Increase in muscle endurance in mice by dietary Yamabushitake mushroom (Hericium erinaceus) possibly via activation of PPARδ

Skeletal muscle fiber is largely classified into two types: type 1 (slow‐twitch) and type 2 (fast‐twitch) fibers. Meat quality and composition of fiber types are thought to be closely related. Previous research showed that overexpression of constitutively active peroxisome proliferator‐activated receptor (PPAR)δ, a nuclear receptor present in skeletal muscle, increased type 1 fibers in mice. In this study, we found that hexane extracts of Yamabushitake mushroom (Hericium erinaceus) showed PPARδ agonistic activity in vitro. Eight‐week‐old C57BL/6J mice were fed a diet supplemented with 5% (w/w) freeze‐dried Yamabushitake mushroom for 24 hr. After the treatment period, the extensor digitorum longus (EDL) muscles were excised. The Yamabushitake‐supplemented diet up‐regulated the PPARδ target genes Pdk4 and Ucp3 in mouse skeletal muscles in vivo. Furthermore, feeding the Yamabushitake‐supplemented diet to mice for 8 weeks resulted in a significant increase in muscle endurance. These results indicate that Yamabushitake mushroom contains PPARδ agonistic ligands and that dietary intake of Yamabushitake mushroom could activate PPARδ in skeletal muscle of mice. Unexpectedly, we observed no significant alterations in composition of muscle fiber types between the mice fed control and Yamabushitake‐supplemented diets.     

猪lncRNA TCONS_00791383对骨骼肌卫星细胞增殖分化的影响

为了探究lncRNA TCONS_00791383对猪骨骼肌卫星细胞增殖和分化的影响。本研究利用qRT-PCR技术检测出生7 d内大白仔猪6种组织（心、脾、肺、肾、背肌和腿肌）以及猪骨骼肌卫星细胞增殖分化前后TCONS_00791383的表达水平；通过设计反义核苷酸（antisense oligonucleotides，ASO）片段在猪骨骼肌卫星细胞中对TCONS_00791383进行敲低，检测敲低TCONS_00791383之后增殖分化标志基因的表达量变化；通过trans （co-expression）对TCONS_00791383进行靶基因预测，使用DAVID对其进行GO富集和KEGG通路分析。结果显示，TCONS_00791383在猪心脏中表达量最高，在脾和肾组织中不表达。在骨骼肌卫星细胞从增殖到分化的过程中，TCONS_00791383的表达量逐渐上升，且在分化后30 h表达量达到最高。在使用ASO片段敲低TCONS_00791383之后，与对照组相比，在分化24 h，增殖标志基因Pax3、Pax7表达量显著或极显著降低（P<0.05，P<0.01），分化标志基因MyoG表达量极显著降低（P<0.01），在分化48 h，增殖标志基因Pax3表达量极显著降低（P<0.01），Pax7表达量显著降低（P<0.05），分化标志基因MyHC表达量显著降低（P<0.05）。预测得到的相关靶基因富集到AMPK、ATP等多个与骨骼肌卫星细胞增殖和分化过程相关的重要信号通路。本研究表明，lncRNA TCONS_00791383可能促进猪骨骼肌卫星细胞的增殖和分化。     

Comparative analysis of mechanical stretch-induced activation activity of back and leg muscle satellite cells in vitro

It has previously been shown that mechanical stretch induces activation of cultured quiescent satellite cells by rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c‐met receptor. The present study provides evidence that the stretch activation activity varies according to the origin of satellite cells from back and leg skeletal muscles in vitro. Satellite cells were isolated from three muscle groups, back (BK), upper hind limb (UL) and lower hind limb (LL) muscles, of adult male rats and stretch activation activities were compared. In response to stretch, lower hind limb satellite cells showed significantly greater response than upper hind limb and back muscles (LL > UL > BK). Immunoblots of stretched culture media revealed a higher HGF‐releasing capacity of lower hind limb satellite cells than back muscle satellite cells. In addition, lower hind limb satellite cells exhibited a greater activation activity in response to exogenous HGF added to culture media than compared to satellite cells from back and upper hind limb (LL > UL > BK). The increased ability to release HGF and the increased cellular responsiveness might account for higher stretch activation activities of lower hind limb satellite cells. Electrophoretic analysis of myosin heavy chain isoforms verified a higher content of slow muscle fibers in lower limb muscles (LL > UL > BK), suggesting a difference in stretch‐induced activation activity between satellite cells associated with fast and slow muscle fibers.     

