miR-889-3p靶向Runx2抑制脂肪间充质干细胞的成骨分化

背景：研究表明miR-889-3p可参与调控多种细胞的增殖分化过程,但是其对脂肪间充质干细胞成骨分化的影响尚不明确。目的：探讨miR-889-3p对脂肪间充质干细胞成骨分化的调节作用。方法：体外分离、培养SD大鼠脂肪间充质干细胞,转染miR-889-3p模拟物(miR-889-3p mimic)、miR-889-3p抑制剂(miR-889-3p inhibitor)和阴性对照(miR-NC)并诱导成骨分化。RT-PCR和Western blot检测成骨诱导14 d后的碱性磷酸酶活性、Runx2、骨钙素、骨桥素、Osterix等成骨相关mRNA和蛋白的表达。将野生型Runx2、突变型Runx2分别与miR-889-3p模拟物和miR-NC共转染脂肪间充质干细胞,通过双荧光素酶报告基因检测miR-889-3p与Runx2的靶向关系。结果与结论：(1)miR-889-3p表达水平随成骨诱导时间延长而逐渐减低;(2)成骨诱导第7天和第14天miR-889-3p inhibitor组的矿化结节数目、大小、颜色深浅明显超过miR-889-3p mimic组和miR-NC组(P <0.05);...     

miR-146a调控骨髓间充质干细胞成骨分化的机制研究

目的探讨miR-146a调控骨髓间充质干细胞(BMSC)成骨分化的作用及其分子机制。方法贴壁法分离培养小鼠BMSC,检测成骨分化早期标志物Runx 2的变化,观察BMSC体外成骨分化,利用miRNA特异性的聚合酶链式反应(miRNAspecific qPCR)观察miR-146a的变化情况,并干预miR-146a表达,明确miR-146a对BMSC成骨分化的调控作用。结果成功建立了稳定的BMSC体外培养体系,该细胞能够成功分化为脂肪细胞和成骨细胞;在成骨诱导培养条件下,随着成骨分化,miR-146a水平降低,过表达miR-146a,成骨分化早期标志分子Runx 2表达降低;转染miR-146a拮抗体antago-miR-146a可以补救Runx 2表达的降低。结论 miR-146a负向调控BMSC成骨分化,拮抗miR-146a可以补救BMSC成骨分化的降低。     

微重力对骨髓间充质干细胞成骨分化的影响

骨髓间充质干细胞（BMSCs）是一种多能成体于细胞,是组织工程重要的种子细胞来源之一.微重力对BMSCs成骨分化具有抑制作用,可使骨量减少和骨微结构改变,从而导致骨质疏松症.这一过程受到多条信号通路的调控,如MAPK信号通路、Notch信号通路和Wnt/β-catenin信号通路等,它们协同调节微重力下BMSCs向成骨细胞方向的分化.研究微重力对BMSCs成骨分化的影响,可以阐明骨质流失机理,为相关疾病的治疗提供新的靶点,促进我国太空宇航事业的发展.     

miR-195对人骨髓间充质干细胞成骨分化的影响

BACKGROUND:Previous studies have found that the expression level of miR-195 in differentiated human bone marrow mesenchymal stem cells (hBMMSCs) is significantly higher than that in undifferentiated hBMMSCs. However, miR-195 effect during this differentiation process and possible mechanism remain unclear. OBJECTIVE:To explore the effect of miR-195 on osteogenic differentiation of hBMMSCs and possible mechanism. METHODS:hBMMSCs were isolated and cultured in vitro. Alkaline phosphatase activity, Runx2, osteopontin and SMAD7 protein expression and miR-195 expression level during osteogenic differentiation of hBMMSCs were determined by alkaline phosphatase kit, western blot and real-time PCR, respectively. miR-195-downexpressed hBMMSCs were constructed by lipofection transfection, and were used to investigate the effect of miR-195 was on osteogenic differentiation of hBMMSCs. Dual luciferase reporter assay was used to identify whether the 3’UTR of SMAD7 mRNA was a binding target of miR-195. In addition, we transfected hBMMSCs with SMAD7 cDNA (pcDNA-SMAD7), and investigated the effect of SMAD7 on osteogenic differentiation of hBMMSCs. RESULTS AND CONCLUSION:The isolated and cultured hBMMSCs had good osteogenic differentiation ability in vitro. Expression level of miR-195 was increased with the increasing of induction time, and the expression level of SMAD7 was reversed. miR-195 promoted osteogenic differentiation of hBMMSCs. Luciferase assay confirmed that miR-195 targeted SMAD7 directly, and overexpression of SMAD7 inhibited the osteogenic differentiation of hBMMSCs. Taken together, miR-195 promotes osteogenic differentiation of hBMMSCs by targeting SMAD7.     

