Muscle satellite cells:one of the important factors in the occurrence and development of sarcopenia

Sarcopenia,or muscle loss,has been one of the hot topics in the medical field in recent years.Due to limited attention and effective treatments for sarcopenia in the past,many patients,especially the elderly,suffered irreversible damage to their motor function caused by sarcopenia.However,recent scientific studies have found that the occurrence and development of sarcopenia are closely related to the function and quantity of muscle satellite cells.This article briefly discusses the relationship between muscle satellite cells and sarcopenia.

Effects of Soybean Isoflavones on In vitro Antioxidative Capacity of Satellite Cells of Porcine Skeletal Muscles

A synthetic isoflavone （ISO-S） or genistein was added in culture medium at different concentrations （0, 10, 20, 30, 40, and 80 p.mol L^-1） to investigate the effects of soybean isoflavones on antioxidative capacity of porcine skeletal muscle satellite cells. After 48 h incubation, the suspension was cryopreserved for the determination of superoxide dismutase （SOD）, catalase （CAT）, glutathione peroxidase （GSH-Px） activities, and malondialdehyde （MDA） content. The mRNA levels of SOD, CAT, and GSH-Px gene in cells were detected with Taqman fluorescent probe method. The results showed that the content of MDA and the activities and the mRNA levels of SOD of porcine skeletal muscle satellite cells were influenced by supplemented soybean isoflavone （P〈0.05） when adding 10-80 μmol L^-1 ISO-S or genistein in the medium. The MDA contents, SOD and CAT activities and their mRNA expression levels of porcine skeletal muscle cells responded quadratically （P〈 0.05） as the level of ISO-S or genistein increased. Pre-incubation of porcine skeletal muscle satellite cells with ISO-S or genistein at 10-40 pmol L-1 elevated the activities and the mRNA expression levels of SOD and CAT in cells concurrently and decreased the cellular content of MDA （P〈 0.05）. The results indicated that pre-incubation of ISO-S or genistein at 10- 40μmol L^-1 could improve the antioxidative capacity of porcine skeletal muscle satellite cells.

MicroRNA-22 inhibits proliferation and promotes differentiation of satellite cells in porcine skeletal muscle

Pig is an important economic animal in China. Improving meat quality and meat productivity is a long time issue in animal genetic breeding. Micro RNAs(mi RNAs) are short non-coding RNAs that participate in various biological processes, such as muscle development and embryogenesis. mi R-22 differentially expresses in embryonic and adult skeletal muscle. However, the underlying mechanism is unclear. In this study, we investigated mi R-22 function in proliferation and differentiation of porcine satellite cells(PSCs) in skeletal muscle. Our data show that mi R-22 expressed in both proliferation and differentiated PSCs and is significantly upregulated(P<0.05) during differentiation. After treated with the mi R-22 inhibitor, PSCs proliferation was significantly increased(P<0.05), as indicated by the up-regulation(P<0.01) of cyclin D1(CCND1), cyclin B1(CCNB1) and down-regulation(P<0.05) of P21. Conversely, over-expression of mi R-22 resulted in opposite results. Differentiation of PSCs was significantly suppressed(P<0.05), evidenced by two major myogenic markers: myogenin(Myo G) and myosin heavy chain(My HC), after transfecting the PSCs with mi R-22 inhibitor. Opposite results were demonstrated in the other way around by transfection with mi R-22 mimics. In conclusion, the data from this study indicated that mi R-22 inhibited the PSCs proliferation but promoted their differentiation.

