方法可培养出高纯度的骨骼肌成肌细胞,操作简单、污染少。     

Application of tissue engineering in the treatment of stress urinary incontinence

Stress urinary incontinence is one of the most common diseases in urology. The main treatments for stress urinary incontinence are pharmacotherapy, physicobehavioral therapy and surgery.However, the results of present methods are not satisfactory. Tissue engineering is a newly emerging technology that may provide a novel method for the treatment of stress urinary incontinence.     

软骨源性形态发生蛋白2基因转染自体成肌细胞表达软骨细胞表型的实验研究

目的研究软骨形态发生蛋白-2(CDMP-2)对小鼠成肌细胞向软骨分化的作用。方法体外培养小鼠成肌细胞,贴壁细胞传代,取第3代细胞,对照组(A组)以无血清L-DMEM培养液培养,实验组(B、C、D组)以无血清L-DMEM培养液培养,分别加入透明质酸钠、CDMP-2(100ng/mL)和CDMP-2基因转染的小鼠成肌细胞,14d后终止培养,倒置相差显微镜观察细胞形态,甲苯胺蓝染色糖胺聚糖(GAG),行Ⅱ型胶原免疫组织化学染色、灰度值分析和Western blot检测。结果培养14d后转染组和诱导组可见成肌细胞形态由梭形向软骨细胞的多边形转变。甲苯胺蓝染色示糖胺聚糖(GAGs)均匀分布于基质中,免疫组织化学染色和Western blot示实验组Ⅱ型胶原表达阳性,且CDMP-2基因转染组细胞表达Ⅱ型胶原水平高于单独给予CDMP-2,且具有统计学意义;对照组和透明质酸钠组未见阳性表达。结论 CDMP-2基因转染的小鼠成肌细胞能够更好的表达软骨特异性Ⅱ型胶原。