Osteoblastic differentiation of mesenchymal stem cells by mumie extract

Mumie, a plant humus matter from rocks, is known as anabolic and a stimulator of bone regeneration in the Russian and Indian systems of health and medicine. The water‐soluble fraction of mumie from Uzbekistan was characterized using ¹H NMR and infrared spectroscopic methods. The mumie extract has been investigated for its effect on osteoblastic differentiation in cell culture assays of human and murine mesenchymal stem cells. The calcium deposition and expression of alkaline phosphatase, osteocalcin, core binding factor 1 (Cbfa1), and ERK have been studied. During the 14‐day assay period, human bone marrow mesenchymal stem cells (hMSCs) and human fetal osteoblasts cultured with mumie (3–5 µg/ml) underwent a dramatic change in cellular morphology, which was accompanied by a significant increase in alkaline phosphatase activity, calcium deposition, and osteocalcin expression. The expression of core binding factor 1 and ERK were enhanced in hMSCs and murine pluripotent mesenchymal precursor cell line C2C12. Dose‐dependent decrease in TRAP‐positive multinucleated cell formation from macrophage‐like cells RAW 264.7 was observed with increasing concentration of mumie in the presence of RANKL (40 ng/ml) and PD98059 (10 µM), a specific inhibitor of ERK activity. The data suggest that mumie is a potent stimulator of osteoblastic differentiation of mesenchymal stem cells and inhibitor of osteoclastogenesis, hence it maybe of clinical benefit in the treatment for osteoporosis in human. Drug Dev. Res. 57: 122–133, 2002. © 2002 Wiley‐Liss, Inc.     

兔骨髓间充质干细胞诱导分化为成骨细胞的实验研究

目的 探讨兔骨髓间充质干细胞(MSCs)体外培养、定向诱导分化为成骨细胞的途径.方法 抽取兔骨髓,以全骨髓贴壁培养法进行体外培养,贴壁细胞传代.取第3代细胞诱导培养,倒置显微镜下观察细胞形态.培养16 d后,用5-溴-4-氯-3-吲哚磷酸盐(BCIP)/氯化硝基四氮唑蓝(NBT)底物显色试剂检测碱性磷酸酶(AP),茜素红染色检测钙结节.结果 全骨髓贴壁培养法可获得MSCs,原代和传代培养的MSCs具有活跃的增殖能力.诱导培养后,AP检测与钙结节染色均为强阳性.结论 兔骨髓中培养出的MSCs可以定向分化为成骨细胞,可用作骨组织工程中的种子细胞.     

骨髓间充质干细胞旁分泌HGF体外调控肝星状细胞

目的 探讨大鼠骨髓间充质干细胞（BMSCs）与肝星状细胞（HSCs）体外共培养体系中,BMSCs旁分泌肝细 胞生长因子（HGF）对 HSCs 增殖、凋亡、活化的影响。方法 全骨髓贴壁法分离、培养、纯化大鼠 BMSCs,另培养 HSCs。6 孔板半透膜建立上下两层细胞非直接接触共培养体系,实验设 H 组（HSCs 单独培养）、H-H 组（HSCs 与 HSCs共培养）、M-H组（BMSCs与HSCs共培养）、M-H-C组（BMSCs与HSCs共培养并加c-met抑制剂）,各组细胞培 养48 h后,流式细胞仪鉴定BMSCs,检测HSCs凋亡率,MTT法检测HSCs的增殖,免疫荧光共聚焦定量检测HSCs中 α-肌动蛋白（α-SMA）的表达量,ELISA法检测共培养体系上清液中HGF的浓度。结果 MSCs高表达阳性表面分子 CD29、CD90,低表达造血细胞表面标记CD45;BMSCs能明显抑制HSCs的增殖、活化并促进其凋亡,且M-H组上清液 中HGF的浓度明显高于其他组。结论 BMSCs与HSCs共培养过程中,BMSCs通过旁分泌HGF促进HSCs的凋亡,抑 制HSCs的增殖、活化。     