Protective Effect of ATP on Skeletal Muscle Satellite Cells Damaged by H_2O_2

This study investigated the protective effect of ATP on skeletal muscle satellite cells damaged by H2O2 in neonatal rats and the possible mechanism. The skeletal muscle satellite cells were randomly divided into four groups： normal group, model group（cells treated with 0.1 mmol/L H2O2 for 50 s）, protection group（cells treated with 16, 8, 4, 2, 1, 0.5, or 0.25 mmol/L ATP for 24 h, and then with 0.1 mmol/L H2O2 for 50 s）, proliferation group（cells treated with 16, 8, 4, 2, 1, 0.5, or 0.25 mmol/L ATP for 24 h）. MTT assay, FITC＋PI＋DAPI fluorescent staining, Giemsa staining and immunofluorescence were performed to examine cell viability and apoptosis, and apoptosis-related proteins. The results showed that the survival rate of skeletal muscle satellite cells was decreased and the apoptosis rate was increased after H2O2 treatment（P〈0.01）. Different doses of ATP had different effects on skeletal muscle satellite cells damaged by H2O2： the survival rate of muscle satellite cells treated with ATP at 4, 2, or 1 mmol/L was increased. The protective effect was most profound on cells treated with 2 mmol/L ATP. Immunofluorescence showed that ATP could increase the number of Bcl-2-positive cells（P〈0.01） and decrease the number of the Bax-positive cells（P〈0.01）. It was concluded that ATP could protect skeletal muscle satellite cells against H2O2 damage in neonatal rats, which may be attributed to the up-regulation of the expression of Bcl-2 and down-regulation of Bax, resulting in the suppression of apoptosis.

Denervated muscle extract promotes recovery of muscle atrophy through activation of satellite cells. An experimental study

Purpose: The objective of the present study was to determine whether a denervated muscle extract(DmEx) could stimulate satellite cell response in denervated muscle.Methods: Wistar rats were divided into 4 groups: normal rats, normal rats treated with DmEx, denervated rats, and denervated rats treated with DmEx. The soleus muscles were examined using immunohistochemical techniques for proliferating cell nuclear antigen, desmin, and myogenic differentiation antigen(MyoD), and electron microscopy was used for analysis of the satellite cells.Results: The results indicate that while denervation causes activation of satellite cells, DmEx also induces myogenic differentiation of cells localized in the interstitial space and the formation of new muscle fibers. Although DmEx had a similar effect in nature on innervated and denervated muscles, this response was of greater magnitude in denervated vs. intact muscles.Conclusion: Our study shows that treatment of denervated rats with DmEx potentiates the myogenic response in atrophic denervated muscles.

Skeletal muscle atrophy,regeneration,and dysfunction in heart failure:Impact of exercise training

This review highlights some established and some more contemporary mechanisms responsible for heart failure(HF)-induced skeletal muscle wasting and weakness.We first describe the effects of HF on the relationship between protein synthesis and degradation rates,which determine muscle mass,the involvement of the satellite cells for continual muscle regeneration,and changes in myofiber calcium homeostasis linked to contractile dysfunction.We then highlight key mechanistic effects of both aerobic and resistance exercise training on skeletal muscle in HF and outline its application as a beneficial treatment.Overall,HF causes multiple impairments related to autophagy,anabolic-catabolic signaling,satellite cell proliferation,and calcium homeostasis,which together promote fiber atrophy,contractile dysfunction,and impaired regeneration.Although both wasting and weakness are partly rescued by aerobic and resistance exercise training in HF,the effects of satellite cell dynamics remain poorly explored.

Impact of Bovine Skeletal Muscle Satellite Cell Differentiation by Small Interfering RNA Targeting Myogenin Gene

To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.

IGF-1, bFGF EXPRESSION AND VASCULAR REGENERATION IN ACUTE INFARCTED CANINE MYOCARDIUM AFTER AUTOLOGUS SKELETAL MUSCLE SATELLITE CELL IMPLANTATION

Objective:To study the cell growth factor secretion and vascular regeneration in acute in-farcted myocardium after autologus skeletal muscle satellite cell implantation.Methods:Autologous skele-tal muscle satellite cells from adult mongrel canine were implanted into the acute myocardial infarct site via the ligated left anterior descending(LAD)artery.Specimens were harvested at 2,4,8 weeks after implantation for the expression of insulin-like growth factor-1(IGF-1),basic fibroblast growth factor(bFGF) and the vas-cular density.Results:The expression of IGF-1,bFGF and the vascular density in skeletal muscle satellite cell implant group were higher than that in the control group.Conclusion:The skeletal muscle satellite cells,after being implanted into the acute myocardial infarction ,not only showed myocardial regeneration,but also showed the ability to secrete the cell factors,hence representing a positive effect on the regeneration of the infarcted myocardium.