Cold exposure increases slow‐type myosin heavy chain 1 (MyHC1) composition of soleus muscle in rats

The aim of this study was to examine the effects of cold exposure on rat skeletal muscle fiber type, according to myosin heavy chain (MyHC) isoform and metabolism‐related factors. Male Wistar rats (7 weeks old) were housed individually at 4 ± 2°C as a cold‐exposed group or at room temperature (22 ± 2°C) as a control group for 4 weeks. We found that cold exposure significantly increased the slow‐type MyHC1 content in the soleus muscle (a typical slow‐type fiber), while the intermediate‐type MyHC2A content was significantly decreased. In contrast to soleus, MyHC composition of extensor digitorum longus (EDL, a typical fast‐type fiber) and gastrocnemius (a mix of slow‐type and fast‐type fibers) muscle did not change from cold exposure. Cold exposure increased mRNA expression of mitochondrial uncoupling protein 3 (UCP3) in both the soleus and EDL. Cold exposure also increased mRNA expression of myoglobin, peroxisome proliferator‐activated receptor gamma coactivator 1α (PGC1α) and forkhead box O1 (FOXO1) in the soleus. Upregulation of UCP3 and PGC1α proteins were observed with Western blotting in the gastrocnemius. Thus, cold exposure increased metabolism‐related factors in all muscle types that were tested, but MyHC isoforms changed only in the soleus.     

β‐hydroxy‐β‐methyl butyrate promotes leucine metabolism and improves muscle fibre composition in growing pigs

The aim of this study was to investigate the effects of excess leucine (Leu) vs. its metabolites α‐ketoisocaproate (KIC) and β‐hydroxy‐β‐methyl butyrate (HMB) on Leu metabolism, muscle fibre composition and muscle growth in growing pigs. Thirty‐two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed 1 of 4 diets for 45 days: basal diet, basal diet + 1.25% L‐Leu, basal diet + 1.25% KIC‐Ca, basal diet + 0.62% HMB‐Ca. Results indicated that relative to the basal diet and HMB groups, Leu and KIC groups exhibited increased Leu concentrations and decreased concentrations of isoleucine, valine and EAAs in selected muscle (p < 0.05) and had lower mRNA levels of MyHC I and higher expression of MyHC IIx/IIb (p < 0.05), and there was no significant difference between the basal and HMB‐supplemented groups. Moreover, the mRNA expression levels of AMPKα and UCP3 were higher but the myostatin mRNA levels were lower in the soleus muscle of the HMB group than those from other groups (p < 0.05). These findings demonstrated that doubling dietary Leu content exerted growth‐depressing effects in growing pigs; dietary KIC supplementation induced muscular branched‐chain amino acid imbalance and promoted muscle toward a more glycolytic phenotype; while dietary HMB supplementation promoted the generation of more oxidative muscle types and increased muscle growth specially in oxidative skeletal muscle, and these effects of HMB might be associated with the AMPKα‐Sirt1‐PGC‐1α axis and mitochondrial biogenesis.     

去乙酰化酶3对线粒体功能和猪生长发育影响的研究进展

去乙酰化酶3（Sirt3）是去乙酰化酶家族的一员,具有高度的去乙酰基酶活性,对动物体的生长发育、衰老、代谢等生理过程具有调控作用。目前的研究表明,Sirt3可以激活超氧化物歧化酶、调控三羧酸循环和促进电子传递链3个途径,消除细胞内的活性氧（ROS）,避免细胞的氧化损伤和线粒体膜电位的损伤,进而影响细胞凋亡与自噬;并可通过调控ROS、降低各种退行性疾病的发病率等途径减缓衰老。同时,Sirt3通过激活AMP依赖的蛋白激酶（AMPK）等途径影响肌肉的发育。对猪的研究表明,不同Sirt3突变型猪的肌肉色值和失水率等肉质指标存在差异,且Sirt3对脂肪酸氧化和酮体生成有一定的影响。在对与猪Sirt3蛋白有较高同源性的牛进行的研究中发现,不同Sirt3突变型的牛体长、体高、肌肉脂肪含量等指标存在显著差异。作者总结了Sirt3对线粒体的能量代谢和细胞氧化损伤的调控机制及其对细胞凋亡、细胞自噬和机体衰老的影响,阐述了猪Sirt3蛋白的部分理化性质及其对猪生长发育的作用机制,以期为了解和挖掘该基因在猪肌肉和脂肪发育中的生物学作用,以及家畜生产性状改良提供理论依据。     

