氯化锂对人骨髓间充质干细胞迁移的影响

目的:探究氯化锂(Lithium chlorid,LiCl)对人骨髓间充质干细胞(human Mesenchymal Stem Cells,hMSCs)迁移的影响。方法:采用划痕试验、Transwell chamber等方法,在梯度浓度LiCl作用下,观察对hMSCs迁移效果的影响并进行分析。结果:1划痕试验显示hMSCs在梯度浓度LiCl作用下,细胞迁移距离逐渐减少,差异具有统计学意义(P0.05)。2 Transwell chamber实验显示hMSCs在梯度浓度LiCl作用下,穿梭至小室下方的细胞逐渐减少,锂剂作用组迁移细胞数差异与对照组比较有统计学意义(P0.05)。结论:LiCl可抑制hMSCs的迁移且呈浓度依赖性。     

四种中药注射液对大鼠骨髓间充质干细胞增殖作用的影响

目的寻找对骨髓间充质干细胞(MSC)增殖有促进作用的中药。方法通过四甲基偶氮唑盐(MTT)法,Brdu标记MSC,免疫细胞化学法分析4种中药(参麦、生脉、黄芪、刺五加)对大鼠第五代MSC的增殖作用。结果4种中药中除黄芪外都有一定的诱导MSCs增殖的功能,但是刺五加的作用最强。结论刺五加具有较强的促进MSCs增殖的功能。     

骨髓间充质干细胞定向分化的经典WNT机制

骨髓间充质干细胞的定向分化一直是干细胞研究的重点,在其分化过程中有多条信号通路参与和调节。目前,Wnt通路在骨髓间充质干细胞定向分化过程中的作用是国外的研究热点。研究发现经典Wnt通路的激活与骨髓间充质干细胞的定向分化高度相关,故将其近年来的研究综述如下,从而为骨质疏松等疾病的治疗以及骨组织工程的发展提供必要的参考依据。     

骨髓间充质干细胞分化为心肌细胞过程中Wnt信号通路的作用研究

骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BM-MSCs)具有多向分化潜能,可以应用于修复细胞、组织、器官的损伤,通过干细胞移植疗法治疗多种疾病。BM-MSCs在一定的条件下可以诱导产生心肌细胞(cardiomyocytes,CMs),为治疗心血管疾病带来了希望。在心脏的分化过程中,Wnt信号通路发挥着非常重要的作用,因此对BM-MSCs分化为CMs过程中Wnt信号通路所发挥的作用做一简述。     

去甲肾上腺素对骨髓间充质干细胞增殖的影响

目的:观察去甲肾上腺素(norepinephrine,NE)对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖的影响及其作用途径.方法:分离培养正常大鼠BMSCs,采用3H-TdR掺入实验检测不同浓度的NE(10-7-10-4 M)作用8h及10-5M的NE作用不同时间(0-24h)BMSCs细胞增殖情况,real time RT-PCR检测肾上腺素能受体α1A-AR,α1B-AR和α1D-AR mRNA表达变化情况.结果:10-7-10-4M的NE作用8h后均促进了BMSCs细胞的增殖.并且在10-5M时NE对BMSCs的促增殖效应最为显著;正常组BMSCs细胞的α1A-AR,α1B-AR,α1D-AR mRNA表达维持在较低水平,加入10-5M的NE作用后α1-AR三个亚型mRNA表达水平均有不同程度的升高(P<0.05).结论:NE能够促进BMSCs的增殖,并且这种促增殖作用是通过AR依赖的信号通路来调节的.     

转化生长因子-β1对大鼠骨髓间充质干细胞增殖及Slug表达的影响

目的检测转化生长因子-β1(transforming growth factor-β1,TGF-β1)对体外培养大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMMSCs)增殖及Slug表达的影响。方法采用密度梯度离心结合贴壁法分离、培养大鼠BMMSCs,用免疫组织化学方法对培养第3代的细胞进行鉴定。用MTT法检测不同浓度TGF-β1对细胞增殖的影响;免疫荧光和免疫印迹法检测TGF-β1处理前后Slug的表达情况。结果密度梯度离心结合贴壁法能有效分离、纯化大鼠BMMSCs,免疫组织化学法检测显示CD29、CD44表达阳性,而CD34、CD45表达阴性;低浓度TGF-β1对BMMSCs的增殖有促进作用,高浓度却抑制BMMSCs的增殖。TGF-β1处理24 h,Slug蛋白表达明显增强。结论一定浓度的TGF-β1可以促进BMMSCs的增殖,而且引起Slug蛋白增加。     