THE IMPROVEMENT OF INFARCTED MYOCARDIAL CONTRACTILE FORCE AFTER AUTOLOGOUS SKELETAL MUSCLE SATELLITE CELL IMPLANTATION

Objective To study the improvement of infarcted myocardial contractile force after autologous skeletal muscle satellite cell implantation via intracoronary arterial perfusion. Methods Skeletal muscle cells were harvested from gluteus max of adult mongrel dogs and the cells were cultured and expanded before being labeled with DAPI (4’, 6-diamidino-2-phenylindone). The labeled cells were then implanted into the acute myocardial infarct site via the ligated left anterior descending (LAD) coronary artery. Specimens were taken at 2nd, 4th, 8th week after myoblast implantation for histologic and contractile force evaluation, respectively. Results The satellite cells with fluorescence had been observed in the infarct site and also in papi- llary muscle with consistent oriented direction of host myocardium. A portion of the implanted cells had differen- tiated into muscle fibers. Two weeks after implantation, the myocardial contractile force showed no significant difference between the cell implant group and control group. At 4 and 8 week, the contractile force in the cell implant group was better than that in control group. Conclusion The skeletal muscle satellite cells, implanted into infarct myocardium by intracoronary arterial perfusion, could disseminate through the entire infarcted zone with myocardial regeneration and improve the contractile function of the infarcted myocardium.

Barriers in contribution of human mesenchymal stem cells to murine muscle regeneration

AIM: To study regeneration of damaged human and murine muscle implants and the contribution of added xenogeneic mesenchymal stem cells(MSCs).METHODS: Minced human or mouse skeletal muscle tissues were implanted together with human or mouse MSCs subcutaneously on the back of non-obese diabetic/severe combined immunodeficient mice. The muscle tissues(both human and murine) were minced with scalpels into small pieces(< 1 mm3) and aliquoted in portions of 200 mm3. These portions were either cryopreserved in 10% dimethylsulfoxide or freshly implanted. Syngeneic or xenogeneic MSCs were added to the minced muscles directly before implantation. Implants were collected at 7, 14, 30 or 45 d after transplantation and processed for(immuno)histological analysis. The progression of muscle regeneration was assessed using a standard histological staining(hematoxylin-phloxinsaffron). Antibodies recognizing Pax7 and von Willebrand factor were used to detect the presence of satellite cells and blood vessels, respectively. To enable detection of the bone marrow-derived MSCs or their derivatives we used MSCs previously transduced with lentiviral vectors expressing a cytoplasmic LacZ gene. X-gal staining of the fixed tissues was used to detect β-galactosidase-positive cells and myofibers.RESULTS: Myoregeneration in implants of fresh murine muscle was evident as early as day 7, and progressed with time to occupy 50% to 70% of the implants. Regeneration of fresh human muscle was slower. These observations of fresh muscle implants were in contrast to the regeneration of cryopreserved murine muscle that proceeded similarly to that of fresh tissue except for day 45(P < 0.05). Cryopreserved human muscle showed minimal regeneration, suggesting that the freezing procedure was detrimental to human satellite cells. In fresh and cryopreserved mouse muscle supplemented with LacZ-tagged mouse MSCs, β-galactosidase-positive myofibers were identified early after grafting at the wellvascularized periphery of the implants. The contribution of human MSCs to murine myofiber formation was, however, restricted to the cryopreserved mouse muscle implants. This suggests that fresh murine muscle tissue provides a suboptimal environment for maintenance of human MSCs. A detailed analysis of the histological sections of the various muscle implants revealed the presence of cellular structures with a deviating morphology. Additional stainings with alizarin red and alcian blue showed myofiber calcification in 50 of 66 human muscle implants, and encapsulated cartilage in 10 of 81 of murine muscle implants, respectively.CONCLUSION: In mouse models the engagement of human MSCs in myoregeneration might be underestimated. Furthermore, our model permits the dissection of speciesspecific factors in the microenvironment.

