MIP-3α 联合成骨诱导因子诱导大鼠脂肪干细胞向成牙本质样细胞分化*

目的:观察巨噬细胞炎性蛋白-3α(MIP-3α)对大鼠脂肪干细胞(Adipose derived stem cells,ASCs)向成牙本质样细胞体外分化作用的影响。方法:分离、培养并鉴定大鼠ASCs;以MIP-3α联合成骨诱导因子(地塞米松,β-甘油磷酸钠,以及抗坏血酸)诱导第3代大鼠ASCs向成牙本质样细胞定向分化。诱导培养1、4、7d后,分别测定碱性磷酸酶(alkaline phosphatese,ALP)活性,并用RT-PCR及Western Blot检测成牙本质细胞的标志基因dspp及标志物牙本质涎蛋白(DSP)。结果:与单独加入成骨诱导因子相比,MIP-3α与成骨诱导因子联合应用能使ALP活性、dspp的mRNA表达以及DSP升高。结论:本研究显示MIP-3α与成骨诱导因子联合应用可以增强成牙本质细胞相关基因以及蛋白的表达,为牙齿再生种子细胞的寻找开辟了一条新思路。     

BMP9经Smad信号通路调控iSCAP成骨/成牙本质分化

目的：研究BMP9是否能够激活 iSCAP细胞中的Smad信号通路,以及Smad信号通路在BMP9诱导iSCAP细胞成骨/成牙本质向分化过程中的作用。方法：首先,采用Western印迹实验检测Ad-BMP9转染iSCAP后Smad1/5/8蛋白的磷酸化水平。随后,利用dnALK1重组腺病毒和BMP9条件培养基作用于iSCAP,Western印迹实验检测Smad1/5/8蛋白磷酸化水平;采用碱性磷酸酶（ALP）活性检测和染色方法分析早期成骨/成牙本质指标变化,茜素红染色法检测钙盐沉积程度;RT-PCR成骨/成牙本质相关基因Runx2、OCN、OPN和DMP1表达的影响。结果：BMP9可上调iSCAP中Smad1/5/8的磷酸化水平;dnALK1抑制BMP9条件培养基作用后,可抑制Smad1/5/8的磷酸化,iSCAP细胞中早期成骨/成牙本质标志物ALP活性和晚期成骨/成牙本质标志钙盐结节减少,重要成骨转录因子Runx2基因表达减少,成骨/成牙本质相关基因OCN、OPN、DMP1的表达也受到了抑制。结论：Smad信号通路在BMP9诱导iSCAP成骨/成牙本质过程中存在并起着重要作用。     

尿酸影响人骨髓间充质干细胞成骨分化过程中BMP-2 表达

目的:探讨在人骨髓间充质干细胞(h BMSCs)成骨分化过程中,不同浓度尿酸(UA)对骨形态形成蛋白-2(BMP-2)表达的影响。方法:以全骨髓贴壁培养法分离h BMSCs,将生长状态良好的第3代h BMSCs分为5组,分别为空白对照组(加入完全培养基)和成骨诱导组(加入成骨诱导液及含0 mmol/L、0.2 mmol/L、0.4 mmol/L、0.8 mmol/L尿酸的完全培养基)。连续干预诱导14d后,用倒置显微镜观察细胞形态的变化,通过观察茜素红染色情况及检测碱性磷酸酶(ALP)活性进行成骨情况的检测。RT-PCR技术检测各组细胞BMP-2 mR NA的表达情况。结果:第3代h BMSCs大多为形态单一的长梭形,呈旋涡状生长;干预诱导后的细胞逐渐变成不规则的立方形,局部形成团块状结节,以含尿酸浓度为0.8 mmol/L的成骨诱导培养基最为显著。连续干预14d后,空白对照组茜素红染色为阴性,而各成骨诱导组细胞茜素红染色结果为阳性,提示干预诱导后的细胞为成骨细胞。碱性磷酸酶活性随尿酸浓度的增加和干预时间的延长而增强(P<0.05)。RT-PCR检测结果显示,空白对照组无BMP-2 mR NA的表达。成骨诱导组随培养基中尿酸浓度的增加,BMP-2 mR NA表达逐渐增强,呈浓度依赖性(P<0.05)。结论:尿酸上调h BMSCs向成骨细胞分化过程中BMP-2 mR NA的表达。     

