永生化成牙本质细胞样细胞系研究进展

由于成牙本质细胞属于终末分化细胞,在体外生存期短,培养困难,从而限制了它的应用,因此,成牙本质细胞的永生化便有了较重要的意义。近年来随着分子生物学的发展,使正常细胞永生化成为可能。本文综述了永生化成牙本质细胞样细胞系的建立及机制等研究概况。     

牙本质基质蛋白-1与生物矿化

牙本质基质蛋白(DMP)-1是骨、软骨、釉质、牙骨质和牙本质生物矿化所必需的一种酸性非胶原蛋白.蛋白质化学研究显示,DMP-1全基因序列是生物矿化的启动因子,由C末端和N末端组成.体内实验证实DMP-1具有多方面功能,不仅是生物矿化的信号分子,同时也是调节因子,对成骨细胞和成牙本质细胞的成熟至关重要.本文就DMP-1在基因调节、组织细胞中的表达以及成骨、成牙本质中的生物矿化作用作一.     

永生化人成牙本质细胞样细胞系体内矿化特征

目的 研究永生化人成牙本质细胞样细胞系hTERT-hOd-1体内的矿化特征。方法 将细胞和三维支架复合后移植至小鼠背部皮下，采用X线和组织学方法观察复合物生长情况。结果 X线显示复合物阻射像的密度和面积随时间而增加。组织学观察复合物在小鼠体内培养12周可形成矿化组织，部分似牙本质，其中含有牙本质小管样结构。结论 hTERT-hOd-1在体内培养中具矿化能力，可用于牙本质形成和组织工程的研究。     

永生化人成牙本质细胞样细胞系体外矿化的初步观察

目的:探讨永生化人成牙本质细胞样细胞系hTERT-hOd-1的体外矿化特征。方法：在矿化液连续培养细胞5周，采用倒置显微镜、透射电镜进行组织学观察以及Von Kossa染色。结果：细胞在矿化液中生长良好，形成细胞结节，分泌细胞外基质，其中有平行排列的胶原纤维和针状晶体沉积。Von Kossa染色显示细胞结节已发生矿化。结论:hTERT-hOd-1细胞在体外培养条件下具有矿化能力。     

牙本质基质蛋白-1及其特异性表达的研究进展

牙本质基质蛋白-1(DMP-1)是在大鼠cDNA文库中检测到的酸性磷酸化细胞外基质蛋白,是矿化组织中非胶原蛋白的重要组成部分。DMP-1主要表达于牙齿与骨组织,其不仅参与未分化间充质细胞向成牙本质细胞分化,还介导了牙本质的矿化和细胞间的信号转导[1]。     

甲状旁腺激素对人牙乳头细胞牙本质基质蛋白1表达的影响

目的：观察甲状旁腺激素(parathyroid hormone，PTH)对体外培养的人牙乳头细胞(human dental papilla cells，HDPC)牙本质基质蛋白1(dental matrix protein，DMP1)合成分泌的影响。方法：原代方法培养出人牙乳头细胞，用不同浓度的甲状旁腺激素刺激培养的第5代人牙乳头细胞，免疫组化观察结果，并采用图像分析的方法，进行半定量分析。结果：PTH可刺激人牙乳头细胞DMP1的表达，诱导的DMP1主要表达于细胞胞浆内，呈一定的浓度依赖性，0．3μg／mL为刺激最佳浓度。结论：PTH能够刺激人牙乳头细胞产生DMP1，对牙乳头细胞向成牙本质细胞分化有一定促进作用，可能是成牙本质细胞分化的重要介质之一。     