MMP14调控骨骼肌卫星细胞分化的分子机制研究

旨在分析基质金属蛋白酶14(MMP14)调控骨骼肌卫星细胞分化的分子机制。本试验取10只4周龄C57/BL6雌性小鼠,利用胶原酶消化法分离骨骼肌卫星细胞。首先,对骨骼肌卫星细胞进行诱导分化,利用qRT-PCR和Western blot试验分析MMP14在骨骼肌卫星细胞增殖期和分化期表达量的变化。应用siRNA抑制MMP14蛋白表达,分为试验组(si-MMP14)和对照组(si-NC),每组3个重复:首先诱导细胞分化,应用免疫荧光和qRT-PCR分析试验组和对照组细胞分化水平的差异;随后取增殖期细胞进行蛋白组学测序,结合生物信息学分析鉴定差异蛋白,并筛选MMP14调控的关键差异蛋白和通路。本研究结果表明:1)MMP14在卫星细胞增殖期表达上调,在分化期表达下调;抑制MMP14蛋白会抑制肌管生成,表现为肌管融合指数下降。2)通过蛋白组学分析筛选到549个差异蛋白,其中有66个上调蛋白和483个下调蛋白,差异蛋白主要富集在细胞黏附、脂肪酸代谢以及AMPK通路等,参与调控细胞命运决定、组蛋白甲基化和染色质结构等生物学过程。3)通过蛋白互作关系网络分析发现MMP14与肌源性分化相关蛋白、脂肪生成相关蛋白以及异染色质结构调控蛋白直接互作,抑制MMP14可导致肌分化转录因子(PAX7、MYOD)和H3-K9甲基转移酶(SETDB1、SUV39H1)下调,而成脂分化转录因子(JUN、C/EBPβ)上调。本研究初步分析了MMP14调控骨骼肌卫星细胞分化的机制,MMP14可能通过H3-K9组蛋白甲基化参与卫星细胞命运决定以及成肌与成脂分化的转换,且这种调控作用发生在卫星细胞细胞分化启动前。本研究结果为骨骼肌发育研究提供理论依据。

骨骼肌再生过程中卫星细胞调控机制及其生态位信号的作用

背景:卫星细胞是骨骼肌包含的一种特定的成体干细胞群,可促进损伤骨骼肌的再生重建,但其具体机制尚不完善。目的:综述骨骼肌再生过程中卫星细胞调控作用以及卫星细胞与其生态位信号相互作用的机制,旨在总结现有知识的基础上提供新的研究思路和角度。方法:检索Web of Science、PubMed、中国知网(CNKI)、万方、维普等数据库2002年1月至2022年6月发表的文献。英文检索词:muscle,skeletal muscle,muscle injury,stem cells,satellite cells,muscle repair等;中文检索词:骨骼肌,骨骼肌再生,骨骼肌重建,卫星细胞,生态位等。共纳入66篇文献进行整理和分析。结果与结论:(1)卫星细胞存在于骨骼肌中,其既有助于损伤后新肌纤维的形成,亦有助于已存在的成年肌纤维有效生长。(2)卫星细胞中的静止卫星细胞被激活后,骨骼肌再生过程中卫星细胞的增殖、分化和融合形成肌纤维等步骤均会受到其内在不同机制调控作用的影响。(3)卫星细胞可与所处生态位信号中的肌纤维、细胞外基质、骨骼肌交界位、纤维生成祖细胞、免疫细胞以及内皮细胞相互作用促进卫星细胞的激活和增殖和分化,进而实现骨骼肌的有效再生。(4)未来研究可能的突破口:机体内卫星细胞的分裂模式;调控卫星细胞转移相关机制;卫星细胞在体内分化或自我更新的具体时间;卫星细胞和骨骼肌交界位的相互作用机制。(5)此次综述可为骨骼肌损伤重建的领域及其创新提供一定的理论参考价值。

