人牙髓细胞的体外培养

对牙髓细胞的体外培养是研究牙髓的有效工具。随着细胞培养技术的发展,牙髓细胞培养的成功率不断提高。干细胞生物学的研究使得人们认识到,在成体组织中也存在干细胞。目前不仅已分离出牙髓干细胞,而且对其干细胞特征作了进一步的研究,这将给牙髓病治疗和牙齿组织工程带来重大的变化。     

Notch信号通路在牙髓干细胞增殖和分化中的调控作用

Notch信号通路是进化过程中高度保守的信号转导机制,通过细胞与细胞间的相互作用控制细胞的命运。牙髓干细胞是一种具有高度增殖和多向分化潜能的成体干细胞,在组织再生中发挥重要作用。该文就该信号通路的组成及其对牙髓干细胞调控的分子机制作一综述。     

体外人牙髓细胞的矿化特性

为探讨体外人牙髓细胞的生物学特性，采用体外细胞连续培养、钙质染色、Ｘ射线能谱分析与透射电镜观察等方法，对体外人恒牙牙髓细胞的矿化特性进行了研究并与人牙龈成纤维细胞（ｈｕｍａｎｇｉｎｇｉｖａｆｉｂｒｏｂｌａｓｔｓ，ＨＧＦｓ）进行了比较。结果表明，人牙髓细胞在体外可以复层生长并形成细胞结节，ＨＧＦｓ则无此能力，牙髓细胞的碱性磷酸酶活性亦较ＨＧＦｓ者高；细胞结节钙质染色呈阳性，结节内钙磷含量明显增高；人牙髓细胞有许多与成牙本质细胞相似的超微结构特征，胞间基质中可见致密晶状小体。结果提示，体外连续培养的人牙髓细胞有向成牙本质细胞分化的可能，这可为研究人牙髓细胞的分化和矿化提供有价值的思路。     

体外培养的人牙髓细胞表型分析

目的：分离培养来源于人体的牙髓细胞并对其细胞表型进行分析。方法：采用组织块法培养人牙髓细胞,根据牙髓的细胞成分选择特异性抗体,利用免疫组化技术对其细胞表型进行分析。结果：Ⅲ型胶原在体外培养的牙髓细胞中广泛表达,ON和OPN表达也较广泛,阳性细胞呈星形,伸出多个胞浆突起互相连接;α-平滑肌肌动蛋白和CD34表达细胞为集落状,前者呈细长的梭形,后者呈圆形;Ⅰ型胶原、BSP和OC仅在少量细胞中表达,且细胞形态不规则;DSP、NF、CD14及CD45均为阴性。结论：体外培养的人牙髓细胞表型呈现多样性,表达平滑肌、内皮细胞及骨的标志物。     

人牙髓细胞的培养

本实验从人恒牙内分离出牙髓细胞，进行组织培养。培养３天，见组织块边缘有梭形细胞游出，随着培养天数的增多，游出的细胞也逐渐增多。来源于相邻组织块的细胞团逐渐靠近，接触；培养２０天时，细胞长满瓶底，铺成单层，按１：３的比例进行传代，平均６天传代一次，观察细胞的形态，排列及增殖情况，从细胞生长曲线可以看到，接种后第１天，细胞基本无增长，第２～５天，细胞增长旺盛，第６天，细胞生长又趋于缓慢，第７天时，增长     

体外牙髓细胞的生物学特征

体外培养的牙髓成纤维细胞样细胞，在形态上与一般成纤维细胞相似，但在生物学表达上却有类似成骨细胞的特征，将其称为牙髓细胞更为合适。它在培养中可出现钙化，有较高的碱笥磷酸酶活性；对甲状旁腺激素，维生素Ｄ３，前列腺素Ｅ２，胰岛素，转化生长因子β及上皮细胞生长因子招摇撞骗 应性类似于成骨细胞是其特征之一。体外牙髓细胞培养为了解牙髓细胞分化，牙髓钙化及牙本的南形成提供了许多有价值的信息。     

Notch配体Delta1对人牙髓干细胞分化的影响

目的:探讨Notch配体Delta1对体外人牙髓干细胞(dentalpulpstemcells,DPSCs)向成牙本质细胞分化能力的影响。方法:利用逆转录病毒载体建立高表达人Delta1基因的人牙髓干细胞系;实验分三组,正常牙髓干细胞组、转导细胞组及混合组(正常与转导细胞比例为100∶1),分别进行体外分化诱导。倒置显微镜观测各组细胞各生长期出现的时间;VonKossa染色计数各组形成钙化结节数;Westernblot法检测各组细胞牙本质涎磷蛋白表达。结果:与正常牙髓干细胞相比,转导细胞各生长期出现时间明显提前,形成的钙化细胞结节数目显著增加,牙本质涎磷蛋白表达显著升高。结论:Delta1基因转导牙髓干细胞仍保持了体外向成牙本质细胞分化的能力;Notch-Delta1信号与分化诱导因子协同作用可促进人牙髓干细胞向成牙本质细胞分化。     

