定向诱导小鼠ES细胞向心肌细胞的分化

为了提高体外诱导ES细胞向心肌细胞分化的效率 ,对以往的诱导方法加以改进 ,采用直接悬浮培养和 0 8%DMSO诱导 ,建立了简便、高效的定向诱导ES细胞向心肌细胞分化的体系 .诱导第 9d起可见自发性、有节律跳动的类胚体出现 ,第 14d达到高峰 ,约有 70 %的拟胚体产生跳动 .用RT PCR的方法在跳动的拟胚体中检测到心肌细胞特异性标志物的表达 ,采用免疫荧光染色的方法在蛋白水平检测到心肌特异的α辅肌动蛋白 (α actinin)的表达 ,并可见清晰肌小节 ,表明在改进的体外诱导条件下ES细胞可分化为成熟的心肌细胞 .     

骨形态发生蛋白9定向诱导多潜能干细胞成骨分化

为确认和评价骨形态发生蛋白9(bone morphogenetic protein 9, BMP9)定向诱导多潜能干细胞成骨分化的能力,以鼠间充质干细胞C3H10、小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)和骨髓基质细胞(bone marrow stromal cell, BMSC)三种多潜能干细胞为目标细胞,用重组腺病毒的方法将BMP9导入细胞,通过荧光素酶报告基因实验、碱性磷酸酶(alkaline phosphatase,ALP)定量测定、钙盐沉积实验、real time PCR、动物实验和组织化学染色等方法,观察BMP9对于多潜能干细胞成骨分化的定向诱导作用．结果提示,BMP9能诱导C3H10、MEFs和BMSC细胞ALP的表达,且具有剂量依赖性．BMP9在体外能够促进C3H10细胞和MEFs细胞的钙盐沉积．经BMP9刺激后,C3H10细胞成骨相关基因ALP、Runx2、骨桥素(osteopontin, OPN)和骨钙素(osteocalcin, OC)的mRNA水平均增加．荧光素酶报告基因实验证实,BMP9可以活化Smad和成骨关键基因Runx2．动物实验和组织化学染色检查显示,BMP9可以诱导C3H10细胞在裸鼠皮下异位成骨,因此,BMP9具有定向诱导多潜能干细胞成骨分化的能力．     

DJ‐1 Regulates Differentiation of Human Mesenchymal Stem Cells into Smooth Muscle‐like Cells in Response to Sphingosylphosphorylcholine

Although multiple factors contribute to the differentiation of human mesenchymal stem cells (hMSCs) into various types of cells, the differentiation of hMSCs into smooth muscle cells (SMCs), one of central events in vascular remodeling, remains to be clarified. ROS participate in the differentiation of hMSCs into several cell types and were regulated by redox‐sensitive molecules including a multifunctional protein DJ‐1. Here, we investigated the correlation between altered proteins, especially those related to ROS, and SMC differentiation in sphingosylphosphorylcholine (SPC)‐stimulated hMSCs. Treatment with SPC resulted in an increased expression of SMC markers, namely α‐smooth muscle actin (SMA) and calponin, and an increased production of ROS in hMSCs. A proteomic analysis of SPC‐stimulated hMSCs revealed a distinctive alteration of the ratio between the oxidized and reduced forms of DJ‐1 in hMSCs in response to SPC. The increased abundance of oxidized DJ‐1 in SPC‐stimulated hMSCs was validated by immunoblot analysis. The SPC‐induced increase in the expression of α‐SMA was stronger in DJ‐1‐knockdown hMSCs than in control cells. Moreover, the expression of α‐SMA, and the calponin and generation of ROS in response to SPC were weaker in normal hMSCs than in DJ‐1‐overexpressing hMSCs. Exogenous H₂O₂ mimicked the responses induced by SPC treatment. These results indicate that the ROS‐related DJ‐1 pathway regulates the differentiation of hMSCs into SMCs in response to SPC.     