INH-α对BMP9诱导间充质干细胞成骨分化的作用

为了研究抑制素α亚基(inhibinα-subunit INH-α)对骨形态发生蛋白9(bone morphogenetic protein9,BMP9)诱导的间充质干细胞(mesenchymal stem cells,MSCs)成骨分化的影响,本研究采用细胞化学染色法检测第3天、第5天、第7天细胞中碱性磷酸酶(alkaline phosphatase,ALP)活性的变化。利用RT-PCR和Western blotting检测细胞中的成骨分化早期标志物(Runx2)和晚期标志物(OPN)的mRNA含量及蛋白表达水平。茜素红S染色法检测第21天细胞中的钙盐沉积变化。发现BMP9组ALP活性明显增高,INH-α组ALP活性与对照组相比无明显变化,但联合运用BMP9和INH-α组ALP活性较BMP9组明显降低。此外,BMP9组Runx2和OPN的mRNA含量和蛋白表达水平明显增高,而联用BMP9和INH-α组中的Runx2和OPN水平较BMP9组显著下降(p<0.01)。同样,在茜素红S染色实验中,BMP9组钙盐结节明显增多,染色深;而在联合运用BMP9和INH-α组钙盐结节较BMP9组明显减少,染色变浅。说明INH-α能够抑制BMP9诱导间充质干细胞成骨分化作用。     

过表达Atg5通过上调细胞自噬水平抑制瓣膜间质细胞成骨样分化

该文的主要目的是探究过表达自噬相关蛋白Atg5对主动脉瓣膜间质细胞自噬水平以及成骨样分化能力的影响,为钙化性主动脉瓣膜疾病的研究提供新方法和新思路。从猪主动脉瓣上分离原代瓣膜间质细胞,细胞免疫荧光进行表型鉴定后,采用成骨培养基诱导瓣膜间质细胞成骨分化,并用Western blot检测细胞成骨指标Runx2、OPN与自噬指标p62和LC3B-II/I的蛋白表达水平;构建重组质粒pAdTrack-ATG5并通过脂质体转染瓣膜间质细胞,采用Q-PCR、Western blot以及免疫荧光染色检测转染后细胞Atg5的表达水平和细胞自噬水平的变化情况;对成骨诱导培养的主动脉瓣膜间质细胞分别转染空载质粒和过表达Atg5质粒,利用Western blot检测72 h后瓣膜间质细胞早期成骨指标Runx2、OPN的表达,并用碱性磷酸酶染色和茜素红S染色方法检测瓣膜间质细胞晚期成骨样分化能力。结果显示,原代分离培养的瓣膜间质细胞的间质细胞标志物α-SMA和vimentin的染色结果呈阳性,内皮细胞标志物vWF的染色结果呈阴性;与对照组比较,用成骨培养基培养的瓣膜间质细胞Runx2、OPN和p62的蛋白表达水平显著上调,LC3B-II/I的比值显著下调;测序结果显示,过表达质粒pAdTrack-ATG5构建成功;在转染pAdTrack-ATG5后,细胞Atg5的基因表达水平和蛋白表达水平均显著上调,自噬指标p62蛋白表达水平显著下调,LC3B-II/I比值显著上调,免疫荧光染色显示,LC3B聚集增加;与转染空载质粒组相比,转染pAdTrack-ATG5组细胞在成骨培养基培养72 h后成骨指标Runx2、OPN的蛋白表达水平显著下降,转染pAdTrack-ATG5组碱性磷酸酶染色和茜素红S染色结果的阳性程度均弱于转染空载质粒组。综上所述,过表达Atg5能上调细胞自噬水平,并抑制瓣膜间质细胞成骨样分化。     

