肌肉生成抑制素基因的研究进展

肌肉生成抑制素(MSTN)是TGF-β超家族中的成员,又称为GDF-8(Growth differentiation factor 8),是一种骨骼肌生长的负调控因子,由于它在医疗卫生、畜牧生产及水产养殖中潜在的重要应用价值,其基因的研究倍受关注.本文从肌肉生成抑制素基因的发现及不同动物间的保守性、人及不同动物(牛、猪、绵羊、鱼)肌肉生成抑制紊基因的研究进展、肌肉生成抑制素的应用前景三个方面进行阐述.     

抗阻练习模型大鼠骨骼肌中胰岛素生长因子1和肌肉生长抑制素的变化

背景：抗阻练习对人体骨骼肌系统和代谢的良性作用大部分都与其引起的肌肉肥大相关。但抗阻训练对骨骼肌中肌肉生长调节的正向调节因子和负向调节因子的影响尚不十分清楚。 目的：采用大鼠负重爬梯抗阻练习模型,探讨抗阻练习对大鼠骨骼肌局部胰岛素生长因子1和肌肉生长抑制素的影响。 方法：SD大鼠随机分成实验组和对照组。采用负重爬梯的抗阻练习模型,每周训练3次,负重从体质量的30%逐渐增加到200%。训练10周后取左侧腓肠肌,用免疫组织化学法和RT-PCR法分别测定肌肉中胰岛素生长因子1多肽含量和肌肉生长抑制素的表达。 结果与结论：10周抗阻练习后,实验组大鼠腓肠肌中胰岛素生长因子1多肽含量明显增加,肌肉生长抑制素 mRNA的表达显著下降。大鼠骨骼肌中的胰岛素生长因子1和肌肉生长抑制素都对抗阻练习非常敏感,在肌肉对抗阻练习的适应过程中分别起到正向和负向调节作用。     

肌肉生长抑制素基因多态性与肌肉生长敏感性的关联

背景：肌肉生长抑制素(growth differentiation factor 8, GDF-8)基因在人类中对于调节肌肉生长起重要的作用。但关于中国人群该基因的研究甚少。 目的：实验从肌肉生长抑制素基因多性AluⅠ酶切位点限制性酶切位点入手,观察GDF-8基因多性与人体肌肉生长的关联。 方法：选取天津体育学院无训练汉族学生92名,进行为期2个月的力量训练,用B超测定运动前后肱二头肌和股四头肌的肌肉厚度。测试训练前后形态学各指标包括身高、体质量、体脂肪含量、瘦体质量的变化、运动前后肱二头肌和股四头肌的肌肉厚度变化趋势以及在GDF-8 AluⅠ酶切位点限制性酶切位点上的差异。含AluⅠ位点片段由引物扩增后长度为135 bp,定义为A,经AluⅠ限制性酶切割后,含有AluⅠ酶切位点的等位基因出现80,55 bp的片段,定义为T,这样即出现3种基因型AA,AT和TT。 结果与结论：从形态学指标上受试者群体在AluⅠ位点上A/T杂合子及T/T纯合子在身高,训练前后的体质量,瘦体质量,肌肉厚度和肱二头肌及股四头肌直径厚度上均高于A/A纯合子(P  < 0.05或P < 0.01)。结果证实,AluⅠ位点多态性与人体身高,体质量及瘦体质量关联,等位基因T为肌肉生长的先天敏感因子。     

血管生成抑制素和反应停对淋巴管内皮细胞生成的影响

目的：探讨血管生成抑制素和反应停对淋巴管内皮细胞增殖和游走的影响及作用机制。方法：淋巴管内皮细胞取自猪的胸导管。采用划线法和MTT法观察血管生成抑制素和反应停对淋巴管内皮细胞增殖和游走的影响。Hoechst染色检测细胞凋亡。结果．划线法和MTT法均显示血管生成抑制素和反应停对淋巴管内皮细胞的增殖和游走有明显的抑制作用。Hoechst染色证实,经血管生成抑制素和反应停处理后的淋巴管内皮细胞,核周围有凋亡小体。结论：血管生成抑制素和反应停对淋巴管内皮细胞的增殖和游走具有明显的抑制作用,且有剂量依赖性。血管生成抑制素和反应停具有促进细胞凋亡的作用。     

