人牙周韧带细胞成骨分化的差异表达蛋白质组学研究

目的 获得人牙周韧带细胞诱导成骨分化通路中的调节蛋白,探讨牙周韧带细胞诱导成骨分化的分子机制.方法 应用胶内差异双向电泳结合基质辅助激光解吸电离-飞行时间质谱鉴定等蛋白质组学技术筛选人牙周韧带细胞诱导成骨分化的差异表达蛋白质组.结果 获得人牙周韧带细胞诱导成骨分化7 d的差异表达蛋白质图谱,确认有显著差异的蛋白质斑点61个,质谱鉴定29个蛋白质,包括细胞骨架蛋白及细胞骨架相关蛋白、膜结合蛋白、核蛋白、基质合成、代谢酶和信号传导等蛋白.其中一组细胞骨架蛋白及细胞骨架相关蛋白同时发生改变,与牙周韧带细胞成骨分化早期细胞骨架重组、有丝分裂和细胞迁移等过程紧密相关.结论 建立了人牙周韧带细胞成骨分化早期的差异蛋白质组图谱,为牙周韧带细胞成骨分化的蛋白调控机制进一步研究提供了实验依据.     

人牙髓干细胞向成牙本质细胞定向分化过程中miRNAs表达谱筛选及鉴定

目的:筛选人牙髓干细胞向成牙本质细胞定向分化过程中差异表达的microRNAs(miRNAs)并进行初步鉴定。方法:体外分离、培养和鉴定人牙髓干细胞,miRNAs芯片筛选牙髓干细胞向成牙本质细胞分化过程中差异表达的miRNAs,并通过TargetScan数据库预测靶基因,实时定量PCR法对结果进行初步鉴定。结果:人牙髓干细胞vimentin、nestin、GFAP和I型胶原4种细胞表型均表达;成脂诱导3周后细胞内有油红O染色阳性脂滴出现;成牙本质诱导可见钙结节形成;基因芯片结果显示,牙髓干细胞向成牙本质细胞分化过程中,发生2倍以上表达变化的miRNAs有6条,其中上调3条,下调3条。通过miRNA靶标预测工具预测靶基因,发现hsa-miR-633和hsa-miR-210有与牙髓干细胞分化相关的靶基因;real time-PCR验证hsa-miR-633和hsa-miR-210表达变化与基因芯片结果相符。结论:人牙髓干细胞经诱导向成牙本质细胞分化过程中miRNAs表达谱具有显著变化,为牙髓干细胞分化机制研究提供了新的提示。     

人牙髓干细胞向成牙本质细胞分化中circRNAs差异表达的研究

目的:检测人牙髓干细胞(DPSCs)向成牙本质细胞分化过程中环状RNA(circRNA)的表达差异。方法:临床分离获取健康的牙髓组织,CD酶消化得到牙髓原代细胞,用鼠抗人STRO-1单克隆抗体孵育人牙髓细胞,用免疫磁珠分选出人DPSCs。用茜素红和碱性磷酸酶试剂鉴定DPSCs分化能力;采用Arraystar基因芯片检测circRNAs在DPSCs成牙本质细胞诱导分化过程中的差异,并做生物信息学分析。结果:在成牙本质诱导的DPSCs组中,有42个circRNAs上调,101个circRNAs下调。GO注释显示差异表达的基因主要参与核转运,胆固醇代谢等生物学过程。Pathway注释显示其主要调节泛素介导的蛋白质水解、癌症途径以及甾体生物合成等途径,调控Wnt、PI3K-Akt和MAPK等多种信号通路。结论:差异表达的circRNAs可能影响DPSCs向成牙本质分化。     

双向电泳分析维生素D对牙髓细胞分化的影响

目的：用双向电泳分析牙髓细胞分化过程中产生或分泌的新蛋白质。方法：在体外培养牙髓细胞，给予一定时间的1，25-双羟维生素D3，促使牙髓细胞分化，结合双向电泳技术分析细胞分化前后蛋白质合成、分泌的变化。结果：pH4-8和相对分子量MW20-90kD范围的蛋白斑点分布较多。双向电泳凝胶图像分析软件ImageMaster 2D Platium比较分析，给予维生素D孵育24h后，检测到77个新的蛋白点，同时有17个蛋白点消失。结论：维生素D促使牙髓细胞分化，并产生或分泌了一些新的蛋白质，维生素D和这些蛋白质对牙齿的发育、矿化的过程起调节作用。     