AGEs和TNF-α对人牙周膜干细胞骨向分化能力的协同抑制作用

本实验旨在研究糖基化终末产物(AGE-BSA)和TNF-α对人牙周膜干细胞增殖及骨向分化能力的影响。本实验通过体外组织块酶消化法和有限稀释法克隆化培养牙周膜干细胞,使用流式细胞仪检测细胞表型分子stro-1、CD146、CD44、CD90的表达而对其进行干细胞鉴定后,取第3代人牙周膜干细胞在100μg/mL AGE-BSA及10 ng/mL TNF-α刺激下进行增殖能力检测;同时矿化诱导,设A组(AGE-BSA刺激组),T组(TNF-α刺激组),AT组(AGE-BSA/TNF-α共同刺激组),不含AGE-BSA/TNF-α的常规矿化诱导组作为对照;于诱导的21d茜素红染色观察钙结节形成情况,诱导7 d,碱性磷酸酶染色观察ALP活性、实时定量聚合酶链反应(real time PCR)和Western blotting检测成骨相关基因及蛋白表达情况。流式细胞仪显示细胞阳性表达STRO-1、CD146、CD44、CD90;成骨诱导21 d后茜素红染色和定量分析显示,AT组骨结节形成量最低,A组及T组相对于对照组骨结节形成量存在下降;差异均有统计学意义(p0.05)。成骨诱导7 d后ALP染色,ALP活性变化趋势与茜素红定量分析相同。成骨诱导7 d后RT-PCR检测成骨相关基因BSP、OCN、ALP mRNA表达,AT组表达水平最低,A组及T组有下降趋势,差异均有统计学意义(p0.05)。Western blotting检测显示,各组总蛋白BSP蛋白表达趋势与RT-PCR趋势相同。AGEs与TNF-α均具有对HPDLSC的骨向分化能力的抑制作用,两者共同刺激对HPDLSC骨向分化能力存在协同抑制作用。     

真皮成纤维细胞成骨分化的实验研究

目的比较不同诱导剂对真皮成纤维细胞成骨分化的不同影响,探讨成纤维细胞成骨分化机制。方法取新生大鼠皮肤进行组织块培养,真皮成纤维细胞分离培养及鉴定,并分别由地塞米松、1,25(OH)2D3以及地塞米松和1,25(OH)2D3进行成骨分化诱导。分别于诱导后14d行ALP含量测定,21d行茜素红染色,并进行TAZ表达检测。结果真皮成纤维细胞表达波形蛋白,不表达角蛋白;成纤维细胞诱导14d后,1,25(OH)2D3诱导组及地塞米松+1,25(OH)2D3诱导组ALP含量与对照组有显著差异;成骨诱导21d,地塞米松诱导组仅见少量散在红色钙结节形成,1,25(OH)2D3诱导组钙结节数量增高,地塞米松+1,25(OH)2D3诱导组钙结节数量明显增高,直径变大;1,25(OH)2D3诱导组及地塞米松+1,25(OH)2D3诱导组,经TAZ免疫荧光染色,可见部分细胞核表达TAZ。结论地塞米松可促进1,25(OH)2D3诱导真皮成纤维细胞成骨分化。     

BMP7对小鼠诱导多能干细胞骨向分化作用的研究

目的:探讨骨形态蛋白(bone morphogenetic protein,BMP)超家族成员之一BMP7在小鼠诱导多能干细胞(Induced pluripotent stem cells,iPS)骨向分化过程中的作用。方法:本试验分成三组,分别是自发分化组,骨诱导组和添加BMP7的骨诱导组。每天观察各组细胞形态学特征及生长状况的差异,在诱导第14天通过茜素红染色检测基质矿化情况,判断BMP7在体外骨诱导条件下对小鼠iPS细胞骨向分化过程所发挥的作用。结果:完成了小鼠iPS细胞的培养鉴定,并诱导形成理想状态的拟胚体(Embryoid body,EB)用于分化接种。结果发现,添加BMP7的骨诱导组细胞的矿化结节阳性率明显增加。结论:BMP7在诱导小鼠iPS细胞骨向分化过程中起促进作用,而对非骨向分化的细胞无成骨促进作用。     

