MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4

Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.

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.

Factors influencing myogenic differentiation of adipose-derived stem cells and their application in muscle regeneration

Skeletal muscle regeneration mainly depends on muscle satellite cells;however,these cells are not sufficient for supporting repair and regeneration in volumetric muscle loss(VML),Duchenne muscular dystrophy,and other muscle injuries or muscle diseases.As such,much work has been conducted in recent years to search for myogenic stem cells.Adipose-derived stem cells(ADSCs)have a wide range of sources,rapid growth,and multi-directional differentiation potential,and have become vital candidates for muscle regeneration.Multiple factors influence the myogenic differentiation capacity of ADSCs.This paper reviews the regulatory aspects and possible factors that have been identified in recent years to affect myogenic differentiation of ADSCs.Based on these factors,gene editing,and perfusion concepts,a method was proposed to achieve maximal differentiation efficiency of ADSCs.This study focused on the application of ADSCs in muscle regeneration and disease.Based on the importance of myogenic differentiation of ADSCs for the repair and regeneration of muscle damage,this study provides a basis for future research surrounding the efficient induction of myogenic differentiation of ADSCs in vitro.

Negative effects of Notch1 on the differentiation of muscle-derived stem cells into neuronal-like cells

We cultured rat muscle-derived stem cells in medium containing nerve growth factor and basic fi-broblast growth factor to induce neuronal-like cell differentiation.Immunocytochemical staining and reverse transcription-PCR showed that the differentiated muscle-derived stem cells exhibited processes similar to those of neuronal-like cells and neuron-specific enolase expression,but Notch1 mRNA and protein expression was decreased.Down-regulation of Notch1 expression may facilitate neuronal-like cell differentiation from muscle-derived stem cells.

TGF-β1-induced LPP Expression Dependant on Rho Kinase during Differentiation and Migration of Bone Marrow-derived Smooth Muscle Progenitor Cells

Lipoma preferred partner(LPP) has been identified as a protein which is highly selective for smooth muscle progenitor cells(SMPCs) and regulates differentiation and migration of SMPCs,but mechanisms of LPP expression are not elucidated clearly.The aim of the present study was to discuss the mechanisms by which LPP expression is regulated in the differentiation and migration of SMPCs induced by TGF-β1.It was found that TGF-β1 could significantly increase the expression of LPP,smooth muscle α-actin,smooth muscle myosin heavy chain(SM-MHC),and smoothelin in SMPCs.Moreover,inactivation of Rho kinase(ROK) with ROK inhibitors significantly inhibited LPP mRNA expression in TGF-β1-treated SMPCs and mouse aortic smooth muscle cells(MAoSMCs).At the same time,LPP silencing with short interfering RNA significantly decreased SMPCs migration.In conclusion,LPP appears to be a ROK-dependant SMPCs differentiation marker that plays a role in regulating SMPCs migration.

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.

Skeletal Muscle-derived Stem Cells Exhibit Cardiocyte Competences

Adult stem cells from skeletal muscle cells were induced to differentiate into cardiocytes to see if stem cells from another different but histologically-comparable tissues can differentiate to the target cells. Skeletal muscles-derived stem cells （MDSCs） were isolated from adult skeleton muscle tissues by differential adhesion, and immunocytochemically identified by using Sca-1. In order to induce the proliferation but not differentiation of MDSCs, the cells were cultured in Dulbecco’s modified Eagle’s medium/F12 （DMEM/F12） supplemented with 1：50 B27, 20 ng/mL basic fibroblast growth factor （bFGF）, 20 ng/mL epidermal growth factor （EGF） in a suspension for 6 days. Then these stem cells were treated with 5 μmol/L 5-azacytidine for 24 h in an adherence culture. The characteristics of induced cells were examined by immunocytochemistry, quantitative real time RT-PCR and morphological observation of cell phenotype. Our results showed that the appearance of some cells gradually changed from spindle-shape into polygonal or short-column-shape. Some of these post-treated cells could contract spontaneously and rhythmically. The expression of GATA-4 and cTnT was increased 1 and 2 week（s） after the treatment. And about 16.6% of post-treated cells were cTnT-positive. Therefore, we are led to conclude that skeletal muscle-derived stem cells could differentiate into cardiocyte-like cells, which exhibited some characteristics of cardiocytes.

