丝切蛋白CFL2基因低表达对小鼠成肌细胞C2C12肌纤维信号通路关键成分的影响

探讨低表达丝切蛋白CFL2基因对小鼠成肌细胞C2C12信号通路关键成分的影响。采用RNAi技术,将猪CFL2基因真核表达shRNA重组体稳定转染至小鼠成肌细胞C2C12后提取其总RNA和总蛋白质,采用Real-time PCR和Western blot检测CFL2基因低表达后成肌细胞C2C12信号通路关键成分(MEF2C、sk MLCK、Rho A、AMPKα2、p38和Ca M)的表达情况。结果表明:在3个稳转细胞株中MEF2C、Ca M、p38和AMPKα2的mRNA表达均为极显著下调(P0.01),sk MLCK的mRNA表达为极显著上调(P0.01),其它成分不显著;MEF2C和Ca M的蛋白表达在3个稳转细胞株中均为显著或极显著下调(P0.05或P0.01),其他4种成分(p38、Rho A、AMPKα2和sk MLCK)均差异不显著(P0.05),说明CFL2低表达显著下调信号通路关键成分MEF2C和Ca M的表达,CFL2与Ca M和sk MLCK分别呈显著正相关和负相关。     

猪新基因CFL2真核表达载体的构建及在C2C12细胞中的瞬时表达

从猪的股二头肌中提取总RNA,利用RT-PCR技术从猪骨骼肌中获得CFL2基因的编码区序列,T-A克隆至PMD-18T载体,经HindⅢ和XhoⅠ酶切后定向克隆至真核表达载体pCDNA3.1(+)中。获得的重组质粒pCDNA3.1(+)/CFL2用限制性内切酶酶切分析和DNA测序分析鉴定,并经脂质体瞬时转染C2C12细胞,最后利用PCR检测转基因细胞中CFL2基因的存在及其表达情况。结果显示:猪CFL2基因已克隆到真核表达载体pCDNA3.1(+)中,转染的C2C12细胞中检测到细胞内较高量CFL2的表达。pCDNA3.1(+)/CFL2真核表达载体的成功构建,为深入研究CFL2基因在猪肌肉细胞中的功能奠定基础,为猪肉质品质的改良提供了新的思路。     

利用免疫共沉淀验证肌纤维形成相关蛋白CFL2与MEF2C的相互作用

利用免疫共沉淀技术验证丝切蛋白(CFL2)和肌细胞增强因子-2(MEF2C)之间的相互作用关系。构建带绿色荧光蛋白(GFP)的p EGFP-N1-CFL2和p Lenti6.3_MCS_IRES2-EGFP-his-MEF2C重组载体,经双酶切鉴定正确后,分别将上述重组载体共转染至人宫颈癌细胞(Hela),利用免疫共沉淀方法验证CFL2和MEF2C之间的相互作用关系。结果表明,p EGFP-N1-CFL2和p Lenti6.3_MCS_IRES2-EGFPhis-MEF2C重组载体共转染Hela细胞,分别用抗CFL2和抗MEF2C抗体沉淀蛋白复合物,用MEF2C和CFL2抗体进行Western blot检测,检测到p Lenti6.3_MCS_IRES2-EGFP-his-MEF2C和p EGFP-N1-CFL2的表达。本试验成功构建p EGFP-N1-CFL2和p Lenti6.3_MCS_IRES2-EGFP-his-MEF2C真核表达重组载体,在Hela细胞中表达后利用免疫共沉淀的方法证实了CFL2和MEF2C之间存在相互作用。     

猪CFL2基因启动子的克隆与序列分析

CFL是真核生物肌动蛋白结合蛋白家族的成员。CFL2主要在哺乳动物的骨骼肌和心肌表达,在肌肉纤维的形成过程及肌肉的再生起重要作用。为了深入研究猪CFL2基因转录的调控机制,本研究利用染色体步移法首次获得了CFL2基因起始密码子上游约2.5 kb区域的序列。应用相关生物信息学软件对该序列进行分析,预测了其启动子核心区域及转录起始点,发现在起始密码子上游-508 bp和-453 bp两处存在TATA-box,另外还发现了GC-box、CAAT-box;该序列还包含SP1、AP1、AP2、GATA-1等转录因子结合位点。本试验结果为进一步研究该基因表达调控与肌肉发育的关系奠定了基础。     