bFGF和BMP-2联合应用对体外培养兔骨髓间充质干细胞增殖与骨向分化的影响

目的:研究碱性成纤维细胞生长因子(bFGF)和骨形成蛋白-2(BMP-2)联合应用对体外培养兔骨髓间充质干细胞(BMSCs)增殖与骨向分化的影响.方法:体外培养兔骨髓间充质干细胞,在第2代细胞培养液中加入不同浓度的bFGF和BMP-2,依据加入bFGF和BMP-2浓度的不同分为5个实验组(组1:80 ng/ml bFGF;组2:80 ng/ml BMP-2;组3:30 ng/ml bFGF 30 ng/ml BMP-2;组4:50ng/ml bFGF 50ng/ml BMP-2;组5:80ng/ml bFGF 80ng/ml BMP-2)和对照组(不加任何生长因子),采用绘制生长曲线,四唑盐比色法(MTT),碱性磷酸酶(ALP)活性检测法和碱性磷酸酶(ALP)免疫组化染色法比较各组间差异,观察不同浓度的bFGF和BMP-2联合应用对兔骨髓间充质干细胞增殖与骨向分化的影响.结果:与对照组相比,单独应用80 ng/ml bFGF可显著促进BMSCs的增殖,但对BMSCs的骨向分化显著抑制;单独应用80 ng/ml BMP-2对BMSCs的增殖和骨向分化均有促进作用;30ng/ml bFGF 30 ng/ml BMP-2、50 ng/ml bFGF 50 ng/ml BMP-2和80 ng/ml bFGF 80 ng/ml BMP-2可显著地促进BMSCs增殖和促进BMSCs的骨向分化,且呈正性剂量-效应关系,联合应用两种生长因子较二者单独应用促细胞增殖及骨向分化的效果更为显著.结论:一定浓度范围内,bFGF和BMP-2的联合应用促进BMSCs增殖的同时也促进其骨向分化,两者对BMSCs有明显的协同增强的生物学效应.     

趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用研究

目的:探究趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用和机制,为钙化性主动脉瓣膜疾病的早期干预和治疗提供新思路。方法:取非钙化主动脉瓣(3例)和钙化主动脉瓣(5例),免疫组织化学染色检测成骨相关转录因子Runx2、骨桥蛋白OPN和骨钙蛋白OCN的表达;取3例非钙化的主动脉瓣,采用胶原酶连续消化法分离人主动脉瓣膜间质细胞,观察细胞形态及生长状态,并采用细胞免疫荧光进行表型鉴定。对成骨诱导培养的人主动脉瓣膜间质细胞分别过表达和干扰趋化因子受体CX3CR1,平行设置CM组、OM组和negative control+OM组,采用qPCR和Western blot检测Runx2、OPN和OCN的表达,Western blot检测AKT和p-AKT的表达。茜素红S染色评价晚期钙结节形成情况。结果:临床标本显示钙化的主动脉瓣较非钙化的主动脉瓣高表达CX3CR1(P 0. 05);成功分离人主动脉瓣膜间质细胞,α-SMA和Vimentin阳性,vWF阴性。与CM、OM、negative control组比较,CX3CR1+OM组Runx2、OPN和p-AKT表达上调(P 0. 05),且茜素红S染色可见明显钙结节;与CM、OM、negative siRNA control+OM组比较,si CX3CR1+OM组Runx2、OPN和p-AKT表达下调(P 0. 05),且茜素红S染色可见钙结节减少。结论:趋化因子受体CX3CR1可能通过AKT信号通路促进人主动脉瓣膜间质细胞成骨分化。     

Special AT‐rich sequence‐binding protein 2 (Satb2) synergizes with Bmp9 and is essential for osteo/odontogenic differentiation of mouse incisor mesenchymal stem cells

