同源盒蛋白(Msx1)过度表达抑制成骨细胞碱性磷酸酶表达的实验研究

目的:观察同源盒蛋白(Msx1)过度表达在成骨细胞MC3T3-E1分化培养过程中对碱性磷酸酶的调节作用,以探讨Msx1蛋白对细胞成骨分化过程的调控机制。方法:将成骨细胞MC3T3-E1分为5组:未转染病毒组作为对照组1(A组);空白腺病毒载体组作为对照组2(B组);未分化组作为空白对照组(C组);分化前1 d转染携带同源盒基因Msx1的腺病毒载体组(D组);分化后1 d转染Msx1腺病毒载体组(E组)。在成骨分化培养基中培养4 d后,检测成骨细胞MC3T3-E1在成骨分化培养过程中碱性磷酸酶(alkaline phosphatase,ALP)的活性。结果:A、B两组成骨细胞MC3T3-E1在成骨分化培养基培养4 d后,ALP活性呈强阳性;C组中未分化成骨细胞MC3T3-E1无ALP活性,D、E两组的成骨细胞MC3T3-E1在成骨分化培养4 d后的ALP活性明显较A、B组减低,而且D、E两组间ALP活性无区别。结论:过度表达同源盒蛋白(Msx1)能抑制成骨细胞MC3T3-E1的ALP表达,在其成骨分化的过程中起一定的抑制作用。     

Rotary culture enhances pre-osteoblast aggregation and mineralization

Three-dimensional environments have been shown to enhance cell aggregation and osteoblast differentiation. Thus, we hypothesized that three-dimensional (3D) growth environments would enhance the mineralization rate of human embryonic palatal mesenchymal (HEPM) pre-osteoblasts. The objective of this study was to investigate the potential use of rotary cell culture systems (RCCS) as a means to enhance the osteogenic potential of pre-osteoblast cells. HEPM cells were cultured in a RCCS to create 3D enviroments. Tissue culture plastic (2D) cultures served as our control. 3D environments promoted three-dimensional aggregate formations. Increased calcium and phosphorus deposition was significantly enhanced three- to 18-fold (P < 0.001) in 3D cultures as compared with 2D environments. 3D cultures mineralized in 1 wk as compared with the 2D cultures, which took 4 wks, a decrease in time of nearly 75%. In conclusion, our studies demonstrated that 3D environments enhanced osteoblast cell aggregation and mineralization.     

IGF-1信号通路相关蛋白在大鼠髁突软骨细胞增殖分化中的表达

目的:研究胰岛素样生长因子-1(IGF-1)信号转导通路在大鼠髁突软骨细胞增殖分化调控过程中相关基因的表达。方法:体外单层培养并鉴定出生后1、7、14、28 d共4组SD大鼠髁突软骨细胞。免疫组织化学方法检测细胞内IGF-1表达情况,Real-time PCR及Western印迹法检测细胞内IGF-1R、Bcl-2、Bax mRNA及蛋白表达。饥饿培养24 h后,实验组加入质量浓度为100 ng/mL的重组大鼠IGF-1细胞因子(rrIGF-1)继续培养24、48 h,CCK-8检测细胞增殖情况,Real-time PCR技术检测各组髁突软骨细胞内IGF-1R、IGF-2R、Raf1、GSK-3、IGFBP3、NF-κB、Bcl-2、Bax、integrin、TGF-βmRNA表达情况;对照组培养液不加rrIGF-1。结果:各鼠龄组SD大鼠髁突软骨细胞内IGF-1表达均阳性,IGF-1R mRNA及蛋白表达相对平稳,Bcl-2、Bax mRNA及蛋白表达逐渐增强,在出生14 d后表达下降(P<0.05)。加入100 ng/mL rrIGF-1后,髁突软骨细胞增殖速度显著增加,细胞凋亡减少,Real-time PCR结果显示实验组IGF-1R、GSK-3、Bax、integrin mRNA表达整体呈下降趋势;IGF-2R、Raf1、IGFBP3、NF-κB、Bcl-2、TGF-βmRNA表达水平总体呈上调趋势,差异有统计学意义。结论 :IGF-1介导的信号转导途径参与了髁突软骨细胞的增殖、分化以及软骨形成早期的调控,并可能与integrin、TGF-β等其他蛋白相互作用,共同参与髁突发育过程。     