重组人白细胞介素-1β诱导人牙髓细胞蛋白质组的差异分析

目的分析重组人白细胞介素-1β（rhIL-1β）诱导前后牙髓细胞蛋白质的差异,鉴定2组间差异表达的蛋白质。方法采用双向凝胶电泳技术分离牙髓细胞全蛋白,获得对照组和诱导组牙髓细胞蛋白质图谱,Image Master2D Elit 5.0软件分析确认差异表达蛋白。通过基质辅助的激光解吸飞行时间质谱对差异表达的蛋白质点进行质谱鉴定,得到肽质量指纹图谱。结果比较对照组和诱导组蛋白质图谱,发现了39个蛋白质点差异明显。其中15个蛋白质点在诱导组高表达,新增13个蛋白质点,7个蛋白质点低表达,4个蛋白质点仅在对照组中表达;质谱鉴定后,10个蛋白得到确认。结论牙髓细胞对rhIL-1β的应答反应是一个非常复杂的过程,多种蛋白质分子涉及其中。鉴定了牙髓细胞中与rhIL-1β作用密切相关的2个差异蛋白,为探索早期牙髓炎的应答机制提供了新的线索和思路。     

CD146在人牙髓干细胞中表达的研究

目的:研究CD146在人牙髓干细胞及其诱导分化过程中的表达情况。方法:体外培养人牙髓干细胞,免疫荧光及流式细胞术检测CD146的表达。矿化诱导人牙髓干细胞分化,检测牙本质唾蛋白的表达,从mRNA及蛋白水平检测诱导过程中CD146的表达。结果:免疫荧光及流式细胞术证明人牙髓干细胞中CD146表达阳性。使用矿化诱导液培养人牙髓干细胞,通过检测到牙本质唾蛋白的表达,证明细胞已向成牙本质细胞方向分化;在此诱导过程中,CD146在人牙髓干细胞中的表达逐渐下调。CD146在人牙髓干细胞中有较特异性的表达,有可能作为其特异性标志物。     

人牙髓细胞在离体牙髓腔内向成牙本质细胞样细胞分化的研究

目的观察人牙髓细胞在离体牙髓腔内向成牙本质细胞样细胞分化的潜能。方法将原代培养的人牙髓细胞接种至处理过的离体牙髓腔内,2周后固定、脱钙、包埋、切片、染色,显微镜下观察其生长及分化情况。结果牙髓细胞在牙本质表面生长良好,部分细胞伸出胞质突伸入牙本质小管中,表现出成牙本质细胞样细胞的形态。结论牙本质可以诱导牙髓细胞向成牙本质细胞样细胞分化,可为组织工程化牙髓的研制提供实验依据。     

小分子整联蛋白结合配体N-连接糖蛋白家族成员在牙髓干细胞分化过程中的作用

体外诱导牙髓干细胞向成牙本质细胞分化并观察其过程,是目前研究牙髓干细胞分化机制和寻找其分化标志的主要方法。在成牙本质细胞分化和矿化过程中,小分子整联蛋白结合配体N-糖蛋白(SBLING)家族成员发挥着重要作用。下面就SBLING家族成员牙本质涎磷蛋白、牙本质基质蛋白-1和细胞外基质磷酸糖蛋白在牙髓干细胞分化过程中的作用作一综述。     

矿化液对人牙髓干细胞生物学特性的影响

目的 探讨矿化液对人牙髓干细胞(DPSCs)生长特性的影响.方法 用含一定浓度的β-甘油磷酸钠、抗坏血酸和地塞米松的矿化液诱导人牙髓干细胞,通过倒置相差显微镜、透射电镜、免疫细胞化学染色、von Kossa染色方法,观察和检测矿化液诱导后细胞形态、超微结构、表型和矿化基质分泌的变化,以未诱导组为对照.结果 矿化液连续培养7d,人DPSCs细胞形态明显改变,呈牙本质样极化细胞表现,另一侧为增大的胞体;超微结构显示,诱导后的细胞体积增大,核浆比例下降,胞浆细胞器丰富;诱导前细胞不表达牙本质涎磷蛋白(DSPP)和骨钙素(OC),诱导后均表达;连续培养21d,诱导组细胞形成多个矿化结节.结论 矿化液能诱导人DPSCs向成牙本质细胞样细胞分化并产生矿化基质.本研究从细胞水平上揭示了人DPSCs向成牙本质细胞分化机理,为人DPSCs应用于牙髓损伤的修复再生提供了依据.     