不同制动时相兔骨骼肌萎缩与细胞凋亡的实验研究-原位缺口末端标记法(TUNEL)的观察检测

探讨制动后骨骼肌萎缩的发生机制与骨骼肌细胞凋亡的关系。按照 Sievanen法制备不同时相的制动实验组 ,2 4只实验兔随机分为 4组 ,每组 6只 ,实验侧用管形石膏固定 ,自身未固定侧做对照组。在制动后 3、7、14和 2 8d取材 ,应用末端转移酶介导的 U TP缺口末端标记法 (Td T— mediated d UTP nick end labeling,TUNEL )检测萎缩骨骼肌中有无凋亡的骨骼肌细胞 ,并与形态学的观察结果相对照。对各组的数据进行统计学处理。结果表明不同制动时相所致的萎缩骨骼肌中均可发现有程度不一的骨骼肌细胞发生凋亡 ,以 14 d组最为显著。不同时相凋亡的肌细胞在空间的分布上呈不一致性。制动所致萎缩的骨骼肌中有时可见呈假阳性 TU NEL染色 ,但与凋亡的肌细胞的表现是不同的。我们认为 (1)制动后骨骼肌萎缩有骨骼肌细胞凋亡的参与。 (2 )骨骼肌细胞凋亡的多少与制动时相密切相关 ,以制动后 14 d最为明显。 (3)凋亡的骨骼肌细胞在空间分布上随制动时相不同而不同 ,呈不一致性。 (4 )制动后骨骼肌萎缩的程度与肌细胞凋亡的程度密切相关。     

血管抑制素和内皮抑制素在大肠杆菌中的融合表达与鉴定

血管抑制素（AS）和内皮抑制素（ES）是两种具有较强活性的内源性血管抑制剂，联合应用具有协同作用。本研究通过大肠杆菌表达AS-ES融合蛋白，观察其对血管生成的抑制作用。首先用RT-PCR方法分别获得AS和ES基因，通过基因拼接获得融合基因，构建了含有该融合基因的原核表达质粒——pET-42（b）／AS-ES。表达菌株经IPTG诱导后目的蛋白以包涵体形式表达，表达量为14％，分子量约65KD。Western blotting检测表明表达产物可分别与AS和ES抗体产生特异的免疫反应。经复性、肝素亲和层析柱纯化的表达产物对鸡胚尿囊膜血管有明显抑制作用。本研究获得了AS、ES在大肠杆菌中的融合表达．目的蛋白具有特异的免疫反应性和血管抑制活性。     

血管抑制素和内皮抑制素的协同作用

血管抑制素和内皮抑制素是内源性的血管生成抑制剂,对肿瘤有明显的抑制作用,具有良好的应用前景,近年来,对于它们之间协同作用的研究已经成为新的热点。本文除对两者做了结构、性质、功能等方面的一般性介绍外,重点介绍了它们之间的协同作用。与血管抑制素和内皮抑制素单体的作用相比,它们之间的联合使用具有更强的抑制血管生成和肿瘤生长的作用。     

血管生成抑制素对胃癌细胞的影响

目的探讨血管生成抑制素对胃癌细胞增殖和游走的影响及对胃癌细胞分泌血管内皮生长因子A和C(VEGF-A和VEGF-C)的影响并分析其作用机制。方法采用MTT法和划线法观察0、0.5、1.0、1.5μg/ml浓度的血管生成抑制素对胃癌细胞增殖和游走的影响。免疫组化的方法检测0、0.5、1.0、1.5μg/ml浓度的血管生成抑制素对胃癌细胞内血管内皮生长因子(VEGF)-A和VEGF-C的影响。结果 MTT法显示不同浓度的血管生成抑制素对胃癌细胞增殖无明显影响(p0.05),同样,划线法测得的胃癌细胞迁移距离同对照组相比无明显差异(p0.05)。但免疫组化法结果显示,0.5、1.0、1.5μg/ml浓度的血管生成抑制素组胃癌细胞VEGF-A和VEGF-C的表达同对照组相比明显减少(p0.05)。结论血管生成抑制素明显减少胃癌细胞内的VEGF-A和VEGF-C的分泌,但对胃癌细胞的增殖和游走无明显作用。     

血管内皮抑制素的研究进展

实体瘤当生长到一定的体积时必然伴随着血管生成。 1971年 ,Ｆｏｌｋｍａｎ[1] 最早提出肿瘤生长是血管依赖性的。他认为肿瘤血管生成是肿瘤生长和转移的形态学基础。它不仅向肿瘤提供充足的营养 ,同时也向机体输出肿瘤细胞 ,导致肿瘤细胞的恶性生长和转移。因此 ,Ｆｏｌｋｍａｎ[2 ] 提出抗血管疗法 ,设想抑制肿瘤血管生成 ,使肿瘤细胞因缺血、缺氧而大部分死亡 ,从而可延缓晚期肿瘤生长 ,延长病人带瘤生存期 ,并可抑制亚临床转移灶生长 ,推迟复发。这一设想为越来越多的证据所支持 ,并使这一领域成为肿瘤研究的热点及肿瘤治疗的新策略。人…     

