Melatonin promotes osteoblast differentiation and mineralization of MC3T3‐E1 cells under hypoxic conditions through activation of PKD/p38 pathways

Osteoblastic differentiation and bone‐forming capacity are known to be suppressed under hypoxic conditions. Melatonin has been shown to influence cell differentiation. A number of in vitro and in vivo studies have suggested that melatonin also has an anabolic effect on bone, by promoting osteoblastic differentiation. However, the precise mechanisms and the signaling pathways involved in this process, particularly under hypoxic conditions, are unknown. This study investigated whether melatonin could promote osteoblastic differentiation and mineralization of preosteoblastic MC3T3‐E1 cells under hypoxic conditions. Additionally, we examined the molecular signaling pathways by which melatonin mediates this process. We found that melatonin is capable of promoting differentiation and mineralization of MC3T3‐E1 cells cultured under hypoxic conditions. Melatonin upregulated ALP activity and mRNA levels of Alp, Osx, Col1, and Ocn in a time‐ and concentration‐dependent manner. Alizarin red S staining showed that the mineralized matrix in hypoxic MC3T3‐E1 cells formed in a manner that was dependent on melatonin concentration. Moreover, melatonin stimulated phosphorylation of p38 Mapk and Prkd1 in these MC3T3‐E1 cells. We concluded that melatonin promotes osteoblastic differentiation of MC3T3‐E1 cells under hypoxic conditions via the p38 Mapk and Prkd1 signaling pathways.     

MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3' UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo.     

Wnt信号系统对骨髓间充质干细胞向心肌细胞分化的作用

骨髓间充质干细胞（BMSCs）移植治疗冠状动脉粥样硬化性心脏病、慢性心力衰竭等已经成为心脏病治疗学的热门课题,但细胞疗法依赖于干细胞向心肌细胞的定向分化,目前对BMSCs分化为心肌细胞的分子机制了解不多。Wnt信号系统与器官的分化和形成密切相关,大量研究表明,Wnt信号系统对干细胞向心肌细胞的定向分化有重要作用,对该信号系统的控制,在心脏病的细胞疗法中显得尤其重要。本文将就Wnt信号系统在BMSCs的增殖、迁移及心肌定向分化中的调控作用展开论述。     

Wnt信号通路分子在大鼠神经干细胞分化过程中的表达

目的：观察Wnt信号通路主要分子[β-连环蛋白（β-catenin）和糖原合酶激酶-3β（GSK-3β）]在神经干细胞（NSCs）体系的表达及其在增殖和分化过程中的变化,探讨其在神经干细胞分化中可能的调控作用。方法采用原代培养方法,自14～16d大鼠胚胎大脑皮质获得含大量细胞团的NSCs体系。应用含10%胎牛血清培养液诱导NSCs分化。Western印迹检测NSCs在加入胎牛血清12,24,48和72h后,β-catenin与GSK-3β在分化过程中的动态变化。结果免疫细胞化学染色显示NSCs细胞团及散在的单个细胞多呈巢蛋白（nestin）阳性,显示NSCs分化过程中两者存在持续表达,β-catenin分子表达呈现逐渐减少趋势,而GSK-3β分子表达则呈现逐渐增加趋势。结论提示Wnt信号通路与NSCs的增殖和分化过程密切相关,呈现出由活跃到逐步抑制的过程,为研究Wnt信号通路参与NSCs增殖分化调控的机制提供了载体。     

Geometric cues for directing the differentiation of mesenchymal stem cells

Significant efforts have been directed to understanding the factors that influence the lineage commitment of stem cells. This paper demonstrates that cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature—and with each occupying the same area—display different adipogenesis and osteogenesis profiles. The results reveal that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. Taken together, this work points to the role that geometric shape cues can play in orchestrating the mechanochemical signals and paracrine/autocrine factors that can direct MSCs to appropriate fates.     

Calcium signaling of pancreatic acinar cells in the pathogenesis of pancreatitis

Pancreatitis is an increasingly common and sometimes severe disease that lacks a specific therapy.The pathogenesis of pancreatitis is still not well understood.Calcium(Ca2+)is a versatile carrier of signals regulating many aspects of cellular activity and plays a central role in controlling digestive enzyme secretion in pancreatic acinar cells.Ca2+overload is a key early event and is crucial in the pathogenesis of many diseases.In pancreatic acinar cells,pathological Ca2+signaling(stimulated by bile,alcohol metabolites and othercauses)is a key contributor to the initiation of cell injury due to prolonged and global Ca2+elevation that results in trypsin activation,vacuolization and necrosis,all of which are crucial in the development of pancreatitis.Increased release of Ca2+from stores in the intracellular endoplasmic reticulum and/or increased Ca2+entry through the plasma membrane are causes of such cell damage.Failed mitochondrial adenosine triphosphate(ATP)production reduces re-uptake and extrusion of Ca2+by the sarco/endoplasmic reticulum Ca2+-activated ATPase and plasma membrane Ca2+-ATPase pumps,which contribute to Ca2+overload.Current findings have provided further insight into the roles and mechanisms of abnormal pancreatic acinar Ca2+signals in pancreatitis.The lack of available specific treatments is therefore an objective of ongoing research.Research is currently underway to establish the mechanisms and interactions of Ca2+signals in the pathogenesis of pancreatitis.     