Peroxisome proliferator-activated receptor-γ coactivator 1 α (PGC-1 α) expression and the formation of slow-twitch muscle fibers in porcine and bovine skeletal muscles

The peroxisome proliferator‐activated receptor‐γ coactivator‐1 α (PGC‐1 α) induces mitochondria biogenesis in skeletal muscles. To determine the relationships between PGC‐1 α and the muscle fiber types, the expression levels of PGC‐1 α were analyzed in porcine and bovine skeletal muscles. As a first step, the nucleotide sequences of the porcine and bovine PGC‐1 α were determined. The porcine and bovine PGC‐1 α cDNA encoded 796 amino acid sequences and showed 95.1% identity between the two species. The expression levels of the PGC‐1 α mRNA were analyzed in the same 10 skeletal muscles from four pigs and three cattle. The contents of porcine and bovine PGC‐1 α were higher in the tongue, masseter and diaphragm, and lower in the Biceps femoris, semimembranosus, Longissimus thoracis and semitendinosus muscles. The contents of myosin heavy chain slow‐type protein (MyHC‐slow) were also determined in the same muscles by ELISA. The analysis of MyHC‐slow showed results similar to those for the PGC‐1 α contents in all of the muscles except for the tongue. The content of MyHC‐slow in the tongue was the lowest among the porcine muscles, and moderate among the bovine muscles. The results suggest that PGC‐1 α relates to the development of oxidative muscle fibers, but is not the principal factor in determining type I fiber content.     

猪骨骼肌卫星细胞体外分离培养研究进展

猪骨骼肌是动物机体重要的运动组织及人类主要的肉食来源,也是研究肌肉生长发育和疾病的良好模型。猪出生后,骨骼肌的生长发育、损伤修复都需要肌卫星细胞的参与,体外分离培养猪骨骼肌卫星细胞是深入研究骨骼肌生长发育及疾病发生机理的基础,是在细胞水平进行分子功能验证的前提。随着肌肉发育和病理分子机制研究的不断深入,猪骨骼肌卫星细胞的体外分离培养技术也迅速发展起来。背最长肌、后腿肌和半腱肌常用于分离骨骼肌卫星细胞,1日龄猪背最长肌的分离效果最好。常用于分离骨骼肌卫星细胞的酶包括链酶蛋白酶、胶原酶、胰蛋白酶、胶原蛋白酶等,各酶及酶联合消化的时间不同,最优的过滤方式是200目+400目联合过滤,3次离心法可获得纯度较高的细胞。常使用的培养基为DMEM/F12+10%胎牛血清(FBS)+1%青-链霉素(P/S)。骨骼肌肌卫星细胞常见标记物有配对盒基因3(PAX3)、PAX7、生肌决定因子5(Myf5)、Myf4、肌分化因子(MyoD)、肌细胞生成素(MyoG)等。作者通过对猪骨骼肌肌卫星细胞的分离、培养及鉴定等方面进行综述,梳理出各步骤中最佳参数,为建立规范猪骨骼肌卫星细胞分离程序提供参考,以期为肌肉发育和疾病研究提供理论及技术支持。     