不同状态下牙髓组织蛋白质表达的二维电泳分析

目的 利用蛋白质组技术探讨牙髓对中龋和热刺激的反应.方法 用双向电泳得到牙髓在中龋和热刺激状态下的二维电泳图谱,对差异点进行质谱鉴定.结果 经Image Master2-D Platinum 5.0软件分析显示,正常牙髓和中龋牙髓的蛋白表达无显著差异;热损伤牙髓有2个蛋白点缺失,8个蛋白点下调.质谱分析鉴定了7种蛋白质.结论 本实验条件下,中龋牙髓的蛋白质表达与正常牙髓无显著差异,热刺激可造成部分蛋白表达的下调.     

Proteomic analysis of protein components in periodontal ligament fibroblasts

BACKGROUND: Characterization of periodontal ligament (PDL) fibroblast proteome is an important tool for understanding PDL physiology and regulation and for identifying disease-related protein markers. PDL fibroblast protein expression has been studied using immunological methods, although limited to previously identified proteins for which specific antibodies are available. METHODS: We applied proteomic analysis coupled with mass spectrometry and database knowledge to human PDL fibroblasts. RESULTS: We detected 900 spots and identified 117 protein spots originating in 74 different genes. In addition to scaffold cytoskeletal proteins, e.g., actin, tubulin, and vimentin, we identified proteins implicated with cellular motility and membrane trafficking, chaparonine, stress and folding proteins, metabolic enzymes, proteins associated with detoxification and membrane activity, biodegradative metabolism, translation and transduction, extracellular proteins, and cell cycle regulation proteins. CONCLUSIONS: Most of these identified proteins are closely related to the extensive PDL fibroblasts' functions and homeostasis. Our PDL fibroblast proteome map can serve as a reference map for future clinical studies as well as basic research.     

破骨细胞形成过程中在牵张力下的差异蛋白分析

目的：应用比较蛋白质组学的方法分析破骨细胞在牵张应力作用下蛋白质的差异表达,为阐明牵张力对破骨细胞的形成机制提供分子基础。方法：利用人骨髓单核细胞,牵张应力加载培养体系诱导培养破骨细胞,分为加力组与对照组。应用双向电泳分离以上两组样本可溶性蛋白质。应用基质辅助激光解吸电离飞行时间质谱获得差异蛋白点的肽质量指纹图谱;通过SWISS-PROT数据库鉴定蛋白质。结果：得到两张2-DE图谱,初步筛选出在牵张力的作用下,有13个蛋白点的表达存在明显差异。其中新增蛋白点1个,缺失7个,2个蛋白点表达发生明显上调,3个蛋白点发生明显下调。经质谱分析,初步鉴定了2种蛋白质。结论：应用2-DE及MALDI-TOF-MS方法分离并初步鉴定了2种蛋白质,为研究牵张力在破骨细胞形成过程中的机制以及正畸骨改建的发生机理提供线索。     

牙乳头细胞基本特征及其研究进展

牙乳头细胞来源于外胚间充质,具有多向分化潜能,是体内唯一分化为成牙本质细胞的前体细胞。该细胞在牙发育和牙体牙髓损伤修复过程中起重要作用。牙乳头细胞相关的组织工程化研究是近年来的热点。下面就牙乳头细胞的形态及其功能特征、三维细胞培养、组织工程化牙研究进展等方面的研究作一综述。     

The prominent proteins expressed in healthy gingiva: a pilot exploratory tissue proteomics study

Gingiva is a unique tissue which protects the underlying periodontal tissues from consistent mechanical and bacterial aggressions. Molecular analysis of gingiva is likely to improve our understanding of the underlying biological processes at work. The aim of this preliminary exploratory study is to analyze the proteomic profile of healthy gingiva and to detect prominently expressed proteins. Gingival tissue samples were obtained from periodontally healthy individuals who underwent surgical crown lengthening procedure. After protein isolation, two dimensional gel electrophoresis (2DE) gels were prepared for each sample and only protein spots common to all gels were selected to eliminate the bias caused by the effect of individuals on proteomic profile. Following the 2DE; in-gel tryptic digestion and MALDI-TOF/TOF steps were performed for protein identifications. Forty-seven proteins were successfully identified. The identified proteins were classified based on their classes, molecular functions and involvements in biological processes and metabolic pathways. Among them, 14-3-3 protein sigma, Protein DJ-1, Alpha-enolase, Triosephosphate isomerase, Superoxide dismutase, Peroxiredoxin-1, Protein S100-A9, Galectin-7, Annexin A2/A4, Carbonic anhydrase 1 and chaperone proteins are worthy of attention. The proteomic profile of the gingiva reflected its highly dynamic characteristics. Despite complexity of the gingival tissue proteome, 2DE was an effective approach in studying the common protein expression profile of the gingiva. Considering the significance of gingiva in the formation of periodontal diseases, it is important to generate a detailed proteome map of gingival tissue to set up a bridge between molecular events and the disease formation. This study established an initial proteome map of the gingival tissue from healthy individuals.     