大电导钙激活钾通道对大鼠骨髓间充质干细胞增殖的影响

为了观察开放和拮抗大电导钙激活钾通道（bigconductanceCa2＋-activated砧channel．BKca）对大鼠骨髓间充质干细胞（bonemarrowmesenchymalstemcells,BMSCs）增殖的影响并探讨其机制,该研究分离培养了大鼠BMSCs,采用BKca通道特异性开放剂msl619）和拮抗剂（IBTX）干预,MTT、平板克隆测定细胞增殖活力及细胞克隆形成能力;流式细胞术分析细胞凋亡及细胞周期分布;Westernblot、定量PCR检测周期蛋白cyclinD1基因和蛋白表达水平;整细胞膜片钳技术分析细胞膜电生理特性。结果显示,NS1619干预组与对照组相比,BMSCs~胞膜科通道外向电流振幅增大,细胞增殖能力和克隆形成能力增强,凋亡减少。此外,开放BKca通道明显促进细胞从G1期向S期过渡,cyclinD1蛋白7LmRNA表达上调,而拮抗BKca通道则相反。推测,BKca通道通过调节细胞周期进程最终影响细胞增殖,该作用可能与其具有调控细胞膜心电流的电生理特性有关。     

Wnt 信号通路与骨髓间充质干细胞成骨之间的关系

Wnt信号通路是由Wnts诱发的一系列相互作用的分子组成。Wnt信号对骨髓间充质干细胞的影响在所有研究中均证实有明显作用,其可调节干细胞增殖、分化及凋亡。研究表明,抑制Wnt信号通路转导可使成骨细胞分化进程受阻,从而抑制骨形成;若诱导Wnt家族成员表达则可使成骨细胞特异性基因表达增加,促进骨形成。本文就Wnt信号通路的作用过程及其与骨髓间充质干细胞成骨诱导的关系做一综述。     

氯化锂抑制A549细胞增殖及其对Polo-like激酶1转录活性的影响

利用糖原合成酶激酶3的抑制剂氯化锂作用于A549细胞,观察细胞形态与增殖的改变及其对Polo－like激酶1转录活性的影响.采用细胞计数检测细胞增殖,流式细胞术分析细胞周期变化;Western印迹检测磷酸化GSK3以及细胞周期相关蛋白p53、cyclin B1和Plk1的表达变化;RT-PCR检测Plk1 mRNA的表达;荧光素酶报告基因分析氯化锂对Plk1启动子活性的影响.结果显示,5 mmol/L氯化锂作用48 h后,A549细胞即发生明显的形态学改变,细胞增殖减慢并发生G2/M期阻滞;Plk1 mRNA和蛋白表达均升高,p53蛋白表达增强,而cyclin B1的蛋白表达无明显变化.氯化锂作用24 h后,可见pGL2-Plk1转染组中荧光素酶活性增高（与对照质粒相比,P<0.05）,48 h后更明显.以上结果表明, 氯化锂减慢A549细胞增殖,导致G2/M期阻滞,并能增强Plk1的启动子活性,促进Plk1的表达.     

间充质干细胞释放 Dkk-1 抑制乳腺癌细胞 Wnt/β-catenin 途径的实验研究

研究表明,间充质干细胞具有向肿瘤细胞定向迁移并且抑制肿瘤细胞的特性,然而其分子机理目前尚不清楚.为了探讨间充质干细胞抑制肿瘤细胞作用的分子机制,应用BMMS-03人间充质干细胞的条件培养液作用于MCF-7乳腺癌细胞,通过软琼脂克隆形成实验、MTT实验、免疫印迹和免疫荧光染色等技术观察细胞克隆形成、增殖和基因表达的变化.结果显示:在BMMS-03细胞条件培养液作用下,MCF-7细胞的克隆形成和增殖受到了明显的抑制,β-catenin及其下游靶蛋白c-Myc、Bcl-2、PCNA和survivin的表达被明显下调,MCF-7细胞浆和细胞核内β-catenin的表达被明显抑制.BMMS-03细胞中Dkk-1的表达水平与MCF-7细胞相比较高.利用抗Dkk-1的抗体中和BMMS-03细胞条件培养液中的Dkk-1后,可明显拮抗BMMS-03细胞条件培养液对MCF-7细胞中β-catenin及c-Myc表达的抑制作用,基因转染使MCF-7细胞过表达Dkk-1后,MCF-7细胞的β-catenin及c-Myc的表达明显下调.同样经基因转染使BMMS-03细胞过表达Dkk-1后,其条件培养液可进一步下调MCF-7细胞β-catenin及c-Myc的表达.上述结果表明,间充质干细胞BMMS-03对乳腺癌MCF-7细胞的恶性表型具有明显抑制作用,其分子机制与间充质干细胞释放Dkk-1抑制乳腺癌细胞Wnt/β-catenin信号途径有关.     