牵引力介导MC3T3-E1细胞microRNA的差异表达

目的 探讨周期性牵引力作用下小鼠成骨前体细胞microRNA的表达谱,寻找与TGF-β信号通路相关的差异microRNA。 方法 体外培养MC3T3-E1细胞。以正常培养细胞为对照,应力加载组细胞加载12%拉伸应变力3 h,利用microRNA基因芯片技术初步筛选出牵张应力作用下MC3T3-E1细胞差异表达的microRNA,然后采用实时荧光定量PCR技术对与TGF-β信号通路相关的差异表达microRNAs进行验证,生物信息学预测可能受其调控的靶基因。 结果 与对照组相比,应力加载组有41个microRNAs表达差异（P值均小于0.05且差异倍数均大于1.5倍）,其中20个表达上调,21个表达下调。实时荧光定量PCR结果显示：相比于对照组,应力加载组中miR-132-3p的表达水平升高,与芯片结果一致且存在统计学差异（P<0.05）。经软件分析,miR-132-3p的靶基因可能为Smad2。 结论 周期性牵引力作用下成骨细胞microRNA表达谱发生改变,这些差异表达的microRNAs可能通过影响TGF-β信号通路或其相关信号分子来影响成骨细胞的生物学功能。     

TiO_2纳米管对MC3T3-E1前成骨细胞骨功能基因表达变化的影响

目的：研究阳极氧化TiO2纳米管对MC3T3-E1前成骨细胞骨功能基因表达变化的影响。方法：采用阳极氧化法在钛基底表面制备TiO2纳米管,扫描电镜观察其表面微结构,肌动蛋白染色研究细胞在材料表面的粘附生长情况,Real-time PCR技术系统分析材料对细胞骨功能基因表达的影响规律。结果：MC3T3-E1前成骨细胞在TiO2纳米管表面比光滑钛有更大的伸展面积,培养1周后,TiO2纳米管提高了细胞骨功能基因碱性磷酸酶、骨桥蛋白和骨钙蛋白的表达水平（P〈0.05）,培养2周后,骨功能基因表达水平与对照组之间没统计学差异。结论：TiO2纳米管可以促进MC3T3-E1前成骨细胞肌动蛋白组装,同时可以提高细胞骨功能基因的早期表达水平。     

rhBMP-2和FGF-2对成骨细胞矿化及ENPP1、ANK、TNAP表达的影响

目的:研究重组人骨形态发生蛋白(rhBMP-2)和重组人碱性成纤维细胞生长因子(FGF-2)对成骨细胞(MC3T3-E1 Subclone 14)矿化及焦磷酸合成酶(ENPP1)、跨膜蛋白(ANK)和组织非特异性碱性磷酸酶(TNAP)表达的影响,探讨生长因子影响细胞矿化的机制。方法:将MC3T3-E1 Subclone 14细胞分成3组:成骨细胞诱导液(OS)成骨诱导培养组(对照组),OS与rhBMP-2培养组(rhBMP-2组),OS与FGF-2培养组(FGF-2组)。培养12 d后进行ALP活性检测及茜素红染色,实时荧光定量PCR检测矿化相关基因ENPP1、ANK和TNAP表达的差异。结果:rhBMP-2组ALP活性以及钙化结节明显高于对照组,ENPP1、ANK和TNAP均高表达;FGF-2组ALP活性以及钙化结节明显低于对照组,ENPP1和ANK呈高表达,TNAP低表达。结论:rhBMP-2和FGF-2通过调节ENPP1、ANK和TNAP的表达变化来影响骨的矿化。     