利用诱导性多能干细胞技术体外扩增造血干/祖细胞的初步研究

目的 利用诱导性多能干细胞技术体外扩增造血干/祖细胞.方法 利用非整合型载体,将四个转录因子:Sox2、Klf4、Oct4和c-Myc导入脐血来源的造血干/祖细胞(CD34+细胞)重编程获得诱导性多能干细胞(iPSCs),再利用与小鼠骨髓基质细胞OP9共培养法将其定向分化成CD34+造血干/祖细胞.借助iPSCs可以在体外无限传代、大量扩增的特点,实现体外保存及大量扩增造血干/祖细胞的目的.结果 脐血CD34+细胞可以在体外重编程为非整合型iPSCs,并能够高效定向分化成为CD34+细胞,其分化效率比胚胎干细胞(ESCs)有显著提高.结论 利用诱导性多能干细胞技术体外大量扩增脐血造血干/祖细胞是一个可行的方案,具有良好的应用前景.     

碱性成纤维细胞因子对SD大鼠表皮干细胞分化为神经干细胞的影响

目的 研究碱性成纤维细胞因子(bFGF)对大鼠表皮干细胞分化为神经细胞的影响。 方法 分离获得饲养了1~3天的新生SD大鼠表皮基底层组织,利用10 min快速贴壁法消化、分离获得表皮干细胞,于倒置显微镜下观察表皮干细胞的形态。培养表皮干细胞的培养基为K-SFM,然后按照不同密度表皮干细胞进行分组处理(0.1×10⁷/mL,0.3×10⁷/mL,0.5×10⁷/mL,0.1×10⁶/mL),每组加入20 ng/mL的bFGF,利用细胞免疫组织化学法检测细胞标志物Nestin和NSE的变化,以及观察细胞形态发生的变化。 结果 成功分离得到SD大鼠表皮干细胞;bFGF诱导后,0.3×10⁷/mL组和0.1×10⁷/mL组细胞第3天即可见细胞开始伸展生长,在约1周时细胞开始分化成神经细胞;且在细胞形态发生双极化改变的数目趋势上也具有一致性;Nestin和NSE检测均呈阳性表达。     

骨相关细胞来源外泌体miRNA调节骨形成的研究进展

外泌体是一种由细胞分泌的具有脂质双分子层结构的杯状细胞外囊泡,直径约30-150 nm。近年来,研究表明骨组织中的各类骨细胞产生的外泌体不仅可介导生理情况下骨髓微环境内的细胞间通讯,维持骨髓微环境的稳态,还参与骨骼疾病的发病过程。该文将着重探讨骨相关细胞来源的外泌体miRNA对骨形成的调节作用。     

Gold nanoparticles in injectable calcium phosphate cement enhance osteogenic differentiation of human dental pulp stem cells

In this study, a novel calcium phosphate cement containing gold nanoparticles (GNP-CPC) was developed. Its osteogenic induction ability on human dental pulp stem cells (hDPSCs) was investigated for the first time. The incorporation of GNPs improved hDPSCs behavior on CPC, including better cell adhesion (about 2-fold increase in cell spreading) and proliferation, and enhanced osteogenic differentiation (about 2–3-fold increase at 14 days). GNPs endow CPC with micro-nano-structure, thus improving surface properties for cell adhesion and subsequent behaviors. In addition, GNPs released from GNP-CPC were internalized by hDPSCs, as verified by transmission electron microscopy (TEM), thus enhancing cell functions. The culture media containing GNPs enhanced the cellular activities of hDPSCs. This result was consistent with and supported the osteogenic induction results of GNP-CPC. In conclusion, GNP-CPC significantly enhanced the osteogenic functions of hDPSCs. GNPs are promising to modify CPC with nanotopography and work as bioactive additives thus enhance bone regeneration.     

microRNAs在调控结直肠癌肿瘤干细胞相关信号通路中的作用机制

肿瘤干细胞的存在是恶性肿瘤发生、复发、转移及耐药的根本原因。目前临床上并没有针对肿瘤干细胞的有效治疗手段,因此寻找针对肿瘤干细胞的治疗靶点,对指导结直肠癌治疗及改善其预后具有重要意义。大量研究表明微小核糖核酸（microRNAs）在调节结直肠癌肿瘤干细胞中起关键作用。本文归纳总结microRNAs的合成途径与功能以及其通过肿瘤干细胞相关信号通路和上皮间质转化途径调控结直肠癌肿瘤干细胞的作用机制。对结直肠癌肿瘤干细胞相关microRNAs进行深入研究,为结直肠癌的治疗提供新的靶点及切入点。     