鼠肌纤维丝切蛋白CFL2互作蛋白的筛选与鉴定

筛选并鉴定小鼠腿肌组织中与丝切蛋白CFL2相互作用的蛋白质。通过免疫共沉淀技术分离小鼠腿肌组织中CFL2蛋白复合物,对获得的免疫共沉淀复合物进行SDS-PAGE分离,将分离出的差异条带用胰蛋白酶进行酶切,酶切后的肽段进行液相色谱分离,最后通过电喷雾质谱技术分析肽段组成并鉴定蛋白质,获得与CFL2互作的组织蛋白谱。通过Western blot检测,确定在天然状态下小鼠腿肌组织中CFL2与Ndufb7之间存在相互作用。首次利用免疫共沉淀联合质谱技术成功从小鼠腿肌组织中筛选并鉴定出与CFL2相互作用的蛋白Ndufb7,为进一步研究CFL2在骨骼肌肌纤维形成中的作用提供理论基础。     

鸡CFL2基因3'UTR多态性与肉质性状的关联分析

以固始鸡×安卡鸡F2代资源群为研究对象,利用PCR-RFLP方法检测鸡CFL2基因3'UTR多态性,并将不同基因型与肉质性状进行相关性分析.结果表明:在鸡CFL2基因3'UTR区预测到5个microRNA靶位点.在CFL2基因3'UTR区发现G577A位点,该位点为Kpn Ⅰ自然酶切位点,表现为GG、AA和GA 3种基因型,基因型频率分别为0.589、0.349和0.061,经X2适合性检验,该位点在群体中处于Hardy-Weinberg平衡状态.该突变位点对鸡的腿肌肌纤维密度和肌纤维直径具有显著影响(P＜0.05).结果显示,CFL2基因对鸡的腿肌肌纤维性状有一定影响,同时为该基因的标记辅助育种和进一步研究提供参考.     

肌纤维相关基因影响猪肉质性状的研究进展

肌纤维作为骨骼肌的基本单位,其类型、密度、直径、面积等对肉品质有直接的影响。随着消费者对肉品质要求的不断提高,肌纤维的调控成为肉质性状选育改良的研究重点之一。动物肌纤维调控受多种信号通路和转录因子影响,了解肌纤维发育调控和转化的相关基因的作用机制以及对肉质性状的影响,对提高选育速度、培育优良品种至关重要。本文主要综述了肌纤维调控相关的基因功能,以及营养、环境等对基因表达与肉质性状的影响,以期对动物肌纤维调控和育种改良提供理论参考。     

家禽骨骼肌肌纤维类型影响因素及其转化的研究进展

肌纤维是构成肌肉组织的基本单位。肌肉的肌纤维类型组成与肉品质密切相关,并可在机体生长发育、营养和饲养环境等外界刺激下发生变化。近年来,由于消费者和育种学家对肉品质的关注,家禽肌纤维类型及其转换规律已成为本领域的研究热点之一。随着组学研究技术的发展,转录组学、蛋白组学等技术已广泛应用于家禽肌纤维类型调控机理的研究,极大地推动了相关研究的进展。充分了解目前有关肌纤维类型转化的研究,是后续开展相关肉品质与调控机制研究的重要理论基础。在此背景下,作者介绍了肌纤维的结构组成及不同类型的分类方法,简述了肌纤维类型与家禽肌肉品质特征相关性状如肉色、pH、系水力和嫩度之间的关系,并总结了生长、品种、环境和饲养方式等因素对肌纤维类型的影响,重点讨论了现有的分子调控研究进展,旨在为家禽肌纤维类型转换机理的研究提供参考。最后,对未来优质肉鸡的育种方向提出了展望,希望随着现代分子生物技术的发展,单细胞/空间组学技术、基于CRISPR/Cas9系统的基因编辑技术等前沿技术能在肌纤维类型研究领域广泛运用,肌纤维类型转换的机制研究将不断完善,并建立家禽肉质性状的分子选育方法。     

肉用家畜骨骼肌肌纤维类型转化的研究进展

骨骼肌是机体中最为丰富并高度特化的组织,约占全身重量的40%。大多数骨骼肌是由具有收缩、代谢、抗疲劳性以及病理生理情况下表现出不同易损性的各种肌纤维混合组成的。动物出生后肌纤维数目基本恒定,肌纤维的生长发育主要依赖于肌纤维维度的增加与类型的转化,而转化的结果将导致肌肉中的主要肌纤维类型占比发生改变,并直接影响到肉色、肌内脂肪沉积和嫩度等肉质性状,使肉质具有差异性。该文总结了肌纤维的分类及其特性,以及影响肌纤维类型转化的因素和机理,以期为骨骼肌肌纤维的分类及其转化以及家畜肉质的改善提供参考。     

鸡CFL2基因3′UTR多态性与肉质性状的关联分析

以固始鸡×安卡鸡F2代资源群为研究对象,利用PCR-RFLP方法检测鸡CFL2基因3′UTR多态性,并将不同基因型与肉质性状进行相关性分析。结果表明:在鸡CFL2基因3′UTR区预测到5个microRNA靶位点。在CFL2基因3′UTR区发现G577A位点,该位点为KpnⅠ自然酶切位点,表现为GG、AA和GA 3种基因型,基因型频率分别为0.589、0.349和0.061,经χ2适合性检验,该位点在群体中处于Hardy-Weinberg平衡状态。该突变位点对鸡的腿肌肌纤维密度和肌纤维直径具有显著影响(P<0.05)。结果显示,CFL2基因对鸡的腿肌肌纤维性状有一定影响,同时为该基因的标记辅助育种和进一步研究提供参考。     