牙本质基质蛋白1基因转染猪成纤维细胞的实验研究

目的评价牙本质基质蛋白1（dental matrix protein-1，DMP1）转基因修饰猪口腔黏膜成纤维细胞（porcine oral mucosa fibroblasts，POMF）后，对POMF生物学特性及DMP1表达的影响。方法构建pEGFP-DMP1绿色荧光融合蛋白真核表达载体，脂质体介导转染POMF，同时转染猪骨髓间质干细胞（mesenehymal stem cells，MSC）作为对照。检测转染后细胞的DMP1、釉鞘蛋白、牙本质涎蛋白（dentin sialoprotein，DSP）基因表达以及DMP1、DSP蛋白的表达情况，同时检测转染细胞矿化诱导后钙盐染色及转染细胞三维立体培养钙结节的形成情况。结果成功构建了DMP1真核表达载体pEGFP-DMP1，转基因后的POMF及MSC均可见有DMP1、釉鞘蛋白、DSP基因表达及DMP1、DSP蛋白表达阳性。DMP1转染POMF及MSC矿化诱导后钙盐染色，以及DMP1转染细胞三维立体培养石蜡切片HE染色，其矿化结节形成率均高于未转染细胞。结论POMF转基因表达DMP1能增强其矿化能力，诱导牙齿发育相关基因釉鞘蛋白及DSP的表达。     

永生化成牙本质细胞样细胞系研究进展

由于成牙本质细胞属于终末分化细胞，在体外生存期短，培养困难，从而限制了它的应用，因此，成牙本质细胞的永生化便有了较重要的意义。近年来随着分子生物学的发展，使正常细胞永生化成为可能。本文综述了永生化成牙本质细胞样细胞系的建立及机制等研究概况。     

牙本质基质蛋白1基因反义核酸对MDPC-23细胞内、外钙离子浓度的影响

目的观察牙本质基质蛋白1(DMP1)基因反义寡核苷酸作用下成牙本质细胞系MDPC-23细胞内、外钙离子浓度的动态变化,揭示牙本质矿化机制。方法以稳定表达DMP1的MDPC-23细胞为靶细胞,10μmol/L反义DMP1(AS-ODN)为阻断剂,用Western blot法检测不同时间细胞DMP1的表达情况,并观察不同作用时间内MDPC-23细胞内游离钙离子[(Ca2+)if]、总钙离子[(Ca2+)it]和细胞外钙离子[(Ca2+)e]的动态变化。结果Western blot法检测DMP1蛋白在MDPC-23细胞的表达在AS-ODN加入后12 h时减弱,24 h后完全阻断。与正常组和正义核酸组(S-ODN)相比较(平均荧光值为87.46±39.60),AS-ODN组(Ca2+)if先升高(平均荧光值12 h处为104.10±27.06;24 h处为98.46±19.92),AS-ODN作用24 h后,(Ca2+)if又降低(平均荧光值36 h处为77.54±14.95;48 h处为68.43±22.11);(Ca2+)it明显降低,于24 h处至最低值(0.142±0.233)mmol/L(P<0.01);(Ca2+)e呈上升趋势,且与对照组差异无显著性(P>0.05)。结论反义DMP1能够影响MDPC-23细胞内(Ca2+)if和(Ca2+)it浓度,提示DMP1参与调节成牙本质细胞的钙离子代谢和转运过程,可能在牙本质矿化过程中发挥作用。     

人成牙本质细胞样细胞的原代培养

目的：培养人原代牙本质细胞。方法：取引产的8月龄死胎，剥离乳磨牙胚牙乳头，组织块法培养。然后采用滤纸片法挑取了3个与成牙本质细胞形态相似的细胞克隆，扩大培养。对培养细胞从形态学、矿化结节、碱性磷酸酶（alkaline phosphatase,ALP）活性、人牙本质基质蛋白-1（human dentin matrix protein-1,hDMP-1）和人牙本质涎磷蛋白（human dentin sialophosphoprotein,hDSPP）在mRNA水平上的表达等方面进行鉴定。结果：有一个克隆来源的细胞呈梭形、有单侧较长细胞突起，未见核极化，同时它们具矿化功能，表达人成牙本质细胞特异性标志hDMP-1、hDSPPmRNA。结论：该培养细胞为人成牙本质细胞样细胞，为进一步的有关研究奠定了基础。     

Reparative Dentin Formation by Dentin Matrix Proteins and Small Extracellular Vesicles