Targeting microRNAs in epithelial-to-mesenchymal transition-induced cancer stem cells: therapeutic approaches in cancer

Introduction: Epithelial-to-mesenchymal transition (EMT) is a pathological phenomenon of cancer that confers tumor cells with increased cell motility, invasive and metastatic abilities with the acquisition of ‘cancer stem-like cell’ (CSC) phenotype. EMT endows tumor cells with intrinsic/acquired resistant phenotype at achievable doses of anticancer drugs and leads to tumor recurrence and progression. Besides the complex network of signaling pathways, microRNAs (miRNAs) are being evolved as a new player in the induction and regulation of EMT.

Areas covered: In this review article, the author has searched the PubMed and Google Scholar electronic databases for original research and review articles to gather current information on the association of EMT-induced CSCs with therapeutic resistance, tumor growth and metastasis, which are believed to be regulated by certain miRNAs.

Expert opinion: This review outlines not only the perspective on selective targeting of EMT-induced CSCs through altered expression of novel miRNAs and/or the use of conventional drugs that affect the levels of critical miRNAs but also the strategies on overcoming the drug resistance by interfering with EMT and modulating its associated pathways in CSCs that can be considered as potential therapeutic approaches toward eradicating the tumor recurrence and metastasis.     

Sequential exposure to cytokines reflecting embryogenesis: the key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells.

Differentiation of adult bone marrow stem cells (BMSC) into hepatocyte-like cells is commonly performed by continuous exposure to a cytokines-cocktail. Here, it is shown that the differentiation efficacy in vitro can be considerably enhanced by sequential addition of liver-specific factors (fibroblast growth factor-4, hepatocyte growth factor, insulin-transferrin-sodium selenite, and dexamethasone) in a time-dependent order that closely resembles the secretion pattern during in vivo liver embryogenesis. Quantitative RT-PCR analysis and immunocytochemistry showed that, upon sequential exposure to liver-specific factors, different stages of hepatocyte differentiation, as seen during liver embryogenesis, can be mimicked. Indeed, expression of the early hepatocyte markers alpha-fetoprotein and hepatocyte nuclear factor (HNF)3beta decreased as differentiation progressed, whereas levels of the late liver-specific markers albumin (ALB), cytokeratin (CK)18, and HNF1alpha were gradually upregulated. In contrast, cocktail treatment did not significantly alter the expression pattern of the hepatic markers. Moreover, sequentially exposed cells featured highly differentiated hepatic functions, including ALB secretion, glycogen storage, urea production, and inducible cytochrome P450-dependent activity, far more efficiently compared to the cocktail condition. In conclusion, sequential induction of the differentiation process, analogous to in vivo liver development, is crucial for in vitro differentiation of adult rat BMSC into functional hepatocyte-like cells. This model may not only be applicable for in vitro studies of endoderm differentiation but it also provides a "virtually unlimited" source of functional hepatocytes, suitable for preclinical pharmacological research and testing, and cell and organ development.     

Recent progresses in plastic surgery using adipose-derived stem cells,biomaterials and growth factors

Plastic and reconstructive surgery is a distinct specialty, which entails craniofacial and hand surgery; trauma, oncologic and congenital reconstruction; burn care, and aesthetic surgery. However, advances in nanotechnology have significantly affected wound management, skin care, implant and prosthetic design, tissue engineering, and drug delivery systems. Presently, plastic surgeons are applying the efficacy of stem cells (ADSCs), biomaterials and growth factors in different facets of plastic surgery. In this review, we will elucidate the applications of stem cells, biomaterials and growth factors in plastic surgeries.     