Effects of growth hormone treatment on the expression of somatotropic axis genes in the skeletal muscle of lactating Holstein cows

This study focused on the expression of somatotropic axis genes in the skeletal muscle of dairy cattle. A slow-release recombinant bovine growth hormone (GH) (rbGH) formulation was administered to 5 cows, and saline solution (control) was administered to another 5 cows every 2 wk for a total of 10 wk, starting from the peak of lactation. Tissue and blood samples were collected on days 2 and 14 after each rbGH injection. As target genes insulin-like growth factor (IGF)-1, IGF-2, IGFBPs (1, 2, 3, 4, 5, 6), acute labile subunit (ALS), IGF-1 receptor (IGF-1R), GH receptor (GHR), and the known GHR 5′-UTR variants were selected as target genes, and their relative expression was measured using real-time polymerase chain reaction. In GH-treated cows, an increase in expression was observed for GHR 5′-UTR variant 1I on day 14 (P < 0.05), whereas a significant down-regulation of GHR (P < 0.05) was found after comparing values of treated cows between day 2 and day 14. However, only IGF binding proteins (BP)-5 was found to be appreciably up-regulated in GH-treated cows (P < 0.001), which may indicate the importance of this gene in the overall molecular response to GH administration. Our study indicated that GH treatment did not affect the expression of most somatotropic axis genes, despite the marked increase in GH and IGF-1 in blood (P < 0.001). Nor did it have a large impact on the proportion of GHR 5′-UTR variants in the skeletal muscle of lactating cows. Finally, although we observed a significant variation in the expression of some genes, it would appear that the differences between GH-treated cows and controls were not great enough to be considered as reliable indirect indicators of GH treatment in dairy cattle.     

姜黄素对猪脂肪间充质干细胞脂肪合成和分解相关基因mRNA表达的影响

为了探讨姜黄素对猪脂肪间充质干细胞脂肪合成和分解相关基因mRNA表达的影响,从断奶仔猪皮下脂肪分离得到间充质干细胞,增殖培养,诱导分化,并在分化的同时进行姜黄素处理。试验分对照组(诱导分化液+姜黄素溶解试剂DMSO)和处理组(诱导分化液+10μmol/L姜黄素),分化48 h后收集细胞,进行后续检测。结果表明:10μmol/L姜黄素处理能显著降低猪脂肪细胞甘油三酯的含量,而对细胞增殖能力没有明显影响。荧光定量PCR结果显示,脂肪分化过程中的两个重要转录因子过氧化物酶增殖物激活受体-γ(PPAR-γ)和CCAAT增强子结合蛋白-β(C/EBP-β),脂肪合成过程中两个关键酶脂肪酸合成酶(FAS)和乙酰辅酶A羧化酶(ACC)mRNA表达在10μmol/L姜黄素处理组均显著下降,而脂肪分解基因激素敏感脂肪酶(HSL)和脂肪组织三酰甘油水解酶(ATGL)mRNA表达以及酶活在10μmol/L姜黄素处理组均显著升高。结果提示姜黄素降低猪脂肪间充质干细胞脂肪沉积,有可能是通过抑制脂肪合成和增强脂肪分解两个过程来实现的,为姜黄素作为减少猪脂肪沉积的饲料添加剂的应用提供了理论支持。     

Histochemical differentiation of fiber types in neonatal canine skeletal muscle

Differentiation of fiber types in developing canine skeletal muscle was studied, using morphologic, morphometric, and histochemical techniques. Sample collections were made from 6 muscles from the pectoral and pelvic limbs of 16 healthy pups between 1 day and 12 weeks of age. In newborn pups, 90% to 95% of the fibers in the 6 muscles were classified as undifferentiated or type IIC; the remaining fibers were classified either normal or large-size type I. Large-size type I fibers usually accounted for 2% to 4% of the total population and were considered analogous with the B fiber of Wohlfart. These fibers were larger than all other fiber types and disappeared after pups reached 4 to 5 weeks of age. After 2 to 4 weeks, the number of undifferentiated fibers decreased with the appearance of, and the concomitant numerical increases of, normal size type I and type IIA fibers. The percentages of type I and IIA fibers approached proportions of the adult dog by 12 weeks, at which time a type IIA fiber predominance was present in biceps femoris, lateral head of the gastrocnemius, cranial tibial, and long head of the triceps. Type I fibers predominated in medial head of the triceps and superficial digital flexor after 4 to 5 weeks. The mean fiber diameters of type I and IIA fibers were similar to any given muscle throughout the postnatal development. All fiber types stained uniformly with the oxidative stain nicotinamide adeninedinucleotide-tetrazolium reductase during the first 12 weeks of life, whereas a distinction between type I and II fibers was evident after 3 to 4 weeks with the periodic acid-Schiff stain reaction.     