E1A 激活基因阻遏子促进人血管平滑肌细胞分化和迁移

为探讨 E1A 激活基因阻遏子 (cellular repressor of E1A-stimulated genes , CREG) 蛋白在人血管平滑肌细胞株 HITASY 分化和迁移中的调控作用,构建了重组逆转录病毒载体 pLNCX₂( ＋ )/CREG 和 pLXSN( － )/CREG. 以带绿色荧光蛋白 (green fluorescent protein , GFP) 的空载体 pLNCX( ＋ )/GFP 和正常 HITASY 为对照,用磷酸钙共沉淀法将重组逆转录病毒载体转染 293 细胞,包装出完整的逆转录病毒后,感染 HITASY. 经 G418 筛选,获得稳定感染的细胞克隆 . 应用免疫荧光染色、蛋白质印迹等方法检测 CREG 和平滑肌分化标志蛋白α- 肌动蛋白 (SMα -actin) 表达,同时通过刮伤实验、慢速显微摄像技术检测细胞迁移能力以及用明胶酶谱方法分析细胞基质金属蛋白酶 (matrix metalloproteinase , MMPs) 的活性 . 结果表明： pLNCX₂( ＋ )/CREG 稳定感染的 HITASY 中 CREG 和 SMα-actin 蛋白表达上调, MMP-2 和 MMP-9 活性升高,细胞迁移速度加快; pLXSN( － )/CREG 稳定感染的 HITASY 中 CREG 和 SMα -actin 蛋白表达下调, MMP-2 和 MMP-9 活性降低,细胞迁移速度减慢 . 上述研究提示 CREG 在诱导血管平滑肌细胞分化的同时促进细胞迁移 .     

Evening Primrose Extracts Inhibit PDGF-BB-Induced Vascular Smooth Muscle Cell Proliferation and Migration by Regulating Cell-Cycle-Related Proteins

The proliferation and migration of vascular smooth muscle cells (VSMCs) are important factors in the occurrence of cardiovascular diseases, such as blood flow abnormalities, stroke and atherosclerosis. Evening primrose, known as Oenothera biennis, is a plant native to Korea that exerts physiological activities, such as antioxidant effects, the inhibition of lipid accumulation and the prevention of muscle atrophy. However, the function of evening primrose stem (EVP) in the regulation of VSMC proliferation and migration and the underlying mechanisms have not been identified. In this study, the effect of EVP on the platelet-derived growth factor (PDGF)-induced proliferation and migration of VSMCs was investigated. The results show that PDGF-BB-induced proliferation of VSMCs was inhibited by EVP at concentrations of 25, 50 or 100 μg/mL in a concentration-dependent manner, and a migration assay showed that EVP inhibited cell migration. Cell cycle analysis was performed to confirm the mechanism by which cell proliferation and migration was inhibited. The results indicate that proteins involved in the cell cycle, such as cyclin, CDK and phosphorylated Rb, were downregulated by EVP at concentrations of 100 μg/mL, thereby increasing the proportion of cells in the G0/G1 phase and inhibiting cell cycle progression. In the PDGF receptor (PDGFR) signaling pathway, phosphorylation of the PDGFR was inhibited by EVP at concentrations of 100 μg/mL, and PLCγ phosphorylation was also decreased. The PDGF-BB-induced effect of EVP on the proliferation of VSMCs involved the inhibition of Akt phosphorylation and the reduction in the phosphorylation of MAPK proteins such as ERK, P38 and JNK. In conclusion, the results demonstrate that EVP inhibited PDGF-BB-induced VSMC proliferation and migration by regulating cell-cycle-related proteins.     

Smooth Muscle Cell Reprogramming in Aortic Aneurysms

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Single-Cell Analysis of the Muscle Stem Cell Hierarchy Identifies Heterotypic Communication Signals Involved in Skeletal Muscle Regeneration

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A Novel Regulatory Mechanism for Differentiation of Mesenchymal Stem Cell: Redox State of DJ‐1 Matters

Reactive oxygen species (ROS) are multifunctional gas transmitters with diverse biological actions (adverse vs beneficial) dependent on their level. The differentiation of vascular stem cells into smooth muscle cells (SMCs) might be involved in the pathogenesis of cardiovascular disorders including hypertension and atherosclerosis. Therefore, controlling the differentiation of vascular stem cells is a potential strategy for the treatment of vascular diseases. Nonetheless, it remains to be revealed whether ROS could mediate the differentiation of mesenchymal stem cells (MSCs) into SMCs. In addition, there are no redox (reduction–oxidation)‐sensitive molecules identified, which are responsible for the ROS‐induced differentiation of MSCs. In article number 1700208, Baek et al. [Proteomics 2017, 17, Issue 21] found that ROS mediate the differentiation of MSCs into SMCs through the modification of redox states of a multifunctional ROS‐responsive protein, DJ‐1, revealing a novel regulatory mechanism for differentiation of MSCs into SMCs and shedding light into the future development of stem‐cell‐targeted pharmacotherapy.     