骨髓间充质干细胞成骨分化调控的研究进展

骨髓间充质干细胞具有较强的自我更新能力和多向分化潜能，其应用是目前国际上组织工程领域中重要的研究内容之一。近年来，许多实验室从分子，生化，物理等水平对其成骨分化调控进行了深入研究，取得了较大的进展。     

骨髓间充质干细胞成骨分化调控的研究进展

骨髓间充质干细胞具有较强的自我更新能力和多向分化潜能 ,其应用是目前国际上组织工程领域中重要的研究内容之一。近年来 ,许多实验室从分子、生化、物理等水平对其成骨分化调控进行了深入研究 ,取得了较大的进展     

骨髓间充质干细胞诱导成骨的研究进展

骨髓间充质干细胞因其具有自我复制和多向分化潜能而成为目前骨组织工程领域研究的重点之一。对这方面近几年的研究成果从诱导因子的作用及调控机制、物理条件、新的研究方法等方面做了简要论述。     

补骨脂素对大鼠骨髓间充质干细胞成骨分化的影响

目的研究不同浓度的补骨脂素对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)成骨分化的影响,为临床应用补骨脂素治疗骨质疏松症提供理论依据。方法选取8周龄健康雄性SD大鼠,取双侧股骨、胫骨,对大鼠的BMMSCs进行分离、原代培养和细胞表型鉴定。取第3代大鼠BMMSCs,体外利用成骨诱导培养基进行不同浓度补骨脂素的药物诱导,MTT法检测不同浓度的补骨脂素对大鼠BMMSCs生长的影响;通过碱性磷酸酶(ALP)活性测定、Western blot法和茜素红染色方法评价不同浓度补骨脂素对大鼠BMMSCs成骨分化能力。结果第3代大鼠BMMSCs表面抗原符合干细胞鉴定标准,成骨诱导后给予15μmol/L补骨脂素对大鼠BMMSCs的增殖作用最佳,ALP活性、Runx-2表达和钙化结节数目均明显高于经典成骨诱导组、5μmol/L和10μmol/L和20μmol/L补骨脂素组。结论15μmol/L补骨脂素成骨诱导促进BMMSCs向成骨分化,从而起到防治骨质疏松的作用。     

miR-155有助于骨髓间充质干细胞成软骨分化

背景：近年发现,miRNA是能够对基因表达产生影响的一种新型调控子,miRNA有助于多能干细胞分化增殖以及自我更新。 目的：探讨miR-155对大鼠骨髓间充质干细胞成软骨分化的调控机制。 方法：12周龄健康SD大鼠60只,随机分为研究组与对照组,每组30只。于麻醉状态下将大鼠处死,获取下肢骨髓,进行骨髓间充质干细胞分离、培养,研究组给予miR-155基因模拟物转染,对照组给予阴性对照序列转染,经成软骨诱导分化后进行RT-PCR检测Sox9、CollagenⅡ、Aggrecan、Collagen X基因的表达,Western blot检测Sox9、Runx2蛋白的表达。 结果与结论：研究组Sox9、CollagenⅡ、Aggrecan基因表达高于对照组,Collagen X基因表达低于对照组,差异均有显著性意义(P < 0.05)。研究组Sox9蛋白表达高于对照组,Runx2蛋白表达低于对照组,差异均有显著性意义(P < 0.05)。结果表明miR-155不仅有助于骨髓间充质干细胞成软骨分化,而且可以抑制骨髓间充质干细胞成软骨分化呈肥大趋势发展。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

力学因素对骨髓间充质干细胞成骨分化的影响

近年来,组织工程学领域发展突飞猛进,已被列为一种修复或再生许多组织和器官的方法。骨髓间充质干细胞具有良好的成骨向分化潜能,在骨组织工程领域中具有广阔的应用前景。骨髓间充质干细胞增殖和成骨向分化受多种力学因素的影响,且不同性质的力学刺激对其定向分化的调节作用不尽相同。目前,许多学者致力于深入探讨力学因素影响骨髓间充质干细胞成骨向分化的具体途径,但其调控机制尚未完全明确。本文综述及讨论力学因素对骨髓间充质干细胞所产生的增殖、定向成骨分化等生物学效应的影响及可能涉及的力化学信号转导通路作用机制,以期丰富研究思路     

骨髓间充质干细胞成脂成骨平衡调控研究进展

骨质疏松症（OP）是一种全身性的骨代谢疾病,主要表现为骨量减少、骨质脆性增加、骨骼微结构退化。随着对骨质疏松症发病机制的深入研究,骨髓间充质干细胞（BMSCs）分化失衡（成骨分化减少,成脂分化增多）是引发骨质疏松症的关键。我们总结了BMSCs成脂成骨分化相关的信号通路,充分理解这些信号通路,有利于更好的阐明骨质疏松症的发病机制,为临床医生有效地治疗骨质疏松症提供帮助。     