Polyclonal antibody production and expression of CREG protein in human vascular smooth muscle cells

Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.

Vascular smooth muscle cell differentiation－2010

Vascular smooth muscle cells have attracted considerable interest as a model for a flexible program of gene expression.This cell type arises throughout the embryo body plan via poorly understood signaling cascades that direct the expression of transcription factors and microRNAs which,in turn,orchestrate the activation of contractile genes collectively defining this cell lineage.The discovery of myocardin and its close association with serum response factor has represented a major break-through for the molecular understanding of vascular smooth muscle cell differentiation.Retinoids have been shown to improve the outcome of vessel wall remodeling following injury and have provided further insights into the molecular circuitry that defines the vascular smooth muscle cell phenotype.This review summarizes the progress to date in each of these areas of vascular smooth muscle cell biology.

Mechanisms simultaneously regulate smooth muscle proliferation and differentiation

Vascular smooth muscle cell （VSMC） differentiation and proliferation are two important physiological proc- esses during vascular development. The phenotypic alteration from differentiated to proliferative VSMC contrib- utes to the development of several major cardiovascular diseases including atherosclerosis, hypertension, resteno- sis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. Since the VSMC phenotype in these pathological conditions resembles that of developing VSMC during embryonic development, understanding of the molecular mechanisms that control VSMC differentiation will provide fundamental insights into the pathological processes of these cardiovascular diseases. Although VSMC differentiation is usually ac- companied by an irreversible cell cycle exit, VSMC proliferation and differentiation occur concurrently during embryonic development. The molecular mechanisms simultaneously regulating these two processes, however, remain largely unknown. Our recent study demonstrates that cell division cycle 7, a key regulator of cell cycle, promotes both VSMC differentiation and proliferation through different mechanisms during the initial phase of VSMC differentiation. Conversely, Kriappel-like factor 4 appears to be a repressor for both VSMC differentia- tion and proliferation. This review attempts to highlight the novel role of cell division cycle 7 in TGF-β-induced VSMC differentiation and proliferation. The role of K141ppel-like factor 4 in suppressing these two processes will also be discussed.

Transforming growth factor-β and smooth muscle differentiation

Transforming growth factor(TGF)-β family members are multifunctional cytokines regulating diverse cel- lular functions such as growth,adhesion,migration, apoptosis,and differentiation.TGF-βs elicit their effects via specific typeⅠand typeⅡserine/threonine kinase receptors and intracellular Smad transcription factors. Knockout mouse models for the different components of the TGF-β signaling pathway have revealed their critical roles in smooth muscle cell(SMC)differentia- tion.Genetic studies in humans have linked mutations in these signaling components to specific cardiovascular disorders such as aorta aneurysm and congenital heart diseases due to SMC defects.In this review,the current understanding of TGF-β function in SMC differentiation is highlighted,and the role of TGF-βsignaling in SMC- related diseases is discussed.

Effects of DHRS3 in C2C12 Myoblast Differentiation and Mouse Skeletal Muscle Injury

Myoblast differentiation is an essential process during skeletal muscle development.C2C12 myoblast is a commonly used experimental model to study muscle cell differentiation in vitro.Dehydrogenase/reductase(SDR family)member 3(DHRS3)is a highly conserved member in short-chain alcohol dehydrogenase/reductase superfamily and has been shown to be involved in the metabolism of retinol.Previous experimental results showed that the expression of DHRS3 increased significantly during the differentiation of myoblasts differentiation.However,the effect of DHRS3 on mouse muscle cell differentiation was unclear.The objective of current study was to determine if DHRS3 affected muscle cell differentiation,and if DHRS3 was involved in muscle regeneration.Protein expression was determined by western blot and immunofluorescence analysis.The activation and inhibition of DHRS3 increased and decreased C2C12 myoblast differentiation respectively,which indicated that DHRS3 could affect C2C12 myoblast differentiation.DHRS3 expression was significantly changed during muscle regeneration,with the regeneration of muscle injury,the expression of DHRS3 tended to increase first and then decrease.It suggested that DHRS3 might be involved in muscle regeneration.In summary,this study confirmed the involvement of DHRS3 in C2C12 myoblast differentiation and mouse skeletal muscle regeneration and provided a theoretical basis for further elucidating the molecular mechanism of muscle development.