ObjectivesMouse incisor mesenchymal stem cells (MSCs) have self‐renewal ability and osteo/odontogenic differentiation potential. However, the mechanism controlling the continuous self‐renewal and osteo/odontogenic differentiation of mouse incisor MSCs remains unclear. Special AT‐rich sequence‐binding protein 2 (SATB2) positively regulates craniofacial patterning, bone development and regeneration, whereas SATB2 deletion or mutation leads to craniomaxillofacial dysplasia and delayed tooth and root development, similar to bone morphogenetic protein (BMP) loss‐of‐function phenotypes. However, the detailed mechanism underlying the SATB2 role in odontogenic MSCs is poorly understood. The aim of this study was to investigate whether SATB2 can regulate self‐renewal and osteo/odontogenic differentiation of odontogenic MSCs.Materials and methods Satb2 expression was detected in the rapidly renewing mouse incisor mesenchyme by immunofluorescence staining, quantitative RT‐PCR and Western blot analysis. Ad‐Satb2 and Ad‐siSatb2 were constructed to evaluate the effect of Satb2 on odontogenic MSCs self‐renewal and osteo/odontogenic differentiation properties and the potential role of Satb2 with the osteogenic factor bone morphogenetic protein 9 (Bmp 9) in vitro and in vivo.Results Satb2 was found to be expressed in mesenchymal cells and pre‐odontoblasts/odontoblasts. We further discovered that Satb2 effectively enhances mouse incisor MSCs self‐renewal. Satb2 acted synergistically with the potent osteogenic factor Bmp9 in inducing osteo/odontogenic differentiation of mouse incisor MSCs in vitro and in vivo.Conclusions Satb2 promotes self‐renewal and osteo/odontogenic differentiation of mouse incisor MSCs. Thus, Satb2 can cooperate with Bmp9 as a new efficacious bio‐factor for osteogenic regeneration and tooth engineering.     

Comparison of Odontogenic Differentiation of Human Dental Follicle Cells and Human Dental Papilla Cells

Classical tooth development theory suggests that dental papilla cells (DPCs) are the precursor cells of odontoblasts, which are responsible for dentin development. However, our previous studies have indicated that dental follicle cells (DFCs) can differentiate into odontoblasts. To further our understanding of tooth development, and the differences in dentinogenesis between DFCs and DPCs, the odontogenic differentiation of DFCs and DPCs was characterized in vitro and in vivo. DFCs and DPCs were individually combined with treated dentin matrix (TDM) before they were subcutaneously implanted into the dorsum of mice for 8 weeks. Results showed that 12 proteins were significantly differential, and phosphoserine aminotransferase 1 (PSAT1), Isoform 2 of hypoxia-inducible factor 1-alpha (HIF1A) and Isoform 1 of annexin A2 (ANXA2), were the most significantly differential proteins. These proteins are related to regulation of bone balance, angiogenesis and cell survival in an anoxic environment. Both DFCs and DPCs express odontogenic, neurogenic and peridontogenic markers. Histological examination of the harvested grafts showed that both DFCs and DPCs form pulp-dentin/cementum-periodentium-like tissues in vivo. Hence, DFCs and DPCs have similar odontogenic differentiation potential in the presence of TDM. However, differences in glucose and amino acid metabolism signal transduction and protein synthesis were observed for the two cell types. This study expands our understanding on tooth development, and provides direct evidence for the use of alternative cell sources in tooth regeneration.     

基于PI3K/AKT通路探究上调miR-210对牙髓干细胞增殖、凋亡能力的影响

摘要 目的：研究基于磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)通路探究上调微小RNA 210(miR-210)对大鼠牙髓干细胞增殖、凋亡能力的影响。方法：选取10只健康Sprague-Dawley（SD）雄性大鼠,颈椎脱臼处死后提取大鼠下切牙牙髓,进行牙髓干细胞培养和鉴定。分为正常组（未进行处理）,miR-210抑制组（给予20 nmol/L的miR-210抑制物）,miR-210对照组（给予20 nmol/L的miR-210模拟物）三组。采用CCK-8法检测牙髓干细胞增殖活性,酶联免疫吸附试验（ELISA）检测ALP活性,流式细胞仪检测细胞凋亡,采用免疫印迹（Western blot）检测PI3K、AKT蛋白。结果：与正常组相比,miR-210抑制组细胞增殖、ALP活性降低,细胞凋亡率升高;miR-210对照组细胞增殖、ALP活性升高,细胞凋亡率降低（P＜0.05）。与miR-210抑制组相比,miR-210对照组细胞增殖、ALP活性升高,细胞凋亡率降低（P＜0.05）。与正常组相比,miR-210抑制组PI3K、p-AKT蛋白表达降低,miR-210对照组PI3K、p-AKT蛋白表达升高（P＜0.05）。与miR-210抑制组相比,miR-210对照组PI3K、p-AKT蛋白表达升高（P＜0.05）。结论：miR-210通过调控PI3K、p-AKT蛋白激活PI3K/AKT通路,促进大鼠牙髓干细胞增殖,抑制牙髓干细胞凋亡。     