MicroRNA-103-3p对小鼠前成骨细胞MC3T3-E1成骨分化早期的影响

目的:探讨miR-103-3p对小鼠前成骨细胞MC3T3-E1成骨分化早期的影响。方法:以小鼠前成骨细胞MC3T3-E1为实验对象,对MC3T3-E1细胞进行成骨诱导,分别在0、3、5、7 d应用实时荧光定量PCR（real-time PCR）检测细胞中Runx2、Osx、ALP、miR-103-3p表达水平,Western免疫印迹（Western blotting）检测Runx2、Osx蛋白表达并进行碱性磷酸酶（ALP）染色。通过脂质体lipofectamine2000瞬时转染miR-103-3p模拟物 （miR-103-3p mimics）及模拟物阴性对照进入MC3T3-E1细胞内,Real-time PCR检测2组细胞miR-103-3p的表达水平,CCK-8试剂盒检测细胞增殖。分别对2组细胞进行成骨诱导,在成骨诱导后0、3、7 d,分别使用Real-time PCR和Western免疫印迹检测2组细胞Runx2、Osx等成骨相关基因mRNA和蛋白的表达变化,并对2组细胞进行ALP染色。实验数据采用SPSS19.0软件包进行统计学分析。结果:MC3T3-E1经成骨诱导0、3、5、7 d后,细胞内Runx2、Osx、ALP转录水平持续显著升高;Runx2、Osx蛋白表达升高。ALP染色逐渐加深。在成骨诱导3、5、7 d的MC3T3-E1细胞中,miR-103-3p水平较诱导前受到持续显著抑制(P<0.05)。瞬时转染miR-103-3p mimics后,MC3T3-E1细胞中的miR-103-3p表达水平较对照组显著上调(P<0.05),细胞增殖受到抑制,Runx2、ALP转录水平显著抑制（P<0.05）,Runx2蛋白表达显著抑制。对转染后的细胞进行成骨诱导3、7 d后,miR-103-3p转染组细胞Runx2、Osx、ALP在转录水平的表达较对照组显著降低,Runx2、Osx在蛋白水平的表达较对照组显著降低,且miR-103-3p转染组细胞ALP活性较对照组显著降低。结论:miR-103-3p可能对小鼠前成骨细胞MC3T3-E1的成骨分化早期起抑制作用。     

Spheroid formation and stemness preservation of human periodontal ligament cells on chitosan films

Objective

The therapeutic potential of periodontal ligament cells for periodontal regeneration gradually decreases when cultured as a monolayer in vitro. Three‐dimensional cell culture models provide an alternative to traditional monolayer cell culture. This study aimed to comparatively evaluate the influence of spheroid culture on periodontal ligament cells.

Materials and Methods

Chitosan films were used to culture three‐dimensional periodontal ligament cell spheroids. The proliferation, self‐renewal, and osteogenic capacity of periodontal ligament cells derived from spheroids were evaluated and compared with cells cultured on a monolayer.

Results

Viable spheroids of periodontal ligament cells were formed on chitosan films. Compared to monolayer cell culture, periodontal ligament cells exhibited decreased proliferation upon spheroid formation. In contrast, their expression of genes related to self‐renewal was significantly higher comparison with cells cultured in a monolayer. Moreover, the formation of periodontal ligament cell spheroids increased their colony‐forming unit ability and osteogenic differentiation capacity.

Conclusion

The results demonstrate the successful use of chitosan films for the culture of periodontal ligament cell spheroids. Compared to cells cultured in monolayer, periodontal ligament cells in spheroids did not proliferate, but exhibited higher self‐renewal gene expression, colony‐forming unit and osteogenic capacity.     

超短波对鼠骨髓间充质干细胞增殖及成骨能力的影响

目的:探讨超短波对鼠骨髓间充质干细胞(rat bone marrow mesenchymal stem cells,rBMSCs)增殖及成骨分化能力的影响。方法:体外分离培养rBMSCs,取第2~4代rBMSCs置于超短波理疗仪上进行处理,1次/d,15 min/次,于1、4、7 d检测细胞增殖能力,3 d后检测细胞碱性磷酸酶(alkaline phosphatase,ALP)分泌量,7 d后检测成骨基因Runx2表达量。结果:每日给予超短波处理的实验组细胞经连续培养7 d后,与不经超短波处理的对照组相比,其增殖能力明显提升(P＜0.05),Runx2表达明显上调(P＜0.05),ALP分泌量增多; 结论:超短波能促进SD大鼠rBMSCs增殖和成骨分化。     

人脐带Wharton's Jelly来源间充质干细胞与人牙周膜干细胞成骨分化能力的对比研究

目的:比较人脐带Wharton's Jelly来源间充质干细胞(human umbilical cord Wharton's Jelly-derived mesechymal stem ceils,hUCWJMSCs)与人牙周膜干细胞(periodontal mesenchymal stem cells,hPDLSCs)成骨分化能力.方法:体外培养hUC-WJMSCs和hPDLSCs.MTT法检测细胞增殖情况;成骨诱导后测定细胞的ALP活性,茜素红染色检测细胞矿化能力,Real-timePCR分析OPN和Runx2基因的表达.结果:hUCWJMSCs增殖能力高于hPDLSCs;经矿化诱导后hPDLSCs ALP表达、矿化结节形成高于hUCWJMSCs(P＜0.05);Runx2在hPDLSCs中表达高于hUCWJMSCs(P ＜0.05);而hUCWJMSCs中OPN表达高于hPDLSCs(P＜0.05).结论:hUCWJMSCs、hPDLSCs均具有成骨分化能力,hPDLSCs成骨分化能力较强.     