转化生长因子-β参与骨骼肌损伤修复的机制探讨及研究进展

骨骼肌损伤是临床上常见的疾病,对其修复机制的深入了解对于制定有效的治疗策略至关重要。本文聚焦于TGF-β在骨骼肌损伤修复中的关键作用,介绍了其家族成员的多样性和信号传导途径,探讨在骨骼肌损伤后TGF-β的表达与调控部分,解析了其早期表达动态和调控因素,深入研究TGF-β对骨骼肌修复的影响,揭示了其在炎症调节、细胞活化与增殖以及纤维化等方面的关键作用。特别关注了其在肌肉再生中的作用机制及在细胞水平上的调控机制。此外,对TGF-β在骨骼肌损伤修复中的潜在临床应用进行了讨论,探索了将其作为治疗靶点和调控剂的发展与应用。然而,当前研究中仍存在争议与不足,如TGF-β的双重作用和个体差异对治疗的影响。未来的研究方向应包括深入挖掘信号通路的细节以及生物标志物的发现。通过克服这些挑战,TGF-β在骨骼肌损伤修复中的潜在临床应用有望迎来新的突破,为患者提供更为个体化和有效的治疗策略。

骨骼肌中卫星细胞衰老生物学机制及潜在的应对策略

背景:卫星细胞是一种肌源性干细胞,位于肌纤维膜与基底膜之间,但尚未有综述完全揭示卫星细胞衰老机制及其潜在应对策略,这对于当前减缓骨骼肌老化的策略应用难以起到有效的指导效果。目的:综述骨骼肌中卫星细胞衰老的机制及其相关减缓其衰老的应对策略。方法:检索各数据库时间截至2023年5月,包括Web of Science、PubMed、中国知网、万方和维普数据库。英文检索词:“Skeletal muscle,satellite cells,aging,mechanism,solution strategye”;中文检索词:“骨骼肌,卫星细胞,衰老,老化,机制,应对策略”。经过严格按照纳入和排除标准进行筛选,最终纳入78篇文献进行综述分析。结果与结论:①卫星细胞位于肌纤维膜与基底膜之间,具有增殖和分化潜能,通常处于静息状态,但在肌肉组织受刺激时会被激活并参与修复和恢复正常组织结构的过程,衰老会导致卫星细胞数量减少,并引发肌力和耐力的下降等症状。②卫星细胞衰老的机制主要涉及再生能力下降、串扰能力随生态位变化、年龄依赖性损失和异质性变化,衰老的卫星细胞数量减少以及活性降低会导致肌肉再生速度变慢和损伤恢复时间增加,且在分化过程中可能出现错误,从而导致肌肉质量下降和功能退化。③针对卫星细胞衰老的应对策略主要包括调节体内卫星细胞的受体环境、外源手段干预促进卫星细胞再生、人体肌肉模型构建和运动和饮食干预诱导卫星细胞增殖等,这些策略具有一定的潜力,可以为卫星细胞的再生和治疗骨骼肌疾病提供新的思路和方法。④未来的研究建议深入探究卫星细胞衰老机制,探究卫星细胞与生态位之间的相互作用,研究卫星细胞与免疫系统和线粒体功能等因素的关系,开发应用人体肌肉模型来提高研究深度和准确性。