胚胎干细胞分化为血管内皮细胞的分子调控机制

胚胎干细胞在不同的诱导条件下具有多向分化的潜能,多种胞内外信号途径参与其分化过程的调控。现就胚胎干细胞向血管内皮细胞分化的诱导条件及分子机制做一综述,并阐明不同阶段的内皮前体细胞所表达的不同分子标志,同时提出胚胎干细胞在再生医学中的应用前景。     

胚胎干细胞分化为肝细胞的研究进展

目前 ,细胞移植作为终末期肝病的辅助治疗方法 ,移植的细胞必须满足在受体肝脏中存活、增殖并可分化为成熟肝细胞两个重要条件 ,但目前应用的肝细胞来源有限 ,其功能随着培养时间的延长而逐渐下降等问题限制了这一治疗策略的广泛开展。作为具有发育全能性和无限增殖能力的细胞 ,胚胎干细胞向肝细胞的分化研究近年来引起了广泛的关注 ,并取得了较大的进展 ,寻找合适、高效的分化诱导方法是目前研究的热点之一。胚胎干细胞向肝细胞的分化研究既可以为临床细胞替代治疗提供合适的细胞来源 ,也可以在药物评估和肝脏发育分化基础研究方面起到重要的作用。通过概括肝脏和拟胚体分化发育的分子机制 ,对体外胚胎干细胞向肝细胞分化的几种诱导体系作了介绍 ,并对分化肝细胞的应用前景和存在的问题进行了讨论。     

脂肪间充质干细胞体外分化成心肌样细胞

探讨大鼠脂肪间充质干细胞（adipose-derived mesenchymal stem cells,AMSC）体外分化成心肌样细胞的潜能,为自体干细胞移植治疗心肌梗死提供理论基础.采用消化法分离大鼠AMSC,培养于RPMI1640生长培养基中,倒置相差显微镜观察细胞形态发现,随着培养时间的延长,细胞形态趋向于心肌细胞,SQ RT-PCR检测表达心肌特异性基因：β-肌球蛋白重链（β-MHC）、α-肌球蛋白重链（α-MHC）、心房利钠肽（ANP）、心肌肌钙蛋白（cTnT）、心肌肌动蛋白、肌肉增强因子和GATA-4;免疫细胞化学和免疫荧光染色检测表达心肌细胞特异性蛋白：结蛋白、横纹肌辅肌动蛋白、心肌肌动蛋白和间隙连接蛋白45(connexin 45);Western印迹检测表达心肌特异性蛋白Nkx2.5. 实验表明,大鼠AMSC在体外培养条件下能分化成心肌样细胞,在组织工程学及干细胞移植领域有着良好的应用前景.     

Lysosomal Signaling Licenses Embryonic Stem Cell Differentiation via Inactivation of Tfe3

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microRNA499 慢病毒载体转染诱导大鼠骨髓间充质干细胞 向心肌样细胞分化*

目的:探讨microRNA 499(miR-499)慢病毒转染对诱导大鼠骨髓来源间充质干细胞(BM-MSCs)向心肌样细胞分化的作用。方法:取第四代Wistar大鼠骨髓来源间充质干细胞进行流式细胞检测,鉴定干细胞表面特异标记物。使用符合干细胞鉴定标准的细胞批次用于后续实验。实验设置miR499慢病毒转染、慢病毒空白转染2个处理组,分别于处理后即日、1d,3d,5d,7d收集细胞进行下列实验:实时荧光定量PCR检测心肌重要转录因子GATA4、NKx2.5和MEF2C的mRNA表达,western-blot检测心肌特异蛋白I(cTnI)的表达。结果:培养第四代Wistar大鼠骨髓来源间充质干细胞表达干细胞表面特异标记物,可用于实验。大鼠骨髓来源间充质干细胞microRNA 499慢病毒载体转染后microRNA 499表达明显升高,且转染后1d,3d,5d,7d,GATA4、NKx2.5和MEF2C的mRNA表达逐渐增强。慢病毒空白转染组未见明显变化。western-blot检测自第3天开始可见cTnI阳性表达条带,慢病毒空白转染组未检测到明显阳性表达条带。结论:microRNA 499可诱导大鼠骨髓来源间充质干细胞向心肌样细胞分化。     