骨髓间充质干细胞调控肝星状细胞增殖和胶原合成的实验研究

目的研究骨髓间充质干细胞（BMSCs）对肝星状细胞（HSCs）增殖、胶原合成的影响。方法 （1）BMSCs对HSCs增殖的影响：利用transwell共培养体系,按1：1细胞比例在6孔塑料培养板上室接种BMSCs（1×105cells/well）,下室接种HSCs;对照组成纤维细胞代替BMSCs接种于上室;空白组为HSCs单独培养。培养24、48、72h后MTT法检测HSCs增殖抑制率;（2）BMSCs对HSCs胶原合成的影响：实验分为3组,A：HSCs组,B：HSCs＋TGF-β1组,C：共培养＋TGF-β1组。按上述方法进行BMSCs、HSCs共培养,培养液中加入TGF-β1诱导活化。培养72h后,取出BMSCs并换液,24h后收集培养液,用ELISA方法测定Ⅰ型胶原浓度,收集HSCs,RT-PCR检测HSCsα-SMA以及Ⅰ型胶原的基因表达。结果培养24、48、72 h后,共培养组较对照组可明显提高HSCs增殖抑制率（P〈0.01）;比较B组,C组Ⅰ型胶原浓度显著下降（P〈0.01）,同时HSCsα-SMA及Ⅰ型胶原基因表达出现显著下调（P〈0.01）。结论 BMSCs在体外可明显抑制HSCs增殖以及抑制TGF-β1诱导的α-SMA表达和Ⅰ型胶原合成。     

低氧培养环境对脐带间充质干细胞增殖及生物学特性的影响

目的 研究低氧环境对人脐带间充质干细胞（hUCMSCs）增殖及生物学特性的影响。方法 分别在5% O₂和21% O₂环境下培养hUCMSCs,使用群体倍增时间（PDT）评价hUCMSCs的增殖情况;流式细胞术检测细胞表面标志CD73、CD90、CD105、CD34、CD45、HLA-DR的表达;培养基诱导分化后,茜素红-S对含钙骨细胞染色检测成骨诱导分化,油红O对脂滴染色检测成脂诱导分化,阿尔新蓝8GX对蛋白多糖检测软骨诱导分化;羧基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）染色法检测hUCMSCs对植物血凝素P（PHA-P）刺激的外周血单个核细胞（peripheral blood mononuclear cell,PBMC）的增殖抑制作用,并应用流式细胞术检测对CD8+T细胞的抑制作用;实时荧光定量PCR（qRT-PCR）法检测低氧诱导因子1α（HIF-1α）、血管内皮生长因子（VEGF）、肝细胞生长因子（HGF）、胰岛素样生长因子2（IGF-2）、转化生长因子β（TGF-β）、碱性成纤维细胞生长因子（bFGF）、基质细胞衍生生长因子1（SDF-1）、神经生长因子（NGF）mRNA表达;试剂盒法检测细胞培养上清中IGF-2浓度。结果 21% O₂和5% O₂环境培养的hUCMSCs表面标志CD73、CD90、CD105的表达均为阳性（＞95%）,CD34、CD45、HLA-DR的表达均为阴性（＜2%）,均具有成骨、成脂、成软骨三系分化的能力;5% O₂组的PDT均显著小于21% O₂组（P＜0.05）;PBMC经PHA-P刺激后观察到细胞增殖聚集,与hUCMSCs共培养后几乎没有聚集出现,与PBMC＋PHA-P组比较,PBMC＋PHA-P＋hUCMSCs（21%或5% O₂）组子代细胞明显减少,PBMC＋PHA-P＋hUCMSCs（5% O₂）组PBMC抑制率为（61.44±0.92）%,与PBMC＋PHA-P＋hUCMSCs （21% O₂）抑制率（60.48±4.00）%相当,无统计学差异,且两组hUCMSCs对CD8+T细胞的抑制作用无统计学差异。与21% O₂组比较,5% O₂组hUCMSCs HIF-1α、IGF-2、SDF-1、HGF、VEGF、bFGF、NGF mRNA表达水平显著升高（P＜0.05、0.001）,TGF-β无明显变化; 5% O₂组hUCMSCs培养上清IGF-2水平显著高于21% O₂组（P＜0.001）。结论 5% O₂环境可使hUCMSCs的增殖能力和相关生长因子的表达增强,尤其是IGF-2,但依然保持着与21% O₂环境培养的hUCMSCs相似的表型、分化能力以及淋巴细胞增殖抑制能力。     