Adipocytes suppress differentiation of muscle cells in a co‐culture system

The development of adipose tissue in skeletal muscle is important for improving meat quality. However, it is still unclear how adipocytes grow in the proximity of muscle fibers. We hypothesized that adipocytes would suppress muscle cell growth so as to grow dominantly within muscle. In this study, we investigated the effect of adipocytes on the differentiation of muscle cells in a co‐culture system. The fusion index of C2C12 myoblasts co‐cultured with 3T3‐L1 adipocytes was significantly lower than that of the control. The expression of myogenin and myosin heavy chain in C2C12 muscle cells co‐cultured with 3T3‐L1 adipocytes was significantly lower than in the control. Furthermore, the expression of Atrogin‐1 and MuRF‐1 was higher in C2C12 muscle cells co‐cultured with 3T3‐L1 adipocytes than the control. These results suggest that 3T3‐L1 adipocytes suppress the differentiation of C2C12 myoblasts. In addition, 3T3‐L1 adipocytes induced the expression and secretion of IL‐6 in C2C12 muscle cells. The fusion index and myotube diameter were higher in C2C12 muscle cells co‐cultured with 3T3‐L1 cells in medium containing IL‐6‐neutralizing antibody than the control. Taken together, there is a possibility that adipocyte‐induced IL‐6 expression in muscle cells could be involved in the inhibition of muscle cell differentiation via autocrine.     

腺病毒与慢病毒感染猪原代细胞的比较研究

本实验旨在探究腺病毒和慢病毒侵染猪原代细胞的最佳感染复数(MOI)和侵染时间,确定较优的侵染方式。实验选择体外分离培养猪骨骼肌卫星细胞、前体脂肪细胞和骨髓间充质干细胞后,分别用腺病毒(MOI=0、50、100、200、500、1 500)和慢病毒(MOI=0、30、50、80、100、300)感染细胞,每隔24 h在荧光显微镜下观察记录细胞中绿色荧光蛋白的表达情况。结果表明:当腺病毒MOI值为500、慢病毒MOI值为80,侵染4 d后,3种细胞荧光强度均较强且细胞形态完好。相比较而言,腺病毒能快速高效侵染猪骨骼肌卫星细胞;而对于猪前体脂肪细胞和猪骨髓间充质干细胞,2种病毒侵染速度相近,但慢病毒的侵染效率和荧光强度更高。因此,对于猪骨骼肌卫星细胞,选取腺病毒作为外源基因载体更为合适;对于猪前体脂肪细胞和猪骨髓间充质干细胞,慢病毒的侵染效果则更好。     

Regulation of the expression of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks: Effects of leucine and glycine–leucine peptide

This study was conducted to analyse the effects of leucine (Leu) and glycine (Gly)‐Leu peptide on expressions of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks. The 4‐day‐old male AA broilers with similar weight were selected to obtain the broiler skeletal muscle satellite cells with the two‐step method of collagenase‐I and trypsin digestion. The satellite cells were subjected to primary culture in vitro, and they were cultured in DMEM medium with the Leu concentration of 0.2 mM and 2 mM as well as with the Gly‐Leu peptide concentration of 0.2 mM and 2 mM. The experiment lasted for 5 days. The results showed that TOR, S6K1 and 4E‐BP1 mRNA expressions in the medium with Leu concentration of 2 mM were significantly higher than that in 0.2 mM group (p < 0.05). There was no difference between the medium with Gly‐Leu concentration of 2 mM and 0.2 mM on the TOR, S6K1 and 4E‐BP1 mRNA expressions (p > 0.05). In conclusion, Leu significantly increases TOR, S6K1 and 4E‐BP1 mRNA expressions of skeletal muscle satellite cells, but Gly‐Leu peptide has no effect on them.     

大白猪不同部位肌肉肌纤维表达特点

取3头本地大白猪4个不同部位的肌肉组织,运用免疫组化方法,测定快肌纤维和慢肌纤维的表达情况。免疫组化结果表明在猪的不同部位肌肉组织中,快肌纤维与慢肌纤维含量之间存在极显著差异(P<0.01)。快肌纤维的表达在股二头肌中最多,其次为背最长肌和内脊肌,头半棘肌最少。慢肌纤维的表达在头半棘肌中最多,其次为内脊肌和股二头肌,在背最长肌中表达最少。通过对猪4种骨骼肌中快肌纤维和慢肌纤维的免疫组化分析显示,二者的表达在猪不同部位的肌肉存在显著差异。     