体外诱导大鼠脂肪间充质干细胞向肝细胞方向分化的研究

目的:探讨大鼠脂肪间充质干细胞(ADMSCs)是否可以在体外被诱导向肝细胞方向分化。方法:从大鼠脂肪组织中分离出干细胞,行体外扩增、传代;用免疫荧光染色法检测其表面标志,用成纤维细胞生长因子(FGF-4)和肝细胞生长因子(HGF)诱导其向肝细胞分化;倒置显微镜观察细胞形态变化,诱导后14天和21天分别用免疫荧光检测法检测肝细胞标志物白蛋白的表达,每次换液时留取培养上清用尿素氮测试盒检测尿素氮含量。结果:免疫荧光检测显示从脂肪组织所获取的细胞表面标志CD29阳性,而CD31和CD145均为阴性;诱导14天后可逐渐观察到肝细胞样形态改变;免疫荧光检测到白蛋白表达;尿素氮检测显示诱导组尿素氮含量随时间延长而逐渐升高。结论:从大鼠脂肪组织中获取的脂肪间充质干细胞能在体外被诱导分化成形态、表型及功能与肝细胞相似的细胞。     

PAX3 Confers Functional Heterogeneity in Skeletal Muscle Stem Cell Responses to Environmental Stress

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骨髓间充质干细胞成肌和成脂分化的调控

骨髓间充质干细胞(mesenchymal stem cells,MSCs)是来源于骨髓基质的一类具有高度自我更新能力和多向分化潜能的成体干细胞.因其具有容易获取、体外扩增方便迅速、移植排斥反应较弱等优点而成为临床应用的理想细胞模型.骨髓间充质干细胞向成肌和成脂的分化对动物机体内肌肉和脂肪的组成具有直接影响,因而与肉品质及人类健康息息相关.本文综述了骨髓间充质干细胞定向分化为骨骼肌细胞和脂肪细胞的过程及其调控机制,并重点分析了关键调控因子PRDM16(PR domain-containing16)和骨形态发生蛋白(bone morphogenetic proteins,BMPs)在骨髓间充质干细胞成肌和成脂分化中的作用.     

黄芩苷通过降低活性氧生成抑制血管平滑肌细胞伪足形成和迁移

已知黄芩苷（baicalin）通过削弱肌动蛋白相关蛋白（actin-related protein, Arp）2/3复合物的活性抑制血管平滑肌细胞（vascular smooth muscle cell, VSMC）伪足形成和迁移,然而,其抑制该信号途径的机制尚不明确。本研究证明,黄芩苷通过抑制VSMC活性氧（reactive oxygen species,ROS）生成降低Arp2/3活性,发挥阻止细胞伪足形成和迁移的功能。分别利用TRITC 鬼笔环肽和ROS荧光探针标记VSMCs,结果显示,黄芩苷能显著抑制血小板源性生长因子（platelet derived growth factor, PDGF）-BB诱导的VSMC伪足形成和迁移,伴有ROS生成减少。用超氧物歧化酶（superoxide dismutase, SOD）清除胞内过氧化物后,PDGF-BB引发的VSMC伪足形成被逆转,且该过程与降低皮层肌动蛋白微丝（F-actin）成核蛋白Arp2/3活性有关。免疫沉淀分析结果进一步表明,黄芩苷降低p47phox磷酸化水平,与ROS生成减少相一致。体内的实验也表明,黄芩苷（70 mg/kg/d）能有效抑制球囊损伤诱导的大鼠颈总动脉ROS生成。以上结果表明,黄芩苷通过抑制NADPH氧化酶介导的ROS生成,降低细胞皮质区F-actin成核活性,阻止细胞伪足形成、迁移,进而发挥血管保护作用。