PI3K／Akt信号通路在骨髓间充质干细胞增殖及成骨分化调控中的作用

目的：研究PI3K/Akt信号通路抑制剂LY294002对骨髓间充质干细胞（mesenchymal stem cells,MSCs）增殖及分化的影响。方法采用贴壁法体外分离人骨髓间充质干细胞（hMSCs）,加入PI3K抑制剂LY294002（1、10μmol/L）,应用MTT法测定细胞增殖,常规成骨诱导分化培养3或7d,采用碱性磷酸酶（ALP）染色观察成骨分化水平,化学比色法测定ALP活性,茜素红染色后观察矿化钙结节数量并定量分析,Westernblot检测磷酸化Akt蛋白表达,应用Realtime-PCR检测各组细胞BMP2、Runx2、OPN及Osterix等成骨分化标记物的基因表达水平。结果从24至72h,LY294002对hMSCs增殖均产生显著抑制,随时间推延,可见抑制增殖效果增强（P＜0.05）。ALP染色和定量测定提示10μmol/L的ALP活性最强,在不同时间显著高于对照组和1μmol/L组（P＜0.05）。成骨诱导培养3和7d,1、10μmol/L组矿化量都显著高于对照组（P＜0.05）。10μmol/L组矿化量在成骨诱导7d也显著高于1μmol/L组（P＜0.05）。Westernblot检测结果证实成骨诱导可激活Akt磷酸化蛋白表达,但LY294002可抑制该蛋白磷酸化。成骨诱导分化7d,1、10μmol/L均明显促进BMP2、Runx2、OPN、Osterix4种基因mRNA表达（均P＜0.05）。结论PI3K/Akt信号通路参与hMSCs增殖和分化过程。成骨分化伴随下游Akt蛋白表达。PI3K抑制剂可抑制hMSCs增殖,但同时促进其向成骨分化和矿化。     

High frequency of cephalic neural crest cells shows coexistence of neurogenic,melanogenic, and osteogenic differentiation capacities

The neural crest (NC) is a vertebrate innovation that distinguishes vertebrates from other chordates and was critical for the development and evolution of a “New Head and Brain.” In early vertebrates, the NC was the source of dermal armor of fossil jawless fish. In extant vertebrates, including mammals, the NC forms the peripheral nervous system, melanocytes, and the cartilage and bone of the face. Here, we show that in avian embryos, a large majority of cephalic NC cells (CNCCs) have the ability to differentiate into cell types as diverse as neurons, melanocytes, osteocytes, and chondrocytes. Moreover, we find that the morphogen Sonic hedgehog (Shh) acts on CNCCs to increase endochondral osteogenesis while having no effect on osteoblasts prone to membranous ossification. We have developed culture conditions that demonstrate that “neural–mesenchymal” differentiation abilities are present in more than 90% of CNCCs. A highly multipotent progenitor (able to yield neurons, glia, melanocytes, myofibroblasts, chondrocytes, and osteocytes) comprises 7–13% of the clonogenic cells in the absence and presence of Shh, respectively. This progenitor is a good candidate for a cephalic NC stem cell.     

Inhibitory effect of alcohol on osteogenic differentiation in human bone marrow-derived mesenchymal stem cells