Satellite cells produce neural chemorepellent semaphorin 3A upon muscle injury

Regenerative mechanisms that regulate intramuscular motor innervation. including configuration of the neuromuscular connections are thought to reside in the spatiotemporal expression of axon‐guidance molecules. Our previous studies proposed a heretofore unexplored role of satellite cells as a key source of a secreted neural chemorepellent semaphorin 3A (Sema3A) expression. In order to verify this concept, there is still a critical need to provide direct evidence to show the up‐regulation of Sema3A protein in satellite cells in vivo upon muscle injury. The present study employed a Sema3A/MyoD double‐immunohistochemical staining for cryo‐sections prepared from cardiotoxin injected gastrocnemius muscle of adult mouse lower hind‐limb. Results clearly demonstrated that Sema3A expression was up‐regulated in myogenic differentiation‐positive satellite cells at 4–12 days post‐injury period, the time that corresponds to the cell differentiation phase characterized by increasing myogenin messenger RNA expression. This direct proof encourages a possible implication of satellite cells in the spatiotemporal regulation of extracellular Sema3A concentrations, which potentially ensures coordinating a delay in neurite sprouting and re‐attachment of motoneuron terminals onto damaged muscle fibers early in muscle regeneration in synchrony with recovery of muscle‐fiber integrity.     

LiCl激活经典Wnt信号通路诱导猪骨骼肌卫星细胞向慢肌分化

Wnt/β-catenin信号通路参与调控细胞的增殖及分化,决定细胞的命运。LiCl通过抑制GSK-3β(glycogen synthetase kinase-3β)稳定细胞内β-catenin的表达。为研究Wnt/β-catenin信号通路在哺乳动物骨骼肌卫星细胞成肌分化过程中的作用。以原代培养猪骨骼肌卫星细胞为实验材料,将其培养在含有1%CEE和20%胎牛血清的GMEM培养液中,再用15 mmol/L LiCl诱导分化,并采用免疫荧光染色和Western blotting等方法检测经典Wnt信号通路中相关因子及成肌分化过程中细胞的蛋白表达。结果表明:LiCl处理猪骨骼肌卫星细胞后促进分化,p-GSK-3β、β-catenin、myosin及慢肌蛋白增加,p-β-catenin、GSK-3β及快肌蛋白减少,并且核内源性β-catenin增多。结果提示,激活Wnt/β-catenin信号通路促进骨骼肌卫星细胞成肌分化,并且诱导其向慢肌分化。     

干扰lnc4351对牛骨骼肌卫星细胞增殖与成肌分化的影响

为探究长链非编码RNA(lncRNA)对牛骨骼肌卫星细胞增殖及分化的影响,本研究以牛骨骼肌卫星细胞及已建立的体外成肌诱导分化模型为基础,以前期高通量测序获得的牛骨骼肌卫星细胞分化前后表达差异倍数较大的一个预测lncRNA为靶标,对其进行生物信息学分析及亚细胞定位,命名为lnc4351。设计合成lnc4351的siRNA,转染牛骨骼肌卫星细胞,采用EdU染色的方法检测干扰lnc4351对细胞增殖的影响;对转染siRNA的牛骨骼肌卫星细胞进行体外成肌诱导分化,观察肌管的形成状态,同时采用实时荧光定量PCR和Western blotting检测分化标志因子MyoG和MHC基因的mRNA及蛋白水平的表达变化,研究干扰lnc4351对细胞分化的影响。结果显示,lnc4351位于牛的14号染色体,不具有蛋白编码潜能,是一个未报道过的lncRNA,在牛骨骼肌卫星细胞的细胞质和细胞核内均有分布,主要存在于细胞核;干扰lnc4351表达后,EdU阳性细胞比率显著下降(P<0.05),说明下调lnc4351表达显著抑制了牛骨骼肌卫星细胞的增殖;下调lnc4351表达后肌卫星细胞经诱导分化产生的肌管量呈现增多趋势,分化标志因子MyoG和MHC的蛋白水平显著或极显著上调(P<0.05;P<0.01),说明干扰lnc4351能够促进牛骨骼肌卫星细胞的成肌分化过程。本研究结果表明,干扰lnc4351表达可以抑制牛骨骼肌卫星细胞的增殖并促进其成肌分化过程,为进一步开展lncRNA对牛骨骼肌发育的调控机制及肌肉发育相关研究提供参考。     