Pluripotent stem cells: An in vitro model for nanotoxicity assessments

The advent of technology has led to an established range of engineered nanoparticles that are used in diverse applications, such as cell–cell interactions, cell–material interactions, medical therapies and the target modulation of cellular processes. The exponential increase in the utilization of nanomaterials and the growing number of associated criticisms has highlighted the potential risks of nanomaterials to human health and the ecosystem. The existing in vivo and in vitro platforms show limitations, with fluctuations being observed in the results of toxicity assessments. Pluripotent stem cells (PSCs) are viable source of cells that are capable of developing into specialized cells of the human body. PSCs can be efficiently used to screen new biomaterials/drugs and are potential candidates for studying impairments of biophysical morphology at both the cellular and tissue levels during interactions with nanomaterials and for diagnosing toxicity. Three‐dimensional in vitro models obtained using PSC‐derived cells would provide a realistic, patient‐specific platform for toxicity assessments and in drug screening applications. The current review focuses on PSCs as an alternative in vitro platform for assessing the hazardous effects of nanomaterials on health systems and highlights the importance of PSC‐derived in vitro platforms. Copyright © 2016 John Wiley & Sons, Ltd.     

不同细胞外基质对神经干细胞分化影响的实验研究

目的:通过观察不同的细胞外基质对神经干细胞(NSCs)分化的影响,寻找促使NSCs向神经元分化的最佳细胞外基质,为NSCs定向分化培养及NSCs共移植体的构建提供实验依据。方法:利用无血清培养和细胞克隆培养技术,从新生大鼠海马分离NSCs,进行体外扩增、悬浮培养、传代;将层粘连蛋白(LN)、Matrigel、多聚赖氨酸与NSCs贴壁混合培养14d;采用免疫细胞化学和免疫荧光技术,观察NSCs的分化情况。结果:通过上述实验表明,在利用不同的细胞外基质混合培养时,LN组MAP2阳性细胞数明显高于其它两组;多聚赖氨酸组和Matrigel组GFAP阳性细胞数明显高于LN组。结论:LN有助于NSCs向神经元定向分化,是NSCs共移植体较理想的细胞外基质。     

Regulation of stem cell differentiation in adipose tissue by chronic inflammation

1. Recent studies suggest that a local hypoxic response leads to chronic inflammation in the adipose tissue of obese individuals. The adipose tissue hypoxia may reflect a compensatory failure in the local vasculature system in response to obesity. 2. Studies suggest that inflammation stimulates angiogenesis and inhibits adipocyte activities in a feedback manner within the obese adipose tissue. Adipose-derived stem cells (ASC) are able to differentiate into multiple lineages of progenitor cells for adipocytes, endothelial cells, fibroblasts and pericytes. Differentiation of ASC into those progenitors is regulated by the adipose tissue microenvironment. 3. As a major factor in the microenvironment, inflammation may favour ASC differentiation into endothelial cells through the induction of angiogenic factors. At the same time, inflammation inhibits ASC differentiation into adipocytes by suppressing peroxisome proliferator-activated receptor γ activity and the insulin signalling pathway. In this context, inflammation may serve as a signal mediating the competition between adipocytes and endothelial cells for the limited source of ASC. 4. It is a new concept that inflammation mediates signals in the competition between adipocytes and endothelial cells for the limited ASC in obesity. There is a lot of evidence that inflammation promotes endothelial cell differentiation. However, this activity of inflammation remains to be established in adipose tissue. The present article reviews the literature in support of this conclusion.     

大鼠骨髓间充质干细胞生物学特性的研究

目的研究体外大鼠骨髓间充质干细胞的生长特性。方法在无菌条件下,取大鼠骨髓,采用密度离心法和全骨髓贴壁法联合培养并结合传代获取、纯化细胞,倒置显微镜观察其形态,流式细胞术检测其表面抗原CD29、CD90及CD34的表达。CCK-8法测定第1、3代细胞的增殖情况,并绘制其生长曲线。结果经全骨髓贴壁法和密度梯度离心联合培养,所得到的原代细胞形态呈椭圆形、圆形、三角形,接近融合状态时可呈现均一的长梭形,排列规则。传至第8代时,细胞的增殖能力减弱,其形态宽大畸形,呈树枝状。经流式细胞仪检测,所分离的细胞CD29的表达率为94.97%,CD90的表达率为88.50%,CD34的表达率为2.23%。CCK-8法显示第3代细胞有较强的增殖能力。结论体外培养的大鼠BMSCs是骨组织工程的优良种子细胞。