长时间血清饥饿胁迫对猪骨骼肌卫星细胞代谢和自噬的影响

为了探究长时间、不同程度饥饿胁迫条件对原代猪骨骼肌卫星细胞(SMSCs)代谢和自噬的影响,本试验通过控制SMSCs培养体系中血清浓度以形成不同程度饥饿胁迫,其中20%血清培养浓度为正常对照组,15%、10%、5%和0%血清培养浓度为试验组。细胞培养96 h后,通过流式细胞术检测各组细胞凋亡率、线粒体膜电位和活性氧(ROS)水平,通过ATP检测试剂盒测定细胞内ATP水平,通过Western blot检测各组细胞蛋白中自噬标志蛋白LC3b、p62以及AMPK-mTOR自噬信号通路中p-AMPK/AMPK和p-mTOR/mTOR蛋白的表达。结果显示,与20%血清组相比,0%血清和5%血清细胞凋亡率极显著上调(P<0.01),10%血清组细胞凋亡率显著上调(P<0.05);各试验组线粒体膜电位均极显著下降(P<0.01),ROS水平和ATP水平均极显著上调(P<0.01);细胞中LC3b蛋白的相对表达量随血清浓度降低而升高,p62蛋白的相对表达量随血清浓度降低而降低;p-mTOR/mTOR比值随血清浓度降低而降低,p-AMPK/AMPK比值随血清浓度降低而升高。结果表明,通过长期减少SMSCs培养体系中血清浓度,可以促进细胞代谢加快和凋亡发生,并且通过AMPK/mTOR信号通路激发细胞发生自噬,上述变化的程度与血清减少的程度呈正相关。本试验为后续探索不同程度饥饿胁迫对动物肌肉发育影响的研究提供理论依据。

SphK1/S1P/S1PR2信号通路促进肌生成:运动改善骨骼肌健康的新视角

背景:近年来,运动改善骨骼肌的健康已成为学者们关注的一个重要研究内容,适宜的运动对骨骼肌具有积极的作用,其中在运动激活鞘氨醇激酶1(sphingosine kinase1,SphK1)/鞘氨醇-1-磷酸(sphingosine-1-phosphate,S1P)/鞘氨醇-1-磷酸受体2(sphingosine-1-phosphate receptor2,S1PR2)信号通路如何改善骨骼肌的健康,正受到科研人员的重视。目的:研究运动经SphK1/S1P/S1PR2信号通路如何改善骨骼肌的健康,探索治疗相关肌肉疾病的新方法,以改善人的骨骼肌健康。方法:检索Web of Science、PubMed、中国知网、万方和维普数据库从建库至今与文章主题相关的文献,以“signaling pathway,SphK1,S1P,S1PR2,skeletal muscle,satellite cell,myogenesis,exercise”为英文检索词,以“信号通路,SphK1,S1P,S1PR2,骨骼肌,卫星细胞,肌生成,运动”为中文检索词,最终纳入69篇文献进行分析。结果与结论:①SphK1/S1P/S1PR2信号通路是一个复杂的调控网络,通过SphK1催化产生的S1P,与S1PR2等受体的相互作用,触发下游信号转导过程,进而调控细胞、组织、器官和系统的多种生物学功能。②SphK1/S1P/S1PR2信号通路能调控卫星细胞增殖和成肌细胞分化,改善肌生成。③文章通过文献资料调研法分析了SphK1/S1P/S1PR2信号通路的生理基础以及运动对其影响的可能性。急性有氧运动可提高骨骼肌中SphK1的表达,人体和动物研究中已证实急性和长期运动均可提高骨骼肌中S1P水平,另外研究表明长期抗阻运动可提高S1PR2在骨骼肌中的表达,部分实验结果表明急性和长期运动对肌肉或者血液中S1P水平无显著影响,出现不同结果的原因可能是选择的研究对象、方式、强度及频率不同,而具体机制尚不明确。④研究认为,运动能够促进SphK1/S1P/S1PR2信号通路在骨骼肌中的表达,调控下游相关信号通路,并且针对这一信号通路的研究可能为骨骼肌疾病的治疗提供新的策略和方法,从而改善骨骼肌健康。⑤未来应深化对SphK1/S1P/S1PR2信号通路与骨骼肌健康关联的研究,进一步揭示其与卫星细胞、成肌细胞的调控关系及与上下游通路的相互作用,挖掘其临床应用价值,制定康复方案时考虑该通路变化,探索不同运动对该通路的影响机制,并将其作为潜在治疗靶点,结合人体肌肉模型提升研究深度和准确性。