Low-intensity pulsed ultrasound promotes the proliferation of human bone mesenchymal stem cells by activating PI3K/AKt signaling pathways

Low-intensity pulsed ultrasound (LIPUS) is a promising therapy that is widely used in clinical applications and fundamental research. Previous research has shown that LIPUS exposure has a positive effect on stem cell proliferation. However, the impact of LIPUS exposure on human bone marrow mesenchymal stem cells (hBMSCs) remains unknown. In our study, the effect and mechanism of LIPUS exposure on the proliferation of hBMSCs were investigated, and the optimal parameters of LIPUS were determined. hBMSCs were obtained and identified by flow cytometry, and the proliferation of hBMSCs was measured using the Cell Counting Kit-8 assay to determine cell cycle and cell count. Expression levels of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKt) pathway proteins and cyclin D1 were determined by western blot analysis. Next, hBMSCs were successfully cultured and identified as multipotent mesenchymal stem cells. We found that LIPUS could promote the proliferation of hBMSCs when the exposure time was 5 or 10 minutes per day. Furthermore, 50 or 60 mW/cm² LIPUS had a more significant effect on cell proliferation, but if cells were irradiated by LIPUS for 20 minutes once a day, an intensity of at least 50 mW/cm² could markedly inhibit cell growth. Cell cycle analysis demonstrated that LIPUS treatment drives cells to enter S and G2/M phases from the G0/G1 phase. LIPUS exposure increased phosphorylation of PI3K/AKt and significantly upregulated expression of cyclin D1. However, these effects were inhibited when cells were treated with PI3K inhibitor (LY294002), which in turn reduced LIPUS-mediated proliferation of hBMSCs. These results suggest that LIPUS exposure may be involved in the proliferation of hBMSCs via activation of the PI3K/AKt signaling pathway and high expression of cyclin D1, and the intensity of 50 or 60 mW/cm² and exposure time of 5 minutes were determined to be the optimal parameters for LIPUS exposure.     

Cholinergic neuronal differentiation of bone marrow mesenchymal stem cells in rhesus monkeys

The purpose of the present study was to determine the best cholinergic neuronal differentiation method of rhesus monkey bone marrow mesenchymal stem cells(BMSCs).Four methods were used to induce differentiation,and the groups were assigned accordingly:basal inducing group(culture media,bFGF,and forskolin);SHH inducing group(SHH,inducing group);RA inducing group(RA,basal inducing group);and SHH+RA inducing group(SHH,RA,and basal inducing group).All groups displayed neuronal morphology and increased expressio...     

Proteomic profiling of distinct clonal populations of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi‐lineage differentiation. The molecular mechanisms governing MSC self‐renewal and differentiation remain largely unknown. The development of sophisticated techniques, in particular clinical proteomics, has enabled researchers in various fields to identify and characterize cell specific biomarkers for therapeutic purposes. This study seeks to understand the cellular and sub‐cellular processes responsible for the existence of stem cell populations in bone marrow samples by revealing the whole cell proteome of the clonal cultures of bone marrow‐derived MSCs (BMSCs). Protein profiling of the MSC clonal populations was conducted by Two‐Dimensional Liquid Chromatography/Matrix‐Assisted Laser Desorption/Ionisation (MALDI) Mass Spectrometry (MS). A total of 83 proteins were identified with high confidence of which 11 showed differential expression between subpopulations, which included cytoskeletal and structural proteins, calcium binding proteins, cytokinetic proteins, and members of the intermediate filament family. This study generated a proteome reference map of BMSCs from the clonal populations, which will be valuable to better understand the underlying mechanism of BMSC self‐renewal and differentiation. J. Cell. Biochem. 106: 776–786, 2009. © 2009 Wiley‐Liss, Inc.     