GSK3β inhibitor promotes myelination and mitigates muscle atrophy after peripheral nerve injury

Delay of axon regeneration after peripheral nerve injury usually leads to progressive muscle atrophy and poor functional recovery. The Wnt/β-catenin signaling pathway is considered to be one of the main molecular mechanisms that lead to skeletal muscle atrophy in the elderly. We hold the hypothesis that the innervation of target muscle can be promoted by accelerating axon regeneration and decelerating muscle cell degeneration so as to improve functional recovery of skeletal muscle following peripheral nerve injury. This process may be associated with the Wnt/β-catenin signaling pathway. Our study designed in vitro cell models to simulate myelin regeneration and muscle atrophy. We investigated the effects of SB216763, a glycogen synthase kinase 3 beta inhibitor, on the two major murine cell lines RSC96 and C2C12 derived from Schwann cells and muscle satellite cells. The results showed that SB216763 stimulated the Schwann cell migra- tion and myotube contraction. Quantitative polymerase chain reaction results demonstrated that myelin related genes, myelin associated glycoprotein and cyclin-D1, muscle related gene myogenin and endplate-associated gene nicotinic acetylcholine receptors levels were stimulated by SB216763. Immunocytochemical staining revealed that the expressions of ^-catenin in the RSC96 and C2C12 cytosolic and nuclear compartments were increased in the SB216763-treated cells. These findings confirm that the glycogen synthase kinase 3 beta in- hibitor, SB216763, promoted the myelination and myotube differentiation through the Wnt/β-catenin signaling pathway and contributed to nerve remyelination and reduced denervated muscle atrophy after peripheral nerve injury.

New insights into the epigenetic control of satellite cells

Epigenetics finely tunes gene expression at a functionallevel without modifying the DNA sequence, thereby contributing to the complexity of genomic regulation. Satellite cells(SCs) are adult muscle stem cells that are important for skeletal post-natal muscle growth, homeostasis and repair. The understanding of the epigenome of SCs at different stages and of the multiple layers of the post-transcriptional regulation of gene expression is constantly expanding. Dynamic interactions between different epigenetic mechanisms regulate the appropriate timing of muscle-specific gene expression and influence the lineage fate of SCs. In this review, we report and discuss the recent literature about the epigenetic control of SCs during the myogenic process from activation to proliferation and from their commitment to a muscle cell fate to their differentiation and fusion to myotubes. We describe how the coordinated activities of the histone methyltransferase families Polycomb group(Pc G), which represses the expression of developmentally regulated genes, and Trithorax group, which antagonizes the repressive activity of the Pc G, regulate myogenesis by restricting gene expression in a time-dependent manner during each step of the process. We discuss how histone acetylation and deacetylation occurs in specific loci throughout SC differentiation to enable the time-dependent transcription of specific genes. Moreover, we describe the multiple roles of micro RNA, an additional epigenetic mechanism, in regulating gene expression in SCs, by repressing or enhancing gene transcription or translation during each step of myogenesis. The importance of these epigenetic pathways in modulating SC activation and differentiation renders them as promising targets for disease interventions. Understanding the most recent findings regarding the epigenetic mechanisms that regulate SC behavior is useful from the perspective of pharmacological manipulation for improving muscle regeneration and for promoting muscle homeostasis under pathological conditions.