内抑制素和血小板因子-4对淋巴管内皮细胞生成的影响

目的寻找安全有效的淋巴管生成抑制剂。方法取猪的胸导管内皮细胞进行培养，进行光镜和电镜观察。设立对照组、内抑制素实验组和血小板因子-4（PF-4）实验组。应用刮线法和MTT法来判定这两种抑制因子对细胞有无抑制作用。结果应用刮线法对Endostatin、PF-4对照组与实验各组的细胞数及细胞游走距离进行比较，均P〈0．05。采用MTT法将内抑制素、PF-4对照组与实验各组吸光光度值相比较，均P〈0．05。结论内抑制素和PF-4对淋巴管内皮细胞的增殖和游走有明显的抑制作用，且有剂量依赖性。     

Estrogen administration attenuates immobilization-induced skeletal muscle atrophy in male rats

We tested the hypothesis that estrogen administration would retard immobilization-induced muscle atrophy in adult male rats. The rats were injected for 24 days with either estrogen (40 microg/kg(-1), beta-estradiol 3-benzoate in olive oil vehicle), or vehicle alone. At day 14 of estrogen treatment, the hindlimb muscles of one leg were immobilized in plantar flexion position by the use of a plaster cast. Following 10 days of immobilzation, the atrophic and the contralateral soleus muscles were both removed and analyzed to determine the level of muscle atrophy along with the measurement of the protein levels of Cu-Zn-superoxide dismutase (Cu-Zn-SOD), heat shock protein 72 (HSP72), and selected proteases. Compared to placebo animals, estrogen treatment significantly reduced (-35%) muscle atrophy. Further, estrogen significantly abridged the expression of the calcium-activated protease, calpain, in the atrophied hindlimb muscle. In contrast, estrogen treatment did not alter the protein levels of HSP72 in the immobilized soleus muscle. These results support the postulate that estrogen attenuates the rate of disuse muscle atrophy, partly because of reductions in immobilization-induced calcium-activated protease levels.     

高选择性神经损伤模型骨骼肌MyoD和myogenin mRNA的表达

为观察成肌调节因子MyoD和myogenin mRNA在单纯运动神经或/和单纯感觉神经损伤后萎缩骨骼肌中的表达,探讨其在失神经骨骼肌萎缩发生发展中的可能作用,我们分别切断大鼠左侧L4～L6脊神经腹根、背根或坐骨神经制备选择性神经损伤模型,并将动物分成腹根切断组(VRT),背根切断组(DRT)和坐骨神经切断组(SNT)。于术后2、4周应用逆转录-聚合酶链式反应(RT-PCR),分别检测和观察腹根、背根及坐骨神经切断组大鼠腓肠肌中MyoD和myogenin mRNA表达的变化。结果显示:与相同时间点正常对照侧相比较,各组大鼠腓肠肌MyoD和myogenin mRNA的表达均增加。4周时不同损伤类型间比较,myogenin mRNA表达差异具有显著性(P<0.05)。以上结果提示,MyoD和myogenin可能参与了神经损伤后骨骼肌的再生修复过程,但两者作用于成肌分化的不同阶段,且作用不同。     

甘草黄酮对运动大鼠骨骼肌Perilipin mRNA及蛋白表达的影响

BACKGROUND: Glycyrrhiza flavonoids can protect muscle tissues against oxidative and inflammatory injuries. OBJECTIVE: To explore the effects of Glycyrrhiza flavonoids on energy storing and release of adipose tissue by studying expressions of perilipin mRNA and protein in skeletal muscle tissues of rats after exhaustive exercise. METHODS: Fifty male healthy Sprague-Dawley rats were used and equally randomized into five groups: quiet control, exercise alone (intragastric administration with saline), exercise combined with low-, moderate-, high-dose Glycyrrhiza flavonoids (intragastric administration with Glycyrrhiza flavonoids) groups, respectively. Perilipin mRNA and protein expressions in skeletal muscle tissues containing gastrocnemius muscle and musculi soleus were determined at 6 weeks after exhaustive exercise. RESULTS AND CONCLUSION: Expression of perilipin mRNA in rat gastrocnemius muscle in quiet control group was significantly decreased compared with exercise alone and all combined intervention groups (P < 0.05 or P < 0.01). Protein expression of perilipin in exercise combined with moderate- or high-dose glycyrrhiza flavonoids group was significantly increased compared with quiet control group (P < 0.05). Expression of perilipin mRNA in rat musculi soleus was significantly decreased compared with exercise combined with moderate-dose glycyrrhiza flavonoids group (P < 0.05). These findings confirm that Glycyrrhiza flavonoids are benefit for improvement of aerobic metabolism capacity of gastrocnemius muscle through regulating lipolysis pathway. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