柚皮苷在促大鼠骨髓间充质干细胞骨向分化过程中对MAPK信号通路的影响

目的: 研究中药单体柚皮苷(NG)对体外培养的大鼠骨髓间充质干细胞(MSCs)向成骨细胞分化过程中MAPK信号通路的影响。方法: 观察在正常、加入p38、ERK和JNK通路抑制剂SB203580、PD98059、SP600125及3种抑制剂全部加入的情况下,各组碱性磷酸酶(ALP)、骨钙素(BGP)、I型胶原(Col I)等骨向分化指标的差异。用Western blotting技术检测各组p38、ERK1/2和JNK蛋白的磷酸化水平,用荧光定量PCR技术检测细胞因子转化生长因子β₁(TGF-β₁)、骨形成蛋白2(BMP-2)和核心结合因子α1(Cbfα1) mRNA的表达。结果: (1)10^-7mol/L为本实验中NG的最佳促骨向分化浓度。(2) NG最佳浓度组的ALP和BGP含量比其它各组都高(P<0.05),Col I含量无明显差异(P>0.05);与NG组相比,加入不同抑制剂组的ALP、BGP和ColⅠ表达量出现不同程度的降低。(3)与空白组相比,NG组JNK蛋白的磷酸化水平升高(P<0.05),p38蛋白的磷酸化水平降低(P<0.01),ERK1/2蛋白的磷酸化水平无明显差异(P>0.05)。与NG组相比,加入不同抑制剂组的p38、ERK1/2和JNK蛋白的磷酸化水平有升高也有降低。(4) NG组上调BMP-2的表达(P<0.05),下调Cbfα1的表达(P<0.05),而对TGF-β₁的表达无明显影响(P>0.05)。与NG组相比,加入不同抑制剂组的TGF-β₁、BMP-2和Cbfα1 mRNA表达量出现不同程度的降低。结论: NG主要通过激活MAPK信号通路中ERK通路、JNK通路以及上调BMP-2的表达,促进MSCs的骨向分化。NG上调BMP-2的表达受MAPK通路中p38通路的影响较大。     

α2-巨球蛋白减轻X射线所致人骨髓间充质干细胞成骨分化障碍

目的:初步探讨α2-巨球蛋白(α2M)对X射线导致的人骨髓间充质干细胞(h BMMSCs)成骨分化障碍的作用。方法:体外培养h BMMSCs,取第4代细胞经8 Gy X射线照射后,进行成骨诱导并以0.5和1.0 g/Lα2M分别作用于照射后的h BMMSCs。成骨诱导的第7天检测碱性磷酸酶(ALP)活性,RT-q PCR法检测Runt相关转录因子2(RUNX2)的mRNA表达;成骨诱导的第14天采用Western blot检测骨甘氨酸(OGN)的蛋白表达;成骨诱导的第21天采用茜素红染色法检测钙结节的形成情况。此外,正常培养的h BMMSCs于8 Gy X射线照射后加入α2M作用24 h,提取细胞检测超氧化物歧化酶(SOD)活力,Western blot检测含锰超氧化物歧化酶(Mn SOD)的蛋白表达。结果:h BMMSCs经8 Gy X射线照射后给予0.5和1.0 g/Lα2M处理,与未加入α2M的单独照射组比较,ALP活性、RUNX2的mRNA表达、OGN和Mn SOD的蛋白表达及SOD活力均升高,钙结节形成增多。结论:α2M可明显提高放射损伤后h BMMSCs的成骨分化能力,上调SOD活力和Mn SOD的蛋白表达水平。     

Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation

Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells were isolated for obtaining mesenchymal cells that had been expanded until osteogenic differentiation was induced. RNA was harvested at seven time points during the osteogenic differentiation period (D0, D+1, D+2, D+7, D+12, D+17 and D+21). Gene expression analysis was performed by the microarray technique and identified several differentially expressed genes. Some important genes for osteoblast differentiation had lower expression in OI patients, suggesting a smaller commitment of these patient's MSCs with the osteogenic lineage. Other genes also had their differential expression confirmed by RT-qPCR. An increase in the expression of genes related to adipocytes was observed, suggesting an increase of adipogenic differentiation at the expense osteogenic differentiation.     

microRNA-25-3p suppresses osteogenic differentiation of BMSCs in patients with osteoporosis by targeting ITGB3