CRISPR/Cas9系统介导的p53基因突变促进树鼩骨骼肌卫星细胞的增殖活性

目的:利用CRISPR/Cas9系统在树鼩骨骼肌卫星细胞中敲除p53基因并检测其增殖活性。方法:通过生物信息学方法对树鼩和15种哺乳动物的p53蛋白氨基酸序列进行同源性分析,根据树鼩基因组信息设计可编辑树鼩p53突变位点sgRNA,构建lenti CRISPR v2-sgp53基因编辑重组质粒,用293T细胞进行慢病毒包装,感染树鼩骨骼肌卫星细胞,嘌呤霉素筛选多克隆细胞并扩增;通过测序确定基因编辑效果。采用CCK8法检测细胞增殖活性,采用western blotting验证p53蛋白表达水平。结果:与人p53和小鼠p53氨基酸序列同源性(96.92%)相比,人p53和树鼩p53的氨基酸序列同源性(98.21%)更高。进化树分析显示,与小鼠相比,人与树鼩的亲缘关系更近。成功构建了3个靶向树鼩p53编辑的重组载体:lenti CRISPR v2-sgp53-g1、lenti CRISPR v2-sgp53-g2、lenti CRISPR v2-sgp53-g3,均成功在树鼩骨骼肌卫星细胞上实现p53基因编辑产生突变,导致p53功能丧失,p53的突变提高了树鼩骨骼肌卫星细胞的增殖能力。经药筛后的树鼩骨骼肌卫星细胞p53基因敲除成功,p53蛋白表达水平显著降低(P<0.01)。结论:本研究利用CRISPR/Cas9系统成功使树鼩骨骼肌卫星细胞p53基因突变、功能丧失,为后续树鼩p53基因敲除模型的建立提供了重要的分子生物学基础。

共培养条件下肌卫星细胞C2C12对软骨细胞ATDC5活性的影响

目的:观察共培养条件下肌卫星细胞C2C12对软骨细胞ATDC5活性的影响。方法:①将肌卫星细胞C2C12分为对照组和地塞米松组,对照组常规培养,地塞米松组采用地塞米松干预,通过CCK8法和BCA法测定地塞米松对C2C12细胞增殖和蛋白合成的影响,以划痕实验和Transwell小室观察地塞米松对C2C12细胞修复和迁移能力的影响。②采用Transwell小室将正常肌卫星细胞C2C12(共培养组)和经地塞米松预处理的肌卫星细胞C2C12(预处理组)分别与软骨细胞ATDC5共培养,对照组Transwell下层小室内接种ATDC5细胞、上层小室内为无细胞的常规培养基,采用CCK8法和二氯二氢荧光素-乙酰乙酸酯荧光探针检测ATDC5细胞增殖率和细胞内活性氧含量。结果:①C2C12细胞增殖率和蛋白含量测定结果。地塞米松组C2C12细胞增殖率和蛋白含量均低于对照组[(78.402±5.401)%,(100.000±3.096)%,t=8.498,P=0.000;(5080.367±296.657)μg·mL-1,(5775.577±150.476)μg·mL-1,t=3.620,P=0.022]。②C2C12细胞伤口修复率测定结果。地塞米松组C2C12细胞伤口修复率低于对照组[(53.173±1.800)%,(79.979±10.176)%,t=4.493,P=0.011]。③C2C12细胞迁移数量测定结果。培养24 h和48 h时,地塞米松组C2C12细胞迁移数量均少于对照组[24 h:(24.200±5.630)个,(57.000±2.449)个,t=11.945,P=0.000;48 h:(57.600±8.820)个,(91.000±4.743)个,t=7.457,P=0.000]。④ATDC5细胞增殖率测定结果。3组ATDC5细胞增殖率比较,差异有统计学意义[(100.000±1.663)%,(116.894±7.917)%,(89.130±2.980)%,F=23.700,P=0.001]。对照组和共培养组ATDC5细胞增殖率均高于预处理组(P=0.037,P=0.006),共培养组ATDC5细胞增殖率高于对照组(P=0.001)。⑤ATDC5细胞内活性氧含量测定结果。3组ATDC5细胞内活性氧含量比较,差异有统计学意义(20.148±5.636,13.959±4.110,40.691±3.146,F=30.096,P=0.001)。预处理组ATDC5细胞内活性氧含量高于对照组和共培养组(P=0.001,P=0.000),对照组和共培养组ATDC5细胞内活性氧含量的差异无统计学意义(P=0.137)。结论:地塞米松可抑制肌卫星细胞C2C12增殖,应用地塞米松干预肌卫星细胞C2C12可体外模拟肌肉萎缩条件下肌卫星细胞的生长状态。正常状态下,肌卫星细胞C2C12的代谢产物可促进软骨细胞ATDC5增殖;肌卫星细胞C2C12活力降低,可抑制软骨细胞ATDC5增殖,同时会增加软骨细胞ATDC5氧化性损伤。

