大鼠骨骼肌卫星细胞诱导分化为胰岛素生成细胞的研究

目的探讨大鼠骨骼肌卫星细胞（MDSCs）定向诱导分化为胰岛素生成细胞（IPCs）,为1型糖尿病的干细胞治疗提供一种新的研究思路。 方法通过二次酶消化法和差速贴壁培养法分离、培养大鼠MDSCs,利用不同的诱导培养液使MDSCs定向分化为IPCs,并对诱导后细胞进行形态观察,通过双硫腙染色和免疫组化染色对MDSCs-IPCs形态进行鉴定,采用Q-PCR和Western Blot方法检测MDSCs-IPCs中C-peptide和Insulin的表达,通过胰岛素释放实验检测MDSCs-IPCs的生物学功能,β细胞和MDSCs-IPCs两组间比较采用t检验。 结果MDSCs在接种4 h后开始贴壁部分细胞伸出小的突起,48 h后绝大多数细胞贴壁呈梭形、胞浆丰富、折光度高。随着培养时间的延长,细胞的梭形形状更为明显且生长迅速。免疫组化结果显示细胞表达Desmin、α-Sarcomeric Actinin、MyoD1、Myf5和PAX7。成胰诱导后MDSCs形成胰岛样的圆形细胞团,双硫腙染色呈猩红色,Insulin免疫组化染色阳性。Q-PCR结果显示MDSCs-IPCs中C-peptide和Insulin mRNA表达量分别是β细胞的0.73倍（P > 0.05）和0.79倍（P > 0.05）。胰岛素释放实验显示,5.6 mmol/L和16.7 nmlol/L葡萄糖刺激培养2 h后,β细胞和MDSCs-IPCs分泌胰岛素量分别为[（20.3±4.2）mU/L]、[（16.1±3.7）mU/L]、[（60.5±9.3）mU/L]和[（40.9±7.3）mU/L],葡萄糖可调节MDSCs-IPCs胰岛素的分泌。 结论MDSCs易于分离培养、增殖能力强,体外可诱导分化为有功能的IPCs,适合作为再生医学的种子细胞。     

Analysis of expression of heavy myosin chains during in vitro differentiation of satellite cells and myoblasts derived from rat skeletal muscles

Differentiation of cultured myogenic progenitor cells (satellite cells and mononucleated myoblasts) derived from hindlimb muscles of rat embryos and newborn animals was studied. Immunocytochemical methods and PCR analysis revealed expression of heavy myosin chains at the earliest stages of myogenesis (in mononucleated myoblasts). Expression of the gene encoding the embryonic form of myosin and a low level of expression of the gene encoding perinatal myosin in cultured progenitor cells derived from embryonic muscles was detected by PCR. Cells derived from muscles of newborn animals also expressed these two myosin forms, though at a lower level. The progenitor cells derived from muscles of rat embryos and newborn animals were found to express myosin 2a, which is characteristic of fast-twitch definitive muscle fibers.     

The effects of aging on satellite cells in skeletal muscles of mice and rats

Summary Myosatellite cells were examined and quantified at the fine structural level of resolution during aging of skeletal muscles in mice and rats. Satellite cells in the soleus and gastrocnemius muscles of animals between eight and 30 months of age appeared, according to morphological criteria, metabolically less active than those examined in immature muscles. In the soleus muscle of the mouse, satellite cells decreased in number from 4.6% at eight months of age to 2.4% at 30 months. This decrease appeared to be due to the passage of some satellite cells into the interstitial space as a result of the formation of external lamina material around the entire satellite cell surface.This study was supported by NIH grant No. 5 S01-RR05356-13Appreciation is extended to Ms. Amy Erisman and Mr. Monroe Sprague for their excellent technical assistance on this project     

Stimulation of post-traumatic regeneration of x-irradiated skeletal muscles in old rats

Regeneration in murine musculus gastrocnemius was studied in animals aged 2-2.5. It was demonstrated that 20Cy irradiation caused the inhibition of necrosis resorption, suppressed the proliferative activity of muscular and connective tissues. Impulse laser therapy employed after irradiation of dissected musculus gastrocnemius potentiated fibrin resorption and markedly stimulated regeneration of muscular and connective tissues. Nonexposed disintegrated muscular tissue, when implanted to the site of injury produced a significant stimulating effect resulting in necrosis resorption, more pronounced than under laser therapy, and recovering the regenerative capacity of the exposed tissue.     