The expression of IGF-I and myostatin mRNAs in skeletal muscle of hypophysectomized and underfed rats during postnatal growth

Aim: To determine the roles of myostatin and insulin‐like growth factor‐I (IGF‐I) during postnatal growth, we examined IGF‐I and myostatin mRNA expression in the skeletal muscles of hypophysectomized and underfed rats during postnatal growth. Methods: Five‐week‐old rats were divided into four groups: freely fed control, moderately underfed, severely underfed and hypophysectomized. Four weeks later, blood and muscle samples were gathered to determine serum IGF‐I, myosin heavy chain (MHC) isoforms, IGF‐I Ea, IGF‐I Eb and myostatin mRNA. Results: The weights of soleus, plantaris and masseter muscles were decreased in underfed and hypophysectomized rats. Hypophysectomy resulted in significant increases of type I MHC at the expense of type IIx in plantaris muscle and of neonatal MHC at the expense of types IIx and IIb in masseter muscle. Serum IGF‐I was decreased by underfeeding and hypophysectomy. Plantaris muscle IGF‐I Ea mRNA in underfed and hypophysectomized rats was significantly lower than in normal controls. Plantaris muscle IGF‐I Eb mRNA in underfed rats was significantly lower than in normal controls. Masseter muscle IGF‐I Eb mRNA in severely underfed rats was significantly lower than in normal control and hypophysectomized rats. Soleus muscle myostatin mRNA in hypophysectomized rats was significantly higher than in normal and significantly underfed rats. No significant differences in plantaris and masseter muscle myostatin mRNA were observed between groups. Conclusion: Suppressed muscle growth caused by hypophysectomy and underfeeding may be attributed mainly to reduced circulating IGF‐I and partially to reduced IGF‐I mRNA, rather than to a change in myostatin.     

Interleukin-6 modifies mRNA expression in mouse skeletal muscle

Aim: The aim of this study was to test the hypothesis that interleukin (IL)‐6 plays a role in exercise‐induced peroxisome proliferator‐activated receptor γ co‐activator (PGC)‐1α and tumor necrosis factor (TNF)‐α mRNA responses in skeletal muscle and to examine the potential IL‐6‐mediated AMP‐activated protein kinase (AMPK) regulation in these responses. Methods: Whole body IL‐6 knockout (KO) and wildtype (WT) male mice (4 months of age) performed 1 h treadmill exercise. White gastrocnemius (WG) and quadriceps (Quad) muscles were removed immediately (0′) or 4 h after exercise and from mice not run acutely. Results: Acute exercise reduced only in WT muscle glycogen concentration to 55 and 35% (P < 0.05) of resting level in Quad and WG respectively. While AMPK and Acetyl CoA carboxylase (ACC) phosphorylation increased 1.3‐fold (P < 0.05) in WG and twofold in Quad immediately after exercise in WT mice, no change was detected in WG in IL‐6 KO mice. The PGC‐1α mRNA content was in resting WG 1.8‐fold higher (P < 0.05) in WT mice than in IL‐6 KO mice. Exercise induced a delayed PGC‐1α mRNA increase in Quad in IL‐6 KO mice (12‐fold at 4 h) relative to WT mice (fivefold at 0′). The TNF‐α mRNA content was in resting Quad twofold higher (P < 0.05) in IL‐6 KO than in WT, and WG TNF‐α mRNA increased twofold (P < 0.05) immediately after exercise only in IL‐6 KO. Conclusion: In conclusion, IL‐6 affects exercise‐induced glycogen use, AMPK signalling and TNF‐α mRNA responses in mouse skeletal muscle.     

Cyclic muscle twitch contraction inhibits immobilization-induced muscle contracture and fibrosis in rats

We investigated the effects of cyclic muscle twitch contraction caused by neuromuscular electrical stimulation (NMES) on immobilization-induced muscle contracture and fibrosis in rats. Twenty-nine rats were divided into control, immobilization, and immobilization with muscle contraction groups. The ankle joints of the immobilization and muscle contraction rats were fixed in full plantar flexion with a plaster cast for 4 weeks. In the muscle contraction group, cyclic muscle twitch contraction of the soleus muscle was induced using a commercial device (1 Hz, 4 ± 2 mA, 60 min/day, 5 times/week) with the ankle joint immobilized. The dorsiflexion range of ankle joint motion in the muscle contraction group was significantly greater than that in the immobilization group. The expressions of fibrosis-related genes (i.e., hypoxia inducible factor-1α, transforming growth factor-β1, α-smooth muscle actin, and types I and III collagen) were significantly decreased in the muscle contraction group compared to the immobilization group. The fluorescence intensities of type I and type III collagen in the perimysium and endomysium in the muscle contraction group were significantly decreased compared to the immobilization group. These results suggest that cyclic muscle twitch contraction induced by NMES might alleviate skeletal muscle fibrosis, reducing immobilization-induced muscle contracture.     