用酶消化法进行鼠原代牙髓细胞的体外培养

目的：试用酶消化法获取鼠原代牙髓细胞，并研究其形态和生长特性。方法：采用０．５％胰酶－０．１％ＥＤＴＡ酶消化法获取ＳＤ鼠原代牙髓细胞，在倒置显微镜下观察其细胞形态和生长情况。结果：细胞形态主要呈梭形和星形，在６ｄ时开始增殖，１３ｄ细胞数达最高，细胞倍增时间为５８．５ｈ（６ｄ～１３ｄ）。结论：用胰酶－ＥＤＴＡ酶消化法可在短期内获得大量的牙髓细胞。     

人牙髓细胞的体外培养

对牙髓细胞的体外培养是研究牙髓的有效工具。随着细胞培养技术的发展，牙髓细胞培养的成功率不断提高。干细胞生物学的研究使得人们认识到，在成体组织中也存在干细胞。目前不仅已分离出牙髓干细胞，而且对其干细胞特征作了进一步的研究，这将给牙髓病治疗和牙齿组织工程带来重大的变化。     

体外牙髓细胞的生物学特征

体外培养的牙髓成纤维细胞样细胞,在形态上与一般成纤维细胞相似,但在生物学表达上却有类似成骨细胞的特征,将其称为牙髓细胞更为合适。它在培养中可出现钙化,有较高的碱性磷酸酶活性;对甲状旁腺激素、维生素D_3、前列腺素E_2、胰岛素、转化生长因子β及上皮细胞生长因子的反应性类似于成骨细胞是其特征之一。体外牙髓细胞培养为了解牙髓细胞分化、牙髓钙化及牙本质形成提供了许多有 价值的信息。     

Notch信号分子于小鼠牙髓干细胞样细胞表达的研究

目的　研究Notch基因在小鼠牙髓干细胞样细胞表达。方法　采用酶消化培养法获得小鼠的单个牙髓细胞悬液,调整细胞密度为1×104个/孔细胞,干细胞培养液培养14 d,挑选细胞克隆扩增,提取细胞的总RNA,反转录聚合酶联反应(RT-PCR)检测Notch基因的表达。结果　小鼠牙髓细胞呈集落状生长,克隆形成率约为1·6 ~2·5 个/104细胞,所形成的集落细胞结合紧密,细胞胞体小、胞核大,RT-PCR结果显示Notch的mRNA在牙髓干细胞样细胞中有表达。结论　培养的集落状生长的小鼠牙髓细胞具有干细胞增殖快的特性,Notch基因于牙髓干细胞中表达,表明Notch信号参与了牙髓干细胞样细胞的早期分化的调控过程。     

Growth and Differentiation Factor-5 Regulates the Growth and Differentiation of Human Dental Pulp Cells

Introduction

Growth and differentiation factor-5 (GDF-5) is a multifunctional protein that regulates the development and repair in many tissues. The purpose of this study was to investigate whether GDF-5 may influence the proliferation, differentiation, and collagen turnover of human dental pulp cells.

Methods

Human dental pulp cells were treated with different concentrations of GDF-5 (0–500 ng/mL). Morphology of pulp cells was observed under a microscope. Cell proliferation was evaluated by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Immunofluorescent assay was used to observe the percentages of cell mitosis. Collagen content was measured by Sircol collagen assay. Tissue inhibitor of metalloproteinase-1 level in the culture medium was measured with enzyme-linked immunosorbent assay and Western blotting. Cell differentiation was evaluated by alkaline phosphatase (ALP) staining and ALP enzyme activity assay.

Results

After exposure of dental pulp cells to various concentrations of GDF-5, cell number was up-regulated significantly in dose-dependent manner. GDF-5 also stimulated mitosis of dental pulp cells as indicated by an increased percentage of binucleated cells from 28% to 35%–45%. GDF-5 did not affect the collagen content and tissue inhibitor of metalloproteinase-1 level of pulp cells. GDF-5 decreased the ALP activity of pulp cells as analyzed by ALP staining and enzyme activity assay, with 14%–44% of inhibition.

Conclusions

GDF-5 revealed mitogenic and proliferative activity to dental pulp cells. GDF-5 showed inhibitory effect on ALP activity but little effect on the collagen turnover. These events are crucial in specific stages of dental pulp repair and regeneration. GDF-5 may be potentially used for tissue engineering of pulp-dentin complex.     