IntroductionVital pulp therapy aims at preserving pulp vitality and regenerating dentin. Therefore, the purpose of this study was to explore the effects of a combination of treated dentin matrix (TDM) proteins and dental pulp cell (DPC)-derived small extracellular vesicles (sEVs) on pulp-dentin complex repair.MethodsWe prepared TDM by chemical demineralization and mechanical disruption of teeth to a powder preparation. The sEVs were isolated from culture supernatants of DPCs and identified by nanoparticle tracking analysis, Western blotting, and transmission electron microscopy. The effect of a combination of TDM proteins and DPC-derived sEVs on DPC proliferation, migration, and odontogenic differentiation was evaluated in vitro. A minipig model of pulp injury was used to compare the clinical outcomes and tissue responses attributed to 4 materials including TDM, sEV-TDM, sEVs, and mineral trioxide aggregate.ResultsThe sEV isolated from the cell supernatant promoted DPC proliferation and migration. The combination of TDM extracts and sEV synergistically promoted the migration of DPCs but suppressed their proliferation. Real-time polymerase chain reaction and Western blot revealed that sEV-TDM enhanced the odontoblast-related protein expressions in DPCs. In in vivo studies, TDM and sEV-TDM promoted the formation of continuous reparative dentin. Furthermore, odontoblastlike high columnar cells were observed on the pulp side of the dentin bridge.ConclusionsThe sEV-TDM complex exhibits intrinsic biological activities, which has potential applications as a bioactive pulp-capping material.     

Dentin matrix proteins

Dentinogenesis consists of highly controlled events occurring a short distance from the periphery of odontoblasts; it involves formation of extracellular collagen fibrils that act as an undergirding for deposition of plate-like carbonate apatite crystals. Odontoblasts also form a set of matrix proteins that are probably secreted at the mineralization front. Although most of these proteins are similar to those of bone, and differ from soft tissue proteins, dentin contains two unique proteins. Dentin phosphoprotein (DPP) is rich in aspartic acid (D) and phosphoserine (S*) and binds large amounts of calcium. DPP contains repeating sequences of DS*S* and S*D in discrete areas of the protein. DS*S* repeats form ridges of phosphates and carboxyllates while the S*D sequences give rise to ridges with phosphates and carboxyllates on opposing sides of the peptide chain. These structures undoubtedly have functional significance since DPP is involved in promotion of mineral initiation and in control of mineral size and shape. Dentin sialoprotein (DSP), found only in dentin, is a 53 kDa glycoprotein rich in aspartic acid, serine, glutamic acid and glycine. DSP is made by odontoblasts and also by pre-ameloblasts, but not by osteoblasts or other cell types. The gene for DSP is now known to be continuous with that of DPP. Thus, DSP and DPP must be secreted as a single protein which is then proteolytically processed to form the individual components found in the dentin matrix. Significantly, the DSP/DPP gene has been localized to human chromosome 4q21 at a site implicated in dentinogenesis imperfecta type II.     

Characterization of Odontoblast-like Cell Phenotype and Reparative Dentin Formation In Vivo: A Comprehensive Literature Review

Introduction

The primary aim was to explore the criteria used in characterization of reparative cells and mineralized matrices formed after treatment of pulp exposures, and the sequence of relative events. The secondary aim was to evaluate whether the reparative events depend on the experimental model species, age, and therapeutic intervention.

脱矿牙本质基质用于根尖诱导成形术的临床研究

目的　观察同种脱矿牙本质基质(DDM)用于根尖诱导成形术的临床效果。方法　将人牙本质去脂、脱矿粉碎、冻干等处理制备成DDM,作为根尖诱导剂应用于临床,并以氢氧化钙糊剂作为对照,共治疗患牙57颗。分别于术后1年、2年拍X线片及临床检查。结果　根尖诱导成形术后1年,患牙X线片有不同程度的根尖屏障形成,或虽无明显的X线改变但根管内探测有明显阻力,此时更换永久性根管充填材料。术后2年的临床观察发现, DDM组的治愈率为92·86%,略高于氢氧化钙组的91·30%,但无显著性差异。结论　脱矿牙本质基质可作为一种新的根尖诱导剂应用于临床。     