In vitro culture and induced differentiation of sheep skeletal muscle satellite cells

Skeletal muscle satellite cells are adult muscle-derived stem cells receiving increasing attention. Sheep satellite cells have a greater similarity to human satellite cells with regard to metabolism, life span, proliferation and differentiation, than satellite cells of the rat and mouse. We have used 2-step enzymatic digestion and differential adhesion methods to isolate and purify sheep skeletal muscle satellite cells, identified the cells and induced differentiation to examine their pluripotency. The most efficient method for the isolation of sheep skeletal muscle satellite cells was the type I collagenase and trypsin 2-step digestion method, with the best conditions for in vitro culture being in medium containing 20% FBS+10% horse serum. Immunofluorescence staining showed that satellite cells expressed Desmin, α-Sarcomeric Actinin, MyoD1, Myf5 and PAX7. After myogenic induction, multinucleated myotubes formed, as indicated by the expression of MyoG and fast muscle myosin. After osteogenic induction, cells expressed Osteocalcin, with Alizarin Red and ALP (alkaline phosphatase) staining results both being positive. After adipogenic induction, cells expressed PPARγ2 (peroxisome-proliferator-activated receptor γ2) and clear lipid droplets were present around the cells, with Oil Red-O staining giving a positive result. In summary, a successful system has been established for the isolation, purification and identification of sheep skeletal muscle satellite cells.     

miR-101a靶向EZH2促进山羊骨骼肌卫星细胞的分化

本实验室前期研究发现,miR-101a对山羊骨骼肌卫星细胞(skeletal muscle satellite cells, SMSCs)分化有促进作用,但其具体作用机制并不清楚。本研究利用PicTar、TargetScan和miRanda软件在线预测miR-101a的靶基因,并通过双荧光素酶报告基因进行实验验证;检测了山羊SMSCs分化不同时期miR-101a和靶基因的表达关系,同时分析了超表达和抑制miR-101a对靶基因表达水平的影响。结果证实,zeste增强子同源物2(enhancer of zeste homologue 2, EZH2)基因mRNA的3°UTR具有miR-101a结合位点,是miR-101a的一个靶基因。在SMSCs分化过程中,随着miR-101a表达水平的升高,EZH2的表达在mRNA和蛋白水平均下调。抑制miR-101a后,EZH2的表达极显著升高(P<0.01),但是在超表达miR-101a条件下,EZH2表达变化在mRNA和蛋白水平均不显著(P>0.05)。以上研究结果表明,miR-101a能通过抑制EZH2的表达来促进山羊SMSCs分化,为进一步阐明miR-101a对SMSCs的调控机制提供了理论依据。     

MiR-499/PRDM16 axis modulates the adipogenic differentiation of mouse skeletal muscle satellite cells

Obesity is associated with increased risks of diverse diseases; brown adipose tissue (BAT) can increase energy expenditure and protect against obesity by increasing the decomposition of white adipose tissue (WAT) to enhance the non-coupled oxidative phosphorylation of fatty acid in adipocytes and contributes to weight loss. However, BAT is abundant in only small rodents and newborn humans, but not in adults. PRDM16 is a key factor that induces the differentiation of skeletal muscle precursors to brown adipocytes and simultaneously inhibits myogenic differentiation. In the present study, we set insulin-induced skeletal muscle satellite cells (SMSCs) adipogenic differentiation model, as confirmed by the contents of adipogenic markers PRDM16, UCP1 and PGC1α and myogenic markers MyoD1 and MyoG. We selected miR-499 as candidate miRNA, which might regulate PRDM16 to affect SMSCs adipogenic differentiation. Possibly through directly binding to PRDM16 3′-UTR, miR-499 negatively regulated PRDM16 expression and hindered SMSCs adipogenic differentiation by reducing adipogenic markers PRDM16, UCP1 and PGC1α and increasing myogenic markers MyoD1 and MyoG. PRDM16 overexpression could partially reverse the effect of miR-499 on the above markers and SMSCs adipogenic differentiation. Taken together, miR-499/PRDM16 axis can affect the balance between SMSC myogenic and adipogenic differentiation, targeting miR-499 to rescue PRDM16 expression, thus promoting SMSCs adipogenic differentiation may be a promising strategy for obesity treatment.     

miR-143 regulates proliferation and differentiation of bovine skeletal muscle satellite cells by targeting IGFBP5

Development of skeletal muscle is a complicated biological process regulated by various regulation factors and signal pathways. MicroRNAs (miRNAs) are novel gene regulators that control muscle cell development. microRNA-143 (miR-143) is highly expressed in skeletal muscle, and we found that miR-143 level is significantly increased during bovine skeletal muscle satellite cells (MSCs) differentiation process through microarray analysis and qRT-PCR detection. However, the function of miR-143 in bovine muscle development remained unclear. In our work, the functions of miR-143 in bovine MSCs myogenic differentiation were investigated. We discovered that IGFBP5 is directly regulated by miR-143 using a dual-luciferase reporter assay. Overexpression of miR-143 led to decreased level of IGFBP5 protein and restrained cell proliferation and differentiation, while downregulation of miR-143 resulted in increased levels of IGFBP5 protein and restrained cell proliferation but improved differentiation. IGFBP5, an important component of IGF signaling pathway, contributes greatly to bovine muscle cell development. A mechanism that miR-143 can regulate the proliferation and differentiation of bovine MSCs through changing expression of IGFBP5 was elucidated by our study.     