BACKGROUND: Alcohol-induced osteoporosis is characterized by a considerable suppression of osteogenesis. The objective of this investigation was to determine the effect of alcohol on gene expression, protein synthesis, and mineralization in human bone marrow-derived mesenchymal stem cells induced toward osteogenic differentiation in vitro. METHODS: Human bone marrow-derived mesenchymal stem cells induced toward osteogenesis were cultured in the presence or absence of 50 mM alcohol. Stem cells were characterized by using SH2 antibody to the cell-surface antigen CD105/endoglin, and their proliferation in the presence of alcohol was quantified. The expression of genes for early, middle, and late markers of the osteogenic lineage was quantified by Northern analysis, and bone matrix protein synthesis was assayed. The effect of alcohol on cell-mediated matrix mineralization in terminally differentiated cultures was determined by von Kossa staining. RESULTS: Fluorescence-activated cell sorting analysis of human mesenchymal stem cells separated with a Percoll gradient proved 99% homogeneity by using SH2 antibody to the surface antigen CD105. Dose-dependent inhibition of proliferation of these stem cells occurred at concentrations greater than 50 mM alcohol. Gene expression of osteoblast-specific factor 2/core binding factor a1 (Osf2/Cbfa1), type I collagen, alkaline phosphatase, and osteocalcin (early, middle, and late markers for osteogenesis, respectively) was analyzed with and without osteogenic induction and treatment with 50 mM alcohol. After induction, Osf2/Cbfa1 levels were unresponsive to alcohol. To determine the effect of alcohol on human mesenchymal stem cell progression along the osteogenic pathway, messenger RNA (mRNA) levels for type I collagen, alkaline phosphatase, and osteocalcin were examined after osteogenic induction. After osteogenic induction, alcohol down-regulated the gene expression of type I collagen and significantly reduced its synthesis. Alcohol did not alter mRNA expression of alkaline phosphatase, a midstage marker for osteogenesis, but significantly decreased its activity compared with osteogenic induction alone. After induction, osteocalcin remained unchanged by alcohol at both the mRNA and protein levels. Histochemistry revealed decreased alkaline phosphatase staining and fewer alkaline phosphatase-positive cells in alcohol-treated human mesenchymal stem cell cultures. von Kossa staining revealed a reduction in the number of mineralizing nodules in stem cell cultures after alcohol treatment. CONCLUSIONS: Collectively, the data suggest that alcohol alters osteogenic differentiation in human bone marrow-derived mesenchymal stem cell cultures during lineage progression and provide further insight into alcohol-induced reduced bone formation.     

二甲双胍靶向Notch信号通路抑制胰腺癌干细胞自我更新

目的探讨二甲双胍对胰腺癌干细胞自我更新及其Notch信号通路活性的影响。方法通过细胞球培养法获取Panc1胰腺癌干细胞。通过CCK-8检测二甲双胍对Panc1普通胰腺癌细胞和胰腺癌干细胞的半数抑制浓度(IC 50)。通过CCK-8检测二甲双胍对Panc1胰腺癌干细胞自我更新能力的抑制;通过实时定量PCR检测二甲双胍对Panc1胰腺癌干细胞中Notch4、DLL1、DLL3、Jagged1和HES1 mRNA水平的影响。结果Panc1普通胰腺癌细胞悬浮培养于干细胞培养基后,全部细胞聚集成球状、团块状;5 d后悬浮培养得到的Panc1胰腺癌干细胞与Panc1普通胰腺癌细胞相比体积小,呈梭形或圆形。二甲双胍对Panc1普通胰腺癌细胞的IC 50为27.97 mmol/L,对Panc1胰腺癌干细胞的IC 50为16.10 mmol/L。在加入20 mmol/L二甲双胍后,体外培养的Panc1胰腺癌干细胞增殖能力明显受到抑制(P<0.01),细胞中Notch4、DLL1、DLL3、Jagged1、HES1 mRNA水平明显下调(P<0.01)。结论二甲双胍对Panc1胰腺癌干细胞的IC 50低于对Panc1普通胰腺癌细胞的IC 50,提示Panc1胰腺癌干细胞对二甲双胍更为敏感;二甲双胍可抑制Panc1胰腺癌干细胞自我更新,并可显著抑制其Notch信号通路活性,提示抑制该通路活性可能是二甲双胍抑制胰腺癌干细胞自我更新的机制之一。     

Melatonin prevents cadmium‐induced bone damage: First evidence on an improved osteogenic/adipogenic differentiation balance of mesenchymal stem cells as underlying mechanism

Melatonin (MLT) plays a role in preserving bone health, a function that may depend on homeostatic effects on both mature osteoblasts and mesenchymal stem cells (MSCs) of the bone tissue. In this study, these functions of MLT have been investigated in rat bone (femur) and in human adipose MSC (hMSC) during chronic exposure to low‐grade cadmium (Cd) toxicity, a serious public health concern. The in vivo findings demonstrate that MLT protects against Cd‐induced bone metabolism disruption and accumulation of bone marrow adipocytes, a cue of impaired osteogenic potential of skeletal MSC niches. This latter symptom was recapitulated in hMSCs in which Cd toxicity stimulated adipogenic differentiation. MLT was found to rescue, at least in part, the osteogenic differentiation properties of these cells. This study reports on a new bone cytoprotection function of MLT pertinent to Cd toxicity and its interfering effect on skeletal MSC differentiation properties that is worth investigating for its possible impact on human bone pathophysiology.     