This study was conducted to investigate the impact of the microRNA (miR)-25–3p/ITGB3 axis on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) from patients with osteoporosis (OP). BMSCs isolated from the bone marrow of healthy controls and OP patients were identified by flow cytometry, in which ITGB3 mRNA and miR-25–3p expression was detected by RT-qPCR and ITGB3, Runx2, OPN, ALP, and OSX protein expression by western blot. The binding between ITGB3 and miR-25–3p was assessed by dual-luciferase reporter gene and Ago2-RIP assays. BMSC osteogenic differentiation was observed by alizarin red staining and ALP activity. The differentiation of BMSCs to adipocytes and chondrocytes was measured by oil red O staining and alcian blue staining, respectively. BMSCs were successfully isolated from the bone marrow of healthy controls (normal-BMSCs) and OP patients (OP-BMSCs). ITGB3, Runx2, OPN, ALP, and OSX expression was poorer and miR-25–3p expression was higher in OP-BMSCs than in normal-BMSCs. Mechanistically, ITGB3 was negatively targeted by miR-25–3p. After osteogenic, adipogenic, and chondrogenic differentiation of BMSCs were successfully induced, adipogenic differentiation increased and osteogenic and chondrogenic differentiation decreased in OP-BMSCs compared with normal-BMSCs. Overexpression of ITGB3 facilitated mineralized nodule formation and elevated ALP activity and Runx2, OPN, and ALP expression in OP-BMSCs. miR-25–3p upregulation diminished mineralized nodule formation, ALP activity, and Runx2, OPN, and ALP expression in OP-BMSCs and normal-BMSCs, which was annulled by additional ITGB3 overexpression. miR-25–3p targets ITGB3, thereby suppressing osteogenic differentiation of BMSCs from OP patients.     

基底硬度与形貌协同对大鼠骨髓间充质干细胞成骨分化的影响

目的探讨基底硬度与形貌协同对大鼠骨髓间充质干细胞(rat bone mesenchymal stem cells,r BMSCs)形态、增殖以及成骨分化的影响。方法分别在硬度为3.5 MPa、槽、脊宽为0.3μm的聚二甲基硅氧烷(polydimethylsiloxane,PDMS)基底;硬度为3.5 MPa、槽、脊宽为1.8μm的PDMS基底;硬度为3.5 MPa的平面PDMS基底;硬度为0.27 MPa、槽、脊宽为0.3μm的PDMS基底;硬度为0.27 MPa、槽、脊宽为1.8μm的PDMS基底;硬度为0.27 MPa的平面PDMS基底上培养r BMSCs,利用倒置荧光显微镜观察r BMSCs的形态,CCK-8试剂盒检测r BMSCs的增殖情况,碱性磷酸酶(alkalinephosphatase,ALP)试剂盒检测r BMSCs的ALP活性,免疫荧光技术检测骨钙蛋白(osteocalcin,OCN)及I型胶原(collagen type I,COL I)的表达,qRT-PCR检测Runx2 mRNA的表达。结果在硬度为3.5 MPa以及槽、脊宽为0.3μm的PDMS上r BMSCs铺展更好、增殖更快,ALP活性更高,OCN、COL I及Runx2mRNA表达量明显多于其他各组。结论基底硬度对r BMSCs的增殖有明显影响,而硬度与形貌能协同促进r BMSCs的增殖及成骨分化。研究结果有助于了解生物物理因素在某些疾病(如骨质疏松)发病机制过程中的作用,并可为骨组织工程新材料的研发提供理论基础。     

Wnt7a在BMSCs成骨分化中的作用和机制

目的 探索Wnt7a在骨髓间充质干细胞(BMSCs)成骨分化中的作用和机制.方法 分离和培养人BMSCs,使用lipo2000在BMSCs中转染干扰Wnt7a和mock寡核苷酸序列,3d后通过Westen Blot 分析寡核苷酸干扰序列siRNA-Wnt7a的干扰效果.诱导培养10d后通过Westen Blot分析BMSCs细胞沉默Wnt7a对骨标志蛋白osteocalcin和骨调控蛋白Runx2表达的影响.诱导培养2周后通过Alizarin S Red染色检测在BMSCs中沉默Wnt7a对骨诱导的影响.结果 在BMSCs中转染寡核苷酸干扰序列Wnt7a和mock 对照序列,3d后Western Blot结果显示寡核苷酸干扰序列Wnt7a可明显降低BMSCs中Wnt7a的表达.诱导培养10d后通过Western Blot试验发现沉默Wnt7a后骨分化标志蛋白osteocalcin表达降低;同时检测到诱导培养10d后,BMSCs沉默Wnt7a组Runx2表达降低.诱导培养2周后通过Alizarin S Red染色显示骨形成受阻.结论 Wnt7a在BMSCs成骨分化中通过激活骨调控蛋白Runx2促进骨分化标志蛋白osteocalcin,在BMSCs成骨形成中扮演着重要角色.