Comparative proteomic analysis of human whole saliva

Human saliva performs a wide variety of biological functions that are critical for the maintenance of the oral health. Various functions include lubrication, buffering, antimicrobial protection, and the maintenance of mucosal integrity. In addition, whole saliva may be analysed for the diagnosis of human systemic diseases, since it can be readily collected and contains identifiable serum constituents. By using proteomic approach, we have established a reference proteome map of human whole saliva allowing for the resolution of greater than 200 protein spots in a single two-dimensional polyacrylamide gel. Fifty-four protein spots, comprised of 26 different proteins, were identifies using N-terminal sequencing, mass spectrometry, and/or computer matching with protein database. Ten proteins, whose levels were significantly different when bleeding had occurred in the oral cavity, were discussed in this study. These 10 proteins include -1-antrypsin, apolipoprotein A-I, cystatin A, SA, SA-III, and SN, enolase I, hemoglobin β-chain, thioredoxin peroxiredoxin B, as well as a prolactin-inducible protein. The proteomic approach identifies candidates from human whole saliva that may prove to be of diagnostic and therapeutic significance.     

Differentially expressed protein profile of human dental pulp cells in the early process of odontoblast-like differentiation in vitro

Dental pulp cells (DPCs) are capable of differentiating into odontoblasts that secrete reparative dentin after pulp injury. The molecular mechanisms governing reparative dentinogenesis are yet to be fully understood. Here we investigated the differential protein profile of human DPCs undergoing odontogenic induction for 7 days. Using two-dimensional differential gel electrophoresis coupled with matrix-assisted laser adsorption ionization time of flight mass spectrometry, 23 protein spots related to the early odontogenic differentiation were identified. These proteins included cytoskeleton proteins, nuclear proteins, cell membrane-bound molecules, proteins involved in matrix synthesis, and metabolic enzymes. The expression of four identified proteins, which were heteronuclear ribonuclear proteins C, annexin VI, collagen type VI, and matrilin-2, was confirmed by Western blot and real-time real-time polymerase chain reaction analyses. This study generated a proteome reference map during odontoblast-like differentiation of human DPCs, which will be valuable to better understand the underlying molecular mechanisms in odontoblast-like differentiation.     

人乳牙牙髓干细胞和恒牙牙髓干细胞的蛋白质组学比较

目的 采用蛋白质组学方法研究人乳牙牙髓干细胞（SHED）和恒牙牙髓干细胞（DPSC）中的蛋白表达差异.方法 应用双向凝胶电泳技术分离SHED和DPSC的细胞总蛋白.通过比较两种细胞的蛋白组学图谱,确定差异表达的蛋白点,而后对差异点进行基质辅助激光解析电离飞行时间质谱分析和蛋白数据库信息检索,对差异蛋白进行功能分类.结果 建立了SHED和DPSC的蛋白质组图谱,经软件分析出45个差异蛋白点,其中26个表达上调,19个表达下调,再经质谱鉴定出48种蛋白,其生物学功能涉及细胞周期、代谢等.结论 SHED与DPSC中蛋白的差异表达体现了两种细胞在结构和功能上的异同性,为进一步研究SHED和DPSC在增殖、分化中的差异,以及牙齿相关干细胞在组织工程和再生医学研究中的应用提供参考.     

胎盘间充质干细胞牙向分化的体外研究

目的探讨胎盘间充质干细胞牙向分化的可能性。方法双酶(胶原酶和胰蛋白酶)消化法获得SD大鼠胎盘间充质干细胞,免疫细胞化学鉴定其表型、成脂成骨诱导鉴定其多向分化能力。SD大鼠胎鼠牙胚细胞条件培养基诱导胎盘间充质干细胞14 d,免疫细胞化学检测DSP和DMP-1,RT-PCR及凝胶电泳检测DSPP和DMP-1基因。结果胎盘间充质干细胞表达CD29、CD44和CD105,而不表达CD31、C34和CD45。成骨和成脂诱导后形成钙结节以及脂滴。牙向诱导后的胎盘间充质干细胞形态无明显改变,表达成牙本质细胞特异性相关蛋白-DMP-1、DSP和特异性基因-DMP-1和DSPP。结论胎盘间充质干细胞具有牙向分化的潜能。