干扰核心蛋白聚糖对牛骨骼肌卫星细胞增殖分化的影响

为探究肌肉生长抑制素(MSTN)对牛骨骼肌生长发育的作用机制,本研究以前期MSTN^+/-蒙古牛与野生蒙古牛腿臀肌肌肉组织定量蛋白质组学与磷酸化蛋白质组学筛选获得的表达差异倍数较大的核心蛋白聚糖(DCN)为靶标,以实验室前期分离培养的牛骨骼肌卫星细胞及建立的体外诱导成肌分化模型为对象,通过对设计合成的3个DCN siRNA干扰效果的筛选,将干扰效果最显著的si-DCN-2(si-DCN)转染牛骨骼肌卫星细胞。采用实时荧光定量PCR和Western blotting方法检测增殖期(GM)牛骨骼肌卫星细胞中增殖标志因子Pax7和MyoD的mRNA水平及蛋白水平的表达变化,以及使用EdU染色的方法检测干扰DCN对细胞增殖的影响。对转染DCN siRNA的牛骨骼肌卫星细胞进行体外成肌诱导分化,通过显微镜观察牛骨骼肌卫星细胞分化第3天(DM3)的肌管形成状态,同时采用实时荧光定量PCR和Western blotting检测分化标志因子MyoG和MyHC的mRNA水平及蛋白水平的表达变化,并对DM3期肌管MyHC进行免疫荧光染色,以研究干扰DCN对细胞分化的影响。结果显示,干扰DCN表达后,增殖期牛骨骼肌卫星细胞中Pax7和MyoD的mRNA水平及蛋白水平都显著或极显著上调(P<0.05;P<0.01),且EdU阳性细胞率显著增加(P<0.05),表明干扰DCN表达显著促进了牛骨骼肌卫星细胞的增殖。干扰DCN表达后,牛骨骼肌卫星细胞分化第3天诱导形成的肌管直径呈现增大趋势,检测成肌分化标志因子MyoG在mRNA和蛋白水平的表达分别极显著和显著高于对照组(P<0.01;P<0.05),MyHC在mRNA水平显著降低(P<0.05),但在蛋白水平上极显著升高(P<0.01),免疫荧光结果显示,下调DCN后肌管融合指数显著高于对照组(P<0.05),说明干扰DCN表达能够促进牛骨骼肌卫星细胞的成肌分化过程。本研究结果表明,干扰DCN可以显著促进牛骨骼肌卫星细胞的增殖和成肌分化过程。研究结果为进一步开展MSTN对牛骨骼肌卫星细胞成肌分化的调控机制研究奠定了基础。     

过表达MyBPC1对牛骨骼肌卫星细胞增殖分化的影响

旨在探究肌球蛋白结合蛋白C1（myosin binding protein C1,MyBPC1）对牛骨骼肌卫星细胞增殖与成肌分化的影响,为进一步研究MyBPC1在细胞分化和肌肉发育过程中的调控作用提供依据。本研究利用西门塔尔胎牛原代牛骨骼肌卫星细胞体外诱导成肌分化模型模拟牛骨骼肌的生长发育过程。采用qRT-PCR和Western blot检测MyBPC1的细胞时序表达谱。试验分为两组。在RNA水平每组4个重复,每个重复20 μL;在蛋白水平每组3个重复,每个重复15 μg。采用qRT-PCR和Western blot检测牛骨骼肌卫星细胞转染MyBPC1的过表达效果,并进一步检测细胞增殖期标志因子Pax7、Ki67以及细胞分化期标志因子MyHC、MyOG的表达变化情况,观察牛骨骼肌卫星细胞肌管形成状态。结果,MyBPC1在牛骨骼肌卫星细胞分化前后表达水平存在极显著差异,牛骨骼肌卫星细胞诱导分化后MyBPC1的mRNA和蛋白表达量均极显著高于增殖期（P<0.01）。过表达MyBPC1后,细胞分化形成的肌管数量明显多于对照组,增殖标志因子Pax7的mRNA水平和蛋白表达水平无显著差异,分化标志因子MyHC的mRNA水平和蛋白表达水平极显著高于对照组（P<0.01）。过表达MyBPC1可以促进牛骨骼肌卫星细胞体外成肌分化,为进一步开展MyBPC1对牛骨骼肌卫星细胞的调控机制奠定基础。     