Potential Roles of Bone Morphogenetic Protein 9 in the Odontogenic Differentiation of Dental Pulp Cells

IntroductionThe differentiation of dental pulp cells (DPCs) plays an important role in the repair of dental pulp injury. Bone morphogenetic protein 9 (BMP9) is one of the most effective BMPs to induce the differentiation of stem cells. However, the role of BMP9 in promoting the odontogenic differentiation of DPCs and dentinogenesis is worth knowing.MethodsFluorescence in situ hybridization and immunohistochemistry staining were performed to detect the BMP9 expression in human dental pulp. BMP9 was overexpressed in human DPCs (hDPCs), and the mineralization of hDPCs was tested by alkaline phosphatase staining and alizarin red staining. The expression of odontogenic differentiation-related genes was examined by quantitative real-time polymerase chain reaction and western blotting. The subcutaneous transplantation experiment was performed to test the odonto-induction ability of BMP9 in vivo. The rat direct pulp-capping experiment was performed to test the function of BMP9 in promoting dentin formation.ResultsBMP9 showed an increased expression in odontoblast layer at both the mRNA and protein levels. BMP9 enhanced the mineralization and induced the expression of odontogenic differentiation-related genes in hDPCs. More mineralized nodules, and increased expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1) were detected in the beta-tricalcium phosphate scaffold/cells composites of BMP9 group compared with the control group. Meanwhile, there was thicker reparative dentin formation in the BMP9 group in the rat pulp exposure experiment.ConclusionsBMP9 participates in the process of DPC differentiation and promotes DPC mineralization and dentinogenesis. BMP9 might be a potential therapeutic target in the repair of dental pulp injury.     

Leptin Induces Odontogenic Differentiation and Angiogenesis in Human Dental Pulp Cells via Activation of the Mitogen-activated Protein Kinase Signaling Pathway

Introduction

Up-regulation of odontogenic differentiation, dentin formation, and angiogenesis in dental pulp are key factors in vital pulp therapy. The aim of this study was to investigate whether leptin could promote odontogenic differentiation and angiogenesis in human dental pulp cells (hDPCs). In addition, the involvement of the intracellular signaling pathway in these effects was determined.

Cell Wall Mannan of Candida Attenuates Osteogenic Differentiation by Human Dental Pulp Cells

IntroductionCandida spp. has recently been introduced to interact with conventional carious bacteria, leading to dental caries progression and virulence ability. Evidence regarding the influence of Candida spp. on human dental pulp cell response remains unknown. This study aimed to investigate the effects of Candida albicans mannans on cytotoxicity, cell proliferation, osteogenic differentiation, and inflammatory-related gene expression in human dental pulp cells (hDPCs).MethodshDPCs were treated with cell wall mannans isolated from C. albicans, Candida krusei, Candida glabrata, Candida tropocalis, Candida parapsilosis, and Candida dubliniensis. Cell viability was performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Osteogenic differentiation– and inflammatory-related gene expression were determined using a real-time polymerase chain reaction. Mineralization was examined using alizarin red S staining.ResultsThe treatment of mannans isolated from C. albicans, C. krusei, C. glabrata, C. tropocalis, C. parapsilosis, and C. dubliniensis at concentrations ranging from 10–100 μg/mL did not affect cytotoxicity or cell proliferation. Mannans isolated from C. albicans, C. glabrata, and C. tropocalis significantly attenuated mineralization. However, cell wall mannans isolated from C. krusei, C. parapsilosis, and C. dubliniensis did not significantly influence mineral deposition in hDPCs. C. albicans cell wall mannans significantly attenuated osteogenic differentiation–related gene expression (RUNX2, ALP, and ENPP1). Interestingly, IL12 messenger RNA expression was significantly upregulated when treated with C. albicans cell wall mannans. The addition of recombinant IL12 significantly decreased mineralization in hDPCs.ConclusionsC. albicans cell wall mannans attenuated osteogenic differentiation in hDPCs and up-regulated inflammatory-related gene IL12 expression.     

人乳牙牙髓干细胞的体外培养观察

目的:体外分离、培养人乳牙牙髓干细胞。方法:采用酶消化法将人乳牙牙髓分离培养,以获得人乳牙牙髓干细胞。有限稀释法分离纯化,测定细胞克隆形成率,细胞计数法测生长曲线,细胞爬片行HE染色、碱性磷酸酶染色、抗波形蛋白(vimentin)免疫组化染色。结果:通过有限稀释法获得了克隆形成率为11～24个/103,细胞群体倍增时间为39.84h,HE染色细胞形态为梭形、细胞体小、胞核大的干细胞。且碱性磷酸酶染色阳性,抗波形蛋白(vimentin)免疫组化染色阳性。结论:人乳牙牙髓中可分离培养出牙髓干细胞。