人类牙乳头细胞的体外培养及细胞生物学性状研究

目的　体外分离培养人类牙乳头细胞,并对传代细胞的生物学特性进行研究。方法　选择3~4月胎龄 的自然流产人胚胎,分离牙乳头,组织块法培养人类牙乳头细胞并鉴定。观察细胞的生长特性,经Ⅰ型胶原、纤维 粘连蛋白和层粘连蛋白免疫细胞化学染色及细胞矿化诱导后的钙盐染色对细胞的生物学性状进行研究。结果　 分离培养的人类牙乳头细胞在体外生长良好,细胞及分泌基质中的Ⅰ型胶原、纤维粘连蛋白和层粘连蛋白表达阳 性。将培养细胞行矿化诱导后,细胞可形成钙化基质。结论　通过机械分离及贴壁法可获得人类牙乳头细胞,其 传代细胞在基质形成和矿化能力方面与体内人牙乳头细胞有相似性,有潜力作为牙组织工程的种子细胞。     

Potential of Treated Dentin Matrix Xenograft for Dentin-Pulp Tissue Engineering

IntroductionThis study aims to develop and characterize the regenerative potential of an atelopeptidized treated dentin matrix xenograft using in vitro and in vivo models.MethodsFreshly extracted bovine dentin was pulverized into 250- to 500-μm particles and demineralized with 17% EDTA for 1, 7, and 13 days. The samples were atelopeptidized with pepsin. The degree of demineralization and the effect of atelopeptidization were assessed using field emission scanning electron microscopy combined with energy-dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy, respectively. The expression of dentin matrix acidic phosphoprotein 1, dentin sialophosphoprotein, and osteopontin was evaluated in dental pulp stem cells using quantitative real-time polymerase chain reaction. The samples were then implanted intramuscularly in rats for 30 days, and the inflammatory cells were quantified histologically.ResultsField emission scanning electron microscopy combined with energy-dispersive X-ray spectroscopy revealed an exposed tubular structure of dentin after 1 and 7 days of demineralization. Fourier transform infrared spectroscopy confirmed the absence of amide peaks at 1260 to 1640/cm after atelopeptidization. The dental pulp stem cell expression of dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein increased in all compared with the untreated control group (P < .05). The maximum expression rates were observed for the 1-day demineralized and atelopeptidized group. The 1-day demineralized group elicited the highest inflammatory response compared with the 7- or 13-day demineralized groups (P < .001). Atelopeptidization significantly decreased the inflammatory response only in the 1-day demineralized dentin group (P < .05).ConclusionsAtelopeptidization of 1-day demineralized dentin xenograft preserved the collagen structure, minimized the immune reaction, and provided sufficient regenerative potential.     

基质金属蛋白酶-1对根面牙本质有机质降解作用的超微结构观察

目的　观察基质金属蛋白酶-1(MMP-1)对根面牙本质有机质的降解作用。方法　收集临床拔除的健康无 龋阻生牙,切取根颈1/3处约5 mm厚的牙体,制成牙本质组织块,随机分为4组。第1组用酸溶液脱矿处理21 d 后,放入MMP-1溶液中孵育7 d;第2组仅用酸溶液脱矿处理21 d;第3组仅用MMP-1溶液酶解7 d;第4组为正常牙 本质标本对照组,不作任何处理。将各标本切割制作样本,脱水干燥,喷金,扫描电镜观察。结果　酸和酶溶液共 同处理的标本牙本质硬组织脱矿明显,牙本质小管管腔失去原有形态,边界不清,周围暴露的胶原纤维断裂不连 续,排列杂乱不规则。酸或酶溶液单独处理组未发生明显的基质纤维降解现象。结论　内源性蛋白酶参与了根面 龋的发生发展过程,MMP-1能够明显降解脱矿后的牙本质有机质。