The Cytomegalovirus Enhancer Induces an Immediate Response to the Myosin Light Chain 2v Promoter during P19CL6 Cell Differentiation

The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhancer and a 250 bp MLC-2v promoter in front of the GFP gene to further evaluate the role of the CMV enhancer. This plasmid (pCBVenh/MLC-2vpro/EGFP) was stably introduced into P19CL6 cells, and the transfectant differentiated into cardiomyocytes with DMSO. Upon DMSO addition, GFP was immediately transcribed (within 2 days) and the amount of the transcript increased with cultivation. Concomitantly, GFP fluorescence was detected in the cells under a microscope. However, native MLC-2v was transcribed later on day 4. This expression time course is different from that of GFP. Clearly the CMV enhancer responded immediately to DMSO. Since GATA DNA-binding proteins play crucial roles in the initiation of cardiomyocyte differentiation, such a response could be ascribed to the presence of multiple GATA motifs in the enhancer sequence but not in the native MLC-2v promoter. Thus the CMV enhancer may be not only useful for gene therapy and monitoring cell differentiation but also the study of the role of GATA transcription factors expressed in P19CL6 cells.

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。

高磷诱导下肢血管平滑肌细胞成骨分化关键差异表达基因筛选及验证

目的:采用mRNA高通量测序技术筛选高磷诱导下肢血管平滑肌细胞(VSMCs)钙化差异表达基因(DEGs),分析VSMCs钙化的关键基因及信号通路。方法:将人VSMCs分为对照组和模型组,模型组细胞中加入高磷培养基,对照组细胞采用含10%胎牛血清的DMEM培养基于相同条件下培养。调整2组稳定转染VSMCs的状态,培养12 d,倒置显微镜下观察细胞形态表现并拍照。采用Hisat2软件筛选DEGs,采用Stringtie软件从生物过程(BP)、分子功能(MF)和细胞成分(CC)3个方面进行基因本体论(GO)功能和京都基因与基因组百科全书(KEGG)信号通路富集分析。Von Kossa染色法观察各组细胞钙化情况,实时荧光定量PCR(RT-qPCR)法检测2组细胞中碱性磷酸酶(ALP)、骨形态发生蛋白2(BMP2)、α-平滑肌肌动蛋白(α-SMA)、肿瘤蛋白53(Tp53)、谷胱甘肽过氧化物酶4(GPX4)、铁蛋白轻链1(Ftl1)和糖基磷脂酰肌醇特异性磷脂酶D1(GPLD1)mRNA表达水平。结果:与对照组比较,模型组共2 524个DEGs,其中1 368个DEGs表达上调,1 156个DEGs表达下调;2组细胞DEGs聚类分离明显。GO功能和KEGG信号通路富集分析表达上调的DEGs主要参与微管细胞骨架组织的调节、细胞极性、蛋白质定位和细胞周期调控等BP,构建细胞膜部分、微管组织、染色体和着丝粒区等CC,发挥与磷脂酰肌醇磷酸盐、 Rho鸟苷酸三磷酸酶(GTPase)蛋白结合、参与跨膜转运和调节蛋白激酶活性等MF;表达下调的DEGs主要参与细胞质翻译、蛋白质膜定位、mRNA代谢和蛋白质内质网定位等BP,构建核糖体亚单位、细胞膜和自噬体等CC,发挥与单链DNA、核糖核蛋白复合物、生长因子结合、调节蛋白激酶活性和催化作用等MF。差异表达上调的基因富集7条信号通路,其中最为显著的是糖基磷脂酰肌醇(GPI)锚定的生物合成;差异表达下调的基因富集18条信号通路,其中最为显著的是铁死亡。RT-qPCR法,与对照组比较,模型组细胞中GPX4、Ftl1和Tp53 mRNA表达水平明显降低(P<0.01),GPLD1 mRNA表达水平明显升高(P<0.01);与对照组比较,模型组细胞中α-SMA mRNA表达水平明显降低(P<0.01),ALP和BMP2 mRNA表达水平明显升高(P<0.01)。结论:钙化的VSMCs与正常细胞存在DEGs,铁死亡和GPI锚定的生物合成信号途径是高磷诱导下肢VSMCs钙化的关键信号通路,主要由GPX4、Ftl1、Tp53和GPLD1共同介导完成。