Timing of transplantation of autologous bone marrow derived mesenchymal stem cells for treating myocardial infarction

It is still unclear whether the timing of intracoronary stem cell therapy affects the therapeutic response in patients with myocardial infarction.The natural course of healing the infarction and the presence of putative homing signals within the damaged myocardium appear to favor cell engraftment during the transendothelial passage in the early days after reperfusion.However,the adverse inflammatory environment,with its high oxidative stress,might be deleterious if cells are administered too early after reperfusion.Here we highlight several aspects of the timing of intracoronary stem cell therapy.Our results showed that transplantation of bone marrow mesenchymal stem cells at 2 4 weeks after myocardial infarction is more favorable for reduction of the scar area,inhibition of left ventricular remodeling,and recovery of heart function.Coronary injection of autologous bone marrow mesenchymal stem cells at 2 4 weeks after acute myocardial infarction is safe and does not increase the incidence of complications.     

Repair mechanisms of bone marrow mesenchymal stem cells in myocardial infarction

The prognosis of patients with myocardial infarction (MI) and resultant chronic heart failure remains extremely poor despite advances in optimal medical therapy and interventional procedures. Animal experiments and clinical trials using adult stem cell therapy following MI have shown a global improvement of myocardial function. Bone marrow-derived mesenchymal stem cells (MSCs) hold promise for cardiac repair following MI, due to their multilineage, self-renewal and proliferation potential. In addition, MSCs can be easily isolated, expanded in culture, and have immunoprivileged properties to the host tissue. Experimental studies and clinical trials have revealed that MSCs not only differentiate into cardiomyocytes and vascular cells, but also secrete amounts of growth factors and cytokines which may mediate endogenous regeneration via activation of resident cardiac stem cells and other stem cells, as well as induce neovascularization, anti-inflammation, anti-apoptosis, anti-remodelling and cardiac contractility in a paracrine manner. It has also been postulated that the anti-arrhythmic and cardiac nerve sprouting potential of MSCs may contribute to their beneficial effects in cardiac repair. Most molecular and cellular mechanisms involved in the MSC-based therapy after MI are still unclear at present. This article reviews the potential repair mechanisms of MSCs in the setting of MI.     

Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells

Pulsed electromagnetic fields (PEMFs) have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. The aim of this study is to investigate the effect of PEMFs on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells (BMMSC). PEMF stimulus was administered to BMMSCs for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the PEMF‐exposed cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm², respectively. Although, based on the kinetic analysis, the growth rates of BMMSC during the exponential growth phase were not significantly affected, 20–60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. These results suggest that PEMF exposure could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase. In addition, according to the cytochemical and immunofluorescence analysis performed, the PEMF‐exposed BMMSC showed multi‐lineage differentiation potential similar to the control group. Bioelectromagnetics 30:251–260, 2009. © 2009 Wiley‐Liss, Inc.     

Sonic hedgehog enhances the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells

MSCs (mesenchymal stem cells) may be promising seed cells for tissue regeneration because of their self-renewal and multi-differentiation potential. Shh (sonic hedgehog) is involved in the skeletal formation during embryo development and skeletal regeneration. However, how Shh regulates the biological characteristics of BM-MSCs (bone marrow-derived MSCs) is poorly understood. We have investigated the effect of rShh-N (recombinant N-terminal Shh) on the proliferation and osteogenic differentiation of rBM-MSCs (rat BM-MSCs) in vitro. rBM-MSCs were treated with rShh-N at concentrations up to 200 ng/ml. Proliferation and colony-forming ability of rBM-MSCs were increased in a dose-dependent manner. rShh-N increased the ratio of cells in S and G2/M phase, as well as the number of Ki-67+ cells. In addition, ALP (alkaline phosphatase) activity and matrix mineralization were enhanced by 200 ng/ml rShh-N. Real-time PCR showed that rShh-N (200 ng/ml) up-regulated the expression of genes encoding Cbfa-1 (core-binding factor α1), osteocalcin, ALP and collagen type I in rBM-MSCs. This information reveals some potential of rShh-N in the therapeutics of bone-related diseases.