胎儿期与成年期蒙古马骨骼肌肌纤维类型转化研究

为探索影响蒙古马胎儿期和成年期肌纤维类型差异机理.本研究选取3匹4月龄胎儿(两母一公)与3匹5岁健康成年母马身体4块分布全身、具有代表性的肌肉组织(长臂三头肌、夹肌、背最长肌、臀中肌)作为一个整体.胎儿期蒙古马肌纤维和成年期蒙古马肌纤维因存在差异各做为一组,试验进行3个生物学重复.首先对蒙古马骨骼肌肌肉样品进行免疫组化...     

Immunocytochemical localisation of carbonic anhydrase isozyme III in equine skeletal muscle

The location of carbonic anhydrase III (CA-III) in frozen sections of biopsies of Thoroughbred horse skeletal muscle was studied. Fibre types were determined by ATP-ase and succinate dehydrogenase staining. CA-III isozyme was detected using a peroxidase conjugated anti-CA-III antibody. CA-III was found to be localised in slow twitch oxidative fibres (ST), but was also present in fast twitch oxidative (FTH) fibres in small amounts. Fast twitch glycolytic (FT) fibres were stained lightly compared with control sections. The concentrations of CA-III in muscle and liver were 70 micrograms/mg protein and 4 micrograms/mg protein, respectively. CA-I and CA-II were not found in muscle extracts by the double immunodiffusion method.     

干扰核心蛋白聚糖对牛骨骼肌卫星细胞增殖分化的影响

为探究肌肉生长抑制素（MSTN）对牛骨骼肌生长发育的作用机制,本研究以前期MSTN^+/-蒙古牛与野生蒙古牛腿臀肌肌肉组织定量蛋白质组学与磷酸化蛋白质组学筛选获得的表达差异倍数较大的核心蛋白聚糖（DCN）为靶标,以实验室前期分离培养的牛骨骼肌卫星细胞及建立的体外诱导成肌分化模型为对象,通过对设计合成的3个DCN siRNA干扰效果的筛选,将干扰效果最显著的si-DCN-2（si-DCN）转染牛骨骼肌卫星细胞。采用实时荧光定量PCR和Western blotting方法检测增殖期（GM）牛骨骼肌卫星细胞中增殖标志因子Pax7和MyoD的mRNA水平及蛋白水平的表达变化,以及使用EdU染色的方法检测干扰DCN对细胞增殖的影响。对转染DCN siRNA的牛骨骼肌卫星细胞进行体外成肌诱导分化,通过显微镜观察牛骨骼肌卫星细胞分化第3天（DM3）的肌管形成状态,同时采用实时荧光定量PCR和Western blotting检测分化标志因子MyoG和MyHC的mRNA水平及蛋白水平的表达变化,并对DM3期肌管MyHC进行免疫荧光染色,以研究干扰DCN对细胞分化的影响。结果显示,干扰DCN表达后,增殖期牛骨骼肌卫星细胞中Pax7和MyoD的mRNA水平及蛋白水平都显著或极显著上调（P<0.05;P<0.01）,且EdU阳性细胞率显著增加（P<0.05）,表明干扰DCN表达显著促进了牛骨骼肌卫星细胞的增殖。干扰DCN表达后,牛骨骼肌卫星细胞分化第3天诱导形成的肌管直径呈现增大趋势,检测成肌分化标志因子MyoG在mRNA和蛋白水平的表达分别极显著和显著高于对照组（P<0.01;P<0.05）,MyHC在mRNA水平显著降低（P<0.05）,但在蛋白水平上极显著升高（P<0.01）,免疫荧光结果显示,下调DCN后肌管融合指数显著高于对照组（P<0.05）,说明干扰DCN表达能够促进牛骨骼肌卫星细胞的成肌分化过程。本研究结果表明,干扰DCN可以显著促进牛骨骼肌卫星细胞的增殖和成肌分化过程。研究结果为进一步开展MSTN对牛骨骼肌卫星细胞成肌分化的调控机制研究奠定了基础。