Characteristics of alpha-aminoisobutyric acid transport in rat skeletal muscles.

alpha-Aminoisobutyric acid (AIB) transport into the intracellular compartment of extensor digitorum longus and soleus muscles was measured (in vitro) after allowance for the equilibration of the amino acid in the extracellular space. The latter was determined with three markers, [14C]inulin, 60Co-EDTA and [3H]mannitol. Net transport of AIB was subsequently divided into its two components, i.e. influx and efflux. Rates of influx were measured as the intracellular accumulation of [14C]AIB after a short incubation (5 min), and efflux was measured as the release of AIB with time (up to maximum of 50 min) from muscles that had previously been preloaded with AIB. This intracellular efflux was resolved into two phases, which probably represent two separate components of exit. The influence of extracellular Na+ on the transport of this neutral amino acid (representing the A system) was investigated. Na+ depletion resulted in lower accumulations of AIB, the effects becoming more pronounced with progressive depletions of external Na+. These changes arose from an inhibition of AIB influx, concomitant with an enhancement of its efflux. In contrast, all components of tyrosine transport (representing the L system) were unaffected by lowering external Na+ concentrations. The net accumulation of AIB was also suppressed by cortisol. This inhibitory effect was, however, Na+-dependent and resulted solely from the steroid's enhancement of AIB efflux, the hormone being without effect on AIB influx.     

The satellite cells of normal anuran skeletal muscle

Population counts and size measurements of satellite cell nuclei and myonuclei were carried out on the normal gastrocnemius muscles of adult Rana pipiens and Rana clamitans. Satellite cell profiles occurred with an observed frequency of about 1.3% in the muscles of the R. pipiens, and with an observed frequency of about 1.6% in the muscles of the R. clamitans. These frequencies were not found to be significantly different. The observed frequencies were corrected for the sampling bias introduced by the difference in the mean size of the satellite cell nuclei and myonuclei. This correction suggests that R. pipiens and R. clamitans both have a true satellite cell frequency of approx 2.7%. Analysis of these data indicates that the satellite cells of the normal anuran gastrocnemius occur in sufficient numbers to account for the regeneration seen after injury to this muscle.     

In vitro differentiation of satellite cells isolated from normal and dystrophic mammalian muscles. A comparison with embryonic myogenic cells

Satellite cells were isolated from skeletal muscles of adult normal and dystrophic mice (C57/6J/dy strain) by sequential digestion of tissue fragments with collagenase, hyaluronidase and trypsin. These cells exhibit in culture similar behaviour to that of embryonic myoblasts, undergoing an initial duplicative period lasting about 2–3 days, followed by a shorter phase (1–2 days) of rapid cell fusion. During the duplicative phase most of the satellite cells appear round-shaped, whereas embryonic myoblasts appear typically spindle-shaped: both cell types actively incorporate [³H] thymidine. During the subsequent days of culture an increasing number of satellite cells becomes spindle-shaped; afterwards the cells contact each other and fuse into multinucleated myotubes. The majority of spindle-shaped satellite cells is unable to incorporate [³H] thymidine, thus behaving as post-mitotic cells. Concomitantly with satellite cell fusion, an increase of about 80-fold of creatine phosphokinase (CPK) specific activity is observed. Satellite cells are able to recognize co-cultured embryonic myoblasts ([³H] thymidine-labelled): hybrid myotubes containing labelled and unlabelled nuclei are formed in these experimental conditions.Satellite cells from dystrophic animals are able to differentiate in culture and do not show appreciable differences as compared to their normal counterparts. In dystrophic myotubes, however, CPK specific activity is almost twice that observed in normal myotubes.Hyman dystrophic satellite cells from biophies of adult muscle cultured in similar conditions grow and fuse into multinucleated myotubes showing a behaviour identical to normal controls.     

Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor-beta

Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with transforming growth factor-beta (TGF-beta). Muscle-specific protein synthesis and satellite cell fusion were used as indicators of muscle differentiation; a dose-dependent inhibition of differentiation was observed in response to TGF-beta. In addition, TGF-beta depressed cell proliferation in a dose-dependent manner. Half-maximal inhibition of differentiation was seen with a TGF-beta concentration of approximately 0.1 ng/ml. Although proliferation was not inhibited, it was depressed and half-maximal suppression of proliferation occurred in response to 0.1-0.5 ng TGF-beta/ml. Neonatal rat myoblasts were also subjected to TGF-beta treatment, and similar results were observed. Neonatal cells, however, were more sensitive to TGF-beta than satellite cells, as indicated by the reduced concentrations of TGF-beta required to inhibit differentiation and reduce the rate of proliferation. Under identical culture conditions proliferation of muscle-derived fibroblasts were also depressed. The differentiation inhibiting effect of TGF-beta on satellite cells was reversible. It has been suggested that TGF-beta could be an important regulator of tissue repair, and its in vitro effects on satellite cells suggest a possible role in regulation of muscle regeneration.     

miR-194-5p negatively regulates the proliferation and differentiation of rabbit skeletal muscle satellite cells

Molecular and Cellular Biochemistry - Skeletal muscle satellite cells (SMSCs), also known as a multipotential stem cell population, play a crucial role during muscle growth and regeneration. In...