The vascular endothelial growth factor expression and vascular regeneration in infarcted myocardium by skeletal muscle satellite cells

Background Myocardial infarction results in tissue necrosis, leading to cell loss and ultimately to cardiac failure. Implantation of skeletal muscle satellite cells into the scar area may compensate for the cell loss and provides a new strategy for infarct therapy. Vascular endothelial growth factor （VEGF） is a promising reagent for inducing myocardial angiogenesis. Skeletal myoblast transplantation has been shown to improve cardiac function in chronic heart failure models by regenerating muscle. We hypothesized that VEGF expression and vascular regeneration increased in infarcted myocardium by skeletal muscle satellite cells, which can promote vascular producing and improve survival environment in infarcted myocardium. Methods The skeletal muscle satellite cells were implanted into the infarcted myocardium in a model through ligated left anterior artery in Louis Inbrad Strain rat. Specimens were got for identifying the expression of VEGF and the density of vascular by immunochemical method at two weeks after implantation. Results The proliferation and differentiation of the skeletal muscle satellite cell was very well. The expression of VEGF was higher in the implanted group （146.83±2.49） than that in the control group （134.26±6.84） （P〈0.05）. The vascular density in the implanted group （13,00± 1.51） was also higher than that in the control （10.68 ± 1.79） （P〈0.05）. Conclusion The implanted satellite cell could excrete growth factor that would induce angiogenesis and improve cell survival environment in infarcted myocardium.

Influence of skeletal muscle satellite cells implanted into infarcted myocardium on remnant myocyte volumes

Objective To study the effects of skeletal muscle satellite cells implanted into infarcted myocardium on the volume of remnant myocytes Methods Thirty six adult mongrel canines were divided randomly into implantation group and control group In the implantation group, skeletal muscle satellite cells taken from the gluteus maximus muscles of the dogs were cultured, proliferated and labeled with 4', 6 diamidino 2 phenylindone (DAPI) in vitro In both groups, a model of acute myocardial infarction was established in every dog In the implantation group, each dog was injected with M199 solution containing autologous skeletal muscle satellite cells The dogs in the control group received M199 solution without skeletal muscle satellite cells The dogs of both groups were killed 2, 4 and 8 weeks after implantation (six dogs in a separate group each time) Both infarcted myocardium and normal myocytes distal from the infracted regions isolated were observed under optical and fluorescent microscope Their volumes were determined using a confocal microscopy image analysis system and analyzed using SAS A P <0 05 was considered significant Results A portion of the implanted cells differentiated into muscle fiber with striations and were connected with intercalated discs Cross sectional area and cell volume were increased in normal myocardium Hypertrophy of remnant myocytes in the infarcted site after skeletal muscle cell implantation was much more evident than in the control group. Cross sectional area, cell area and cell volume differed significantly from those of the control group ( P < 0.05) Hypertrophy of the cells occurred predominantly in terms of width and thickness, whereas cell length remained unchanged Conclusion Skeletal muscle satellite cells implanted into infarct myocardium, could induce the hypertrophy of remnant myocyte cells in the infarcted site and could also aid in the recovery of the contractile force of the infarcted myocardium