Histochemical identification of fiber types in canine skeletal muscle.

Proximal and distal skeletal muscles from pectoral and pelvic limbs were histochemically examined in 18 neuromuscular disease-free dogs. On the basis of the human system of classification and nomenclature and results of standard adenosine triphosphatase (ATPase) and glycine-formaldehyde preincubation procedures, the fiber types identified in immature and mature canine skeletal muscles were I, IIA, and IIC.     

Effects of post-exercise supplements on glycogen repletion in skeletal muscle

Post-exercise carbohydrate supplementation has been routinely used to enhance glycogen concentrations in skeletal muscle, particularly during multiple-day athletic events. Consumption of protein hydrolysates mixed with carbohydrate supplements during the post-exercise period may increase insulin response and cause glycogen repletion in skeletal muscle. A group of Alaskan sled dogs were selected to examine post-exercise supplementation in a paired crossover study design. The dogs were subjected to the same exercise regimen and provided one of three treatments-water, glucose polymers, or glucose polymers with protein hydrolysates-over a 2-month period. Parameters tested at various post-exercise time points included plasma insulin, glucagon and glucose concentrations, and skeletal muscle glycogen content to gain a better understanding of glucose metabolism and glycogen repletion. The results showed an enhanced insulin, glucose, and glucagon response immediately after supplementation and significantly increased glycogen concentrations in skeletal muscle within 24 hours when dogs received either of the glucose-containing supplements compared with water alone. There were no differences in the plasma parameters or skeletal muscle glycogen stores in dogs provided the glucose polymers alone or the glucose polymers plus protein hydrolysates. Thus, post-exercise carbohydrate supplementation increased muscle glycogen repletion, but inclusion of protein hydrolysates in the supplements provided no additional benefits.     

Influence of recombinant duck follistatin protein on embryonic muscle development and gene expressions

Follistatin (FST) acts as a positive regulator of muscle development by inhibiting the activities and expression of myostatin. The recombinant duck FST protein was injected into hatching eggs and was also added to the medium of duck myoblast to study its role on duck embryonic muscle development and gene expressions. Duck embryo weight increased 3.49% (p > 0.05) in FST treatment group as compared with control group, but minor effects were found on leg or breast muscle weights of ducklings at 2 days post‐hatching (p > 0.05). Relative expression of Pax7 was upregulated in both leg and breast muscle tissues (p < 0.05), while MyoD was only upregulated in leg muscle (p < 0.05), and Myf5 was only upregulated in breast muscle (p < 0.05). Relative expression of myostatin was downregulated in both muscle tissues researched (p < 0.05). In vitro studies also showed some maker genes relevant to protein synthesis and degradation, cells’ proliferation and differentiation had significant changes in myoblasts after treated with FST. These results suggested that in ovo feeding of recombinant FST protein to duck hatching eggs had an effect on duck embryo development but have less roles on the duck embryonic muscle development.     

Effects of hypothyroidism on the skeletal muscle blood flow response to contractions

Hypothyroidism is associated with impaired blood flow to skeletal muscle under whole body exercise conditions. It is unclear whether poor cardiac and/or vascular function account for blunted muscle blood flow. Our experiment isolated a small group of hindlimb muscles and simulated exercise via tetanic contractions. We hypothesized that muscle blood flow would be attenuated in hypothyroid rats (HYPO) compared with euthyroid rats (EUT). Rats were made hypothyroid by mixing propylthiouracil in their drinking water (2.35 x 10-3 mol/l). Treatment efficacy was evidenced by lower serum T3 concentrations and resting heart rates in HYPO (both P<0.05). In the experimental preparation, isometric contractions of the lower right hindlimb muscles at a rate of 30 tetani/min were induced via sciatic nerve stimulation. Regional blood flows were determined by the radiolabelled microsphere method at three time points: rest, 2 min of contractions and 10 min of contractions. Muscle blood flow generally increased from rest ( approximately 5-10 ml/min per 100 g) through contractions for both groups. Further, blood flow during contractions did not differ between groups for any muscle (eg. red section of gastrocnemius muscle; EUT, 59.9 +/- 14.1; HYPO, 61.1 +/- 15.0; NS between groups). These findings indicate that hypothyroidism does not significantly impair skeletal muscle blood flow when only a small muscle mass is contracting. Our findings suggest that impaired blood flow under whole body exercise is accounted for by inadequate cardiac function rather than abnormal vascular function.