A comprehensive analysis of human dental pulp cell spheroids in a three-dimensional pellet culture system

ObjectiveThree-dimensional (3D) cell culture methods are of high importance to studies of biological processes. This is particularly the case with spheroid cultures, which create 3D cell aggregates without the use of exogenous materials. Compared to conventional monolayer cultures, cellular spheroid cultures have been demonstrated to improve multilineage potential and extracellular matrix production. To address this issue in depth, we present a more comprehensive analysis of 3D human dental pulp cell (hDPC) spheroids.DesignhDPC spheroids were fabricated by the pellet culture method and were cultured without adding any reagent to induce differentiation. The gene-expression profiles of the 3D and two-dimensional (2D) cultured hDPCs were compared by complementary DNA microarray analysis. Odontoblastic and osteoblastic differentiation marker gene expression was evaluated by quantitative real-time PCR (RT-qPCR). Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) were applied to examine the morphology of hDPC spheroids and extracellular matrix components.ResultsCompared with 2D monolayer culture, microarray analysis identified 405 genes and 279 genes with twofold or greater differential expression after 3 days and 28 days of 3D culture, respectively. In 3D hDPC spheroids, gene ontology analysis revealed upregulation of extracellular matrix-related genes and downregulation of cell growth-related genes. RT-qPCR analysis showed higher expression levels of osteocalcin, dentin sialophosphoprotein, and alkaline phosphatase. TEM revealed the morphological characteristics of the fibrillar collagen-rich matrix and cell-cell interactions.ConclusionsThe present findings provide clues to understanding the mechanisms of pellet-cultured hDPCs and contribute to future research in the comparative studies of different 3D culture methods.     

Patterns of integrin expression in a human mandibular explant model of osteoblast differentiation

Cell-matrix interaction is crucial in regulating osteoblast differentiation and function. These interactions are themselves regulated, at least in part, by integrins. Although there are some data from mammalian models, few studies have compared integrin expression at different stages of the osteoblast lineage. Here, primary human mandibular osteoblast cultures were grown in the presence of epidermal growth factor (EGF), giving a proliferative, less differentiated phenotype, or of vitamin D(3) and hydrocortisone (D+Hc), giving a more differentiated phenotype. These cultures were compared with those of cells prepared in the absence of EGF or D+Hc by fluorescence-activated cell sorter using a panel of monoclonal antibodies to specific integrin heterodimers. To provide in vivo correlation, the same panel of antibodies was used to stain fresh-frozen, undemineralised sections of human mandibular bone. Under baseline conditions the alpha(3), alpha(5), alpha(v), alpha(v)beta(3), beta(3) and beta(1) integrin subunits were expressed strongly by the cells, with low-level expression of the alpha(1), alpha(2) and alpha(4) subunits. In the presence of EGF there was increased alpha(2) expression. With D+Hc, alpha(3) and alpha(5) expression was elevated. Immunohistochemical analysis demonstrated alpha(2), alpha(3), alpha(5), alpha(v)beta(3), beta(1) and beta(3) subunits in cells of the osteoblast lineage; alpha(2) staining was restricted to cells close to the bone surface whilst alpha(v)beta(3) and beta(3) were most frequently localised in the osteocytes. The results provide evidence that cells at successive stages of the osteoblast lineage show different patterns of integrin expression. These integrins may be important in cell-matrix interactions leading to osteoblast differentiation.     

Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells

Objective

Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts.

Methods

We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically.

Results

Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (p < 0.05). Histologically, 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. Proliferating cells with high ALP activity and DMP1 expression were found mainly in the peripheral zone that also showed strong expression of vinculin and integrin signalling-related proteins. In contrast, apoptotic and hypoxic cells were detected in the core zone.

Conclusion

3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling.     