牙源性间充质干细胞对成骨前体细胞成骨分化的影响

目的:探讨牙源性间充质干细胞对成骨前体细胞成骨分化的影响。方法:将小鼠成骨前体细胞MC3T3-E1分为两组,观察组为牙源性间充质干细胞与MC3T3-E1细胞共培养,对照组为单一MC3T3-E1细胞培养。采用CCK-8法检测细胞增殖水平,采用酶联免疫法检测碱性磷酸酶(Alkaline phosphatase,ALP)活性并进行茜素红染色,采用qRT-PCR、Western blot检测ALP与骨桥素(osteopontin,OPN) m RNA与蛋白表达水平。结果:细胞共培养1 d与3 d后,观察组的细胞增殖指数、ALP活性显著高于对照组(P<0.05)。与对照组相比,观察组的矿化结节显著增加,经茜素红染色呈红褐色。细胞共培养1 d与3 d后,观察组的ALP、OPN m RNA与蛋白相对表达水平显著高于对照组(P<0.05)。结论:牙源性间充质干细胞能促进成骨前体细胞的ALP、OPN表达,提高ALP活性,增加细胞增殖能力,诱发矿化,从而促进成骨分化。     

Changes of mitochondrial respiratory function during odontogenic differentiation of rat dental papilla cells

Dental papilla cells (DPCs) belong to precursor cells differentiating to odontoblasts and play an important role in dentin formation and reproduction. This study aimed to explore the changes and and involvement of mitochondrial respiratory function during odontogenic differentiation. Primary DPCs were obtained from first molar dental papilla of neonatal rats and cultured in odontogenic medium for 7, 14, 21 days. DPCs, which expressed mesenchymal surface markers CD29, CD44 and CD90, had the capacity for self-renewal and multipotent differentiation. Odontoblastic induction increased mineralized matrix formation in a time-dependent manner, which was accompanied by elevated alkaline phosphatase (ALP), dentin sialophosphoprotein and dentin matrix protein 1 expression at mRNA and protein levels. Notably, odontogenic medium led to an increase in adenosine-5′-triphosphate content and mitochondrial membrane potential, whereas a decrease in intercellular reactive oxygen species production and NAD⁺/NADH ratio. Furthermore, odontogenic differentiation was significantly suppressed by treatment with rotenone, an inhibitor of mitochondrial respiratory chain. These results demonstrate that enhanced mitochondrial function is crucial for odontogenic differentiation of DPCs.     

In Vitro Reparative Dentin: a Biochemical and Morphological Study

In this study, starting from human dental pulp cells cultured in vitro, we simulated reparative dentinogenesis using a medium supplemented with different odontogenic inductors. The differentiation of dental pulp cells in odontoblast-like cells was evaluated by means of staining, and ultramorphological, biochemical and biomolecular methods. Alizarin red staining showed mineral deposition while transmission electron microscopy revealed a synthesis of extracellular matrix fibers during the differentiation process. Biochemical assays demonstrated that the differentiated phenotype expressed odontoblast markers, such as Dentin Matrix Protein 1 (DMP1) and Dentin Sialoprotein (DSP), as well as type I collagen. Quantitative data regarding the mRNA expression of DMP1, DSP and type I collagen were obtained by Real Time PCR. Immunofluorescence data demonstrated the various localizations of DSP and DMP1 during odontoblast differentiation. Based on our results, we obtained odontoblast-like cells which simulated the reparative dentin processes in order to better investigate the mechanism of odontoblast differentiation, and dentin extracellular matrix deposition and mineralization.Key words: dental tissue, in vitro differentiation, DMP1, DSP, type I collagen