叶酸通过JNK/p38 MAPK信号通路调节小鼠C2C12成肌细胞分化

目的:观察叶酸(folic acid,FA)对小鼠C2C12成肌细胞增殖和分化的影响并探讨其作用机制。方法:(1)在小鼠C2C12成肌细胞增殖阶段,采用不同浓度(0、2.5、5、10和20μmol/L)的FA处理C2C12细胞,显微镜下观察各组细胞的状态,MTT法检测细胞活力,EdU法检测细胞增殖情况。(2)在小鼠C2C12成肌细胞分化阶段,将细胞分为对照(control,Ctrl)组(0μmol/L FA)和FA组(10μmol/L FA),于分化的第2天和第4天,采用免疫荧光染色和Western blot检测成肌细胞分化相关蛋白成肌细胞决定蛋白1(myoblast determination protein 1,MyoD)、成肌蛋白(myogenin,MyoG)和肌球蛋白重链(myosin heavy chain,MyHC)的表达水平,并统计各组细胞肌管形成情况。(3)在小鼠C2C12成肌细胞分化第4天时,加入FA处理0、1、3和6 h,采用Western blot检测各时点c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、磷酸化JNK(phosphorylated JNK,p-JNK)、p38丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和磷酸化p38 MAPK(phosphorylated p38 MAPK,p-p38 MAPK)蛋白水平。(4)将分化4 d的小鼠C2C12成肌细胞分为Ctrl组、FA组、SP600125(JNK特异性抑制剂)组、SB2035805(p38 MAPK特异性抑制剂)组、FA+SP600125组和FA+SB203580组。C2C12成肌细胞先接受10μmol/L的SP600125或SB203580处理1 h,再经10μmol/L的FA处理24 h,FA组经10μmol/L FA处理24 h,Ctrl组不作处理。采用Western blot检测p-JNK、JNK、p-p38 MAPK、p38 MAPK和MyHC蛋白水平。结果:(1)与0μmol/L FA组相比,其余各浓度组的细胞数量明显增多,细胞活力显著提高(P<0.05),EdU阳性细胞率均显著增加(P<0.05)。(2)与Ctrl组相比,FA组MyoD、MyoG和MyHC的表达水平均显著提高(P<0.05),肌管融合指数显著增加(P<0.05或P<0.01)。(3)与0 h组相比,FA处理1、3和6 h后p-JNK/JNK和p-p38 MAPK/p38 MAPK的比值均显著升高(P<0.05或P<0.01),且随着处理时间的延长,p-JNK/JNK和p-p38 MAPK/p38 MAPK的比值呈现逐渐升高的趋势。(4)与Ctrl组相比,FA组p-JNK、p-p38 MAPK和MyHC蛋白水平显著升高(P<0.01);与FA组相比,FA+SP600125组p-JNK和MyHC蛋白水平显著降低(P<0.05),FA+SB203580组p-p38 MAPK和MyHC蛋白水平显著降低(P<0.05或P<0.01)。结论:FA可以通过激活JNK/p38 MAPK信号通路促进小鼠C2C12成肌细胞分化。

面肩肱肌营养不良症患者外周血单个核细胞来源的诱导多能干细胞的构建及骨骼肌分化

目的建立并鉴定面肩肱肌营养不良症(FSHD)患者外周血单个核细胞(PBMC)来源的诱导多能干细胞(iPSCs),初步探讨其骨骼肌分化能力,评估该细胞模型应用于疾病机制研究的可行性。方法收集1例FSHD患者的PBMC,用含4个重编程转录因子(OCT4、SOX2、KLF4和c-MYC)的仙台病毒感染PBMC并获得FSHD患者来源的iPSCs,继续诱导其骨骼肌分化。通过基因组光学图谱技术、核型、免疫荧光染色、实时荧光定量PCR等分析iPSCs和骨骼肌细胞特性。结果成功获得FSHD患者来源的iPSCs,其可表达多能干性标记。FSHD-iPSC核型及D4Z4拷贝数结果与患者的临床背景一致,并在体外可定向诱导分化为骨骼肌细胞,该细胞同时表达DUX4致病基因以及调节基因。结论FSHD患者来源的PBMC可重编程为iPSCs,FSHD-iPSC可分化为疾病相关的肌源组细胞及肌管细胞,为FSHD发病机制的体外研究提供了良好的细胞模型,并为寻找该病的有效治疗手段提供了工具。