神经生长因子对脂多糖诱导的成骨细胞炎症反应的影响

目的研究外源性神经生长因子（NGF）对脂多糖（LPS）诱导的成骨细胞炎症相关基因表达的影响。方法原代培养新生大鼠颅盖骨成骨细胞,Griess试剂法测定培养基上清中NO含量：MTT法检测细胞活力;Real-time定量PCR检测炎症相关基因TNF-α、COX-2和NF-κB mRNA表达。结果 NGF高中剂量（100ng/ml、10ng/ml）可显著抑制LPS诱导的成骨细胞NO的释放,该浓度下细胞活力不受影响;NGF可显著抑制TNF-α、COX-2和NF-κB基因表达。结论 NGF通过减少NO释放,抑制炎症相关基因表达发挥抗炎作用。     

小鼠骨髓间充质干细胞成骨分化过程中Caveolin-1的表达

目的:探讨小鼠骨髓间充质干细胞( bone marrow mesenchymal stem cells,BMSCs)向成骨细胞分化过程中小窝蛋白-1(Caveolin-1)的表达变化。方法:体外培养BMSCs细胞株,分为诱导成骨组和未诱导组,在培养的不同时期(3 d、7 d、10 d、14 d),采用碱性磷酸酶(AKP)活性测定、茜素红染色对BMSCs成骨分化进行鉴定,实时聚合酶链反应(real-time polymerase chain reaction RT-PCR)、蛋白质印迹法(Western Blot)检测各组细胞Caveolin-1 mRNA、蛋白的表达情况。结果:RT-PCR结果显示同一时间点诱导组Caveolin-1 mRNA表达量高于未诱导组,诱导组Caveolin-1 mRNA表达以时间依赖性的方式不断增高;Western Blot结果显示同一时间点诱导组Caveolin-1蛋白水平高于未诱导组,诱导组Caveolin-1蛋白水平以时间依赖性的方式增高。结论:本实验从mRNA、蛋白水平证实BMSCs表达Caveolin-1的量随着成骨分化的进行不断增加。Caveolin-1可能参与调控BMSCs成骨分化过程。     

miR-199a调控IGF1表达对机械刺激下成骨细胞分化的影响

目的： 探讨微小RNA（miR）-199a在机械牵张力刺激下MC3T3-E1细胞中的表达变化及其对牵张力刺激MC3T3-E1细胞成骨分化的作用机制。方法： 对体外培养的MC3T3-E1细胞加载12%牵张力0、3、6、12和24 h后,利用碱性磷酸酶（ALP）活性检测试剂盒检测ALP活性,实时荧光定量PCR检测骨钙素（OCN）、成骨细胞特异性转录因子Osterix（OSX）、Runt相关转录因子2（Runx2） mRNA和miR-199a的表达。将MC3T3-E1细胞分为对照组、牵张力组、牵张力+miR-NC组和牵张力+miR-199a组,加载12%牵张力和转染miR-199a模拟物后,观察miR-199a和OCN、OSX、Runx2 mRNA及蛋白表达以及ALP活性。茜素红S（ARS）染色观察钙结节形成能力。采用双荧光素酶报告基因实验检测miR-199a与胰岛素样生长因子1（IGF1）的靶向关系,实时荧光定量PCR法和免疫印迹法检测miR-199a模拟物对IGF1 mRNA和蛋白表达的影响。采用SPSS 24.0软件包对数据进行统计学分析。结果： 与0 h时间点相比,以机械牵张力刺激3、6、12和24 h后,MC3T3-E1细胞ALP活性和OCN、OSX、Runx2 mRNA表达水平均显著升高,而miR-199a表达水平显著降低（P<0.05）,12 h时变化最为显著。与对照组相比,牵张力组细胞中miR-199a表达水平显著降低,而细胞ALP活性、OCN、OSX、Runx2 mRNA及蛋白表达水平、钙结节形成水平均显著升高（P<0.05）;与牵张力组相比,牵张力+miR-NC组细胞中上述各指标差异均无统计学意义（P>0.05）;与牵张力+miR-NC组相比,牵张力+miR-199a组细胞中miR-199a表达水平显著升高,而细胞ALP活性、OCN、OSX、Runx2 mRNA及蛋白表达水平、钙结节形成水平均显著降低（P<0.05）。miR-199a可与IGF1靶向结合,miR-199a模拟物可使MC3T3-E1细胞中IGF1 mRNA和蛋白表达水平显著降低（P<0.05）。结论： miR-199a可抑制机械牵张力刺激诱导的MC3T3-E1细胞成骨分化,其作用机制可能与靶向调控IGF1表达有关。