被动训练促进失神经肌萎缩模型大鼠骨骼肌结构和功能的恢复

文题释义：肌肉萎缩：是指横纹肌营养障碍,肌肉纤维变细甚至消失等导致的肌肉体积缩小。多由肌肉本身疾患或神经系统功能障碍所致,病因主要有：神经源性肌萎缩、肌源性肌萎缩、失用性肌萎缩和其他原因性肌萎缩。肌肉营养状况除肌肉组织本身的病理变化外,更与神经系统有密切关系。脊髓疾病常导致肌肉营养不良而发生肌肉萎缩。 肌卫星细胞：是一类存在于肌细胞基底膜与肌膜之间的成体干细胞,作为肌源性干细胞在肌肉组织损伤后,能够在激活后发挥良好的增殖、分化能力,在骨骼肌损伤的修复和再生过程中发挥重要作用。 背景：炎症细胞或炎性因子参与失神经损伤后骨骼肌肌卫星细胞的增殖和分化,在失神经骨骼肌肌组织病理过程中起着重要的作用。 目的：研究被动康复训练对失神经萎缩大鼠骨骼肌结构、功能以及肌动蛋白和炎症因子表达的影响。 方法：将30只SD大鼠平均分为假手术组、模型组和训练组,模型组及训练组大鼠暴露坐骨神经并剪断,假手术组只暴露而不剪断坐骨神经。造模后2个月始用自制滚筒对训练组大鼠进行被动康复训练2个月,用肌肉湿质量比和BBB评分评估肌肉萎缩的程度及运动功能,苏木精-伊红染色观察肌纤维微细结构及横截面积,免疫组化染色检测各组腓肠肌肌动蛋白及肿瘤坏死因子α、白细胞介素6及白细胞介素1β表达。实验经沈阳医学院实验动物福利伦理委员会的审批,批准文号为SYYXY2015010601。结果与结论：①训练组BBB评分高于模型组;②训练组腓肠肌湿质量高于模型组但肌纤维的横截面积却低于模型组(P < 0.001,P < 0.05),训练组腓肠肌肌动蛋白表达高于模型组(P < 0.001);③训练组炎症因子肿瘤坏死因子α、白细胞介素6及白细胞介素1β的表达水平低于模型组(P < 0.001或P < 0.05);④结果说明,被动训练有助于失神经萎缩肌肉结构和功能的恢复,降低炎症因子的水平防止肌肉的进一步萎缩,提高骨骼肌的肌力。 ORCID: 0000-0002-9303-8651(王世杨) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Regulation of skeletal muscle PPARδ mRNA expression in twins

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors regulating the expression of genes involved in lipid and glucose metabolism in a complex and to some extent unknown manner. Our aim was to study the impact of different factors on PPAR δ mRNA expression in human skeletal muscle on one side, and the impact of PPAR δ mRNA expression on these factors, including glucose and lipid metabolism, aerobic capacity, fibre type composition and lipid profile, on the other side. PPAR δ mRNA levels were quantified by real-time PCR in muscle biopsies from 176 young and elderly monozygotic and dizygotic twins. Young twins had significantly increased PPAR δ mRNA levels compared with elderly twins. A 2 h hyperinsulinaemic euglycaemic clamp had no significant effect on PPAR δ mRNA levels. Biometric models were calculated for basal PPAR δ mRNA expression to estimate the degree of genetic versus environmental influence. In both young and elderly twins there was a substantial genetic component influencing basal PPAR δ mRNA levels. In a regression model, the muscle PPAR δ mRNA expression was correlated to birth weight, central adiposity and age. The level of PPAR δ mRNA was also positively correlated with markers for oxidative muscle fibres. However, in this apparently healthy study population, we found no correlations between PPAR δ mRNA expression and aerobic capacity, lipid profile or glucose and lipid metabolism. In conclusion, we provide evidence that mRNA expression of PPAR δ in human skeletal muscle is under genetic control but also influenced by factors such as age, birth weight and central adiposity.