体外诱导骨髓间充质干细胞向神经元样细胞分化的研究

目的观察人骨髓间充质干细胞（BM-MSCs）体外扩增和定向诱导分化为神经元样细胞的变化,为其临床应用奠定基础。方法采用密度梯度离心法分离BM-MSCs细胞,用MesencultTM培养基和贴壁培养法培养、纯化和扩增细胞,流式细胞术检测细胞表面CD29和CD90分子表达率。采用20 ng/ml重组碱性成纤维细胞生长因子（bFGF）、20 ng/ml新型人表皮生长因子（hEGF）和20 g/L二甲基亚砜（DMSO）、5 mmol/Lβ-巯基乙醇（BME）分别诱导BM-MSCs;采用免疫组化法检测细胞的神经元特异性烯醇化酶（NSE）、胶原纤维酸性蛋白（GFAP）、波形蛋白（VIM）表达情况。结果BM-MSCs细胞增殖迅速,3周可传代培养;BM-MSCs传十代扩增1－2×10^3倍。分离和扩增BM-MSCs CD29、CD90强表达率分别为95.02%、93.81%。药物诱导后的BM-MSCs发生轴突等神经元样细胞变化,阳性表达NSE、GFAP、VIM分子。结论人BM-MSCs能体外培养和扩增,并能定向诱导分化为神经元样细胞。     

大鼠骨髓Thy-1.1+干细胞诱导分化为肝干细胞样细胞的体外实验研究

目的：分离培养大鼠骨髓Thy-1．1^＋干细胞，探讨其肝分化条件及潜能。方法：分离大鼠骨髓细胞，Percoil密度梯度离心获取骨髓单个核细胞后免疫磁珠分选Thy-1．1^＋干细胞群，流式细胞术测定分选后细胞的纯度与表型。不同条件下诱导分化，用形态学观察、逆转录聚合酶链反应和免疫细胞化学法判定其所处的肝系分化阶段。结果：免疫磁珠分选Thy-1．1^＋细胞纯度达94．2％。Thy-1．1^＋细胞不表达CD34和c-kit；高表达β2微球蛋白和CD45；部分表达Flt-3。Thy-1．1^＋细胞在HGF／FGF-1／FGF-2诱导下呈多角形上皮状克隆样生长，诱导前后持续表达细胞角质素18，诱导过程中白蛋白的表达逐渐减少，甲胎球蛋白的表达逐渐增强。结论：免疫磁珠可有效分选Thy-1．1^＋细胞。Thy-1．1^＋细胞在HGF／FGF．1／FGF-2诱导下呈肝干细胞样细胞表现。     

Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells

Nitric oxide (NO) is involved in number of physiological and pathological events. Our previous studies demonstrated a differential expression of NO signaling components in mouse and human ES cells. Here, we demonstrate the effect of NO donors and soluble guanylyl cyclase (sGC) activators in differentiation of ES cells into myocardial cells. Our results with mouse and human ES cells demonstrate an increase in Nkx2.5 and myosin light chain (MLC2) mRNA expression on exposure of cells to NO donors and a decrease in mRNA expression of both cardiac-specific genes with nonspecific NOS inhibitor and a concomitant increase and decrease in the mRNA levels of sGC α₁ subunit. Although sGC activators alone exhibited an increase in mRNA expression of cardiac genes (MLC2 and Nkx2.5), robust inductions of mRNA and protein expression of marker genes were observed when NO donors and sGC activators were combined. Measurement of NO metabolites revealed an increase in the nitrite levels in the conditioned media and cell lysates on exposure of cells to the different concentrations of NO donors. cGMP analysis in undifferentiated stem cells revealed a lack of stimulation with NO donors. Differentiated cells however, acquired the ability to be stimulated by NO donors. Although, 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b]pyridine (BAY 41-2272) alone was able to stimulate cGMP accumulation, the combination of NO donors and BAY 41-2272 stimulated cGMP levels more than either of the agents separately. These studies demonstrate that cGMP-mediated NO signaling plays an important role in the differentiation of ES cells into myocardial cells.     

5-氮杂胞苷通过PI3K/Akt/mTOR通路介导人脂肪间充质干细胞向心肌细胞定向分化的研究

目的:研究磷脂酰肌醇3激酶(PI3K)及其下游分子丝氨酸/苏氨酸激酶(Akt)和雷帕霉素靶蛋白(mTOR)所组成的信号通路在5-氮杂胞苷(5-aza)诱导人脂肪间充质干细胞(ADMSCs)向心肌细胞定向分化中的作用,探讨其信号转导机制。方法:利用胶原酶法分离、培养ADMSCs,并用10μmol/L的5-aza诱导其向心肌细胞定向分化,采用Western blot的方法分析5-aza诱导前后Akt通路相关蛋白的表达情况。结果:5-aza诱导前ADMSCs内Akt通路相关蛋白的表达水平较低,诱导后增强。PI3K抑制剂Ly294002处理后,Western blot结果显示,细胞内PI3K的磷酸化受到抑制和TnT的表达水平显著降低,具有统计学意义。结论:5-aza能诱导ADMSCs向心肌细胞分化,PI3K/Akt/mTOR信号通路在5-aza诱导ADMSCs向心肌细胞分化中发挥重要的调控作用。