基质细胞衍生因子1在人炎症牙髓组织中表达的实验研究

目的:研究基质细胞衍生因子1(Stromal cell-derived factor-1,SDF-1)及其受体CXCR4在人炎性牙髓组织中的表达,探讨SDF-1/CXCR4轴在牙髓炎症发生发展中的可能作用。方法:采用免疫组织化学染色的方法检测SDF-1和CXCR4阳性细胞在健康、炎症牙髓组织中的分布情况。以实时荧光定量RT-PCR方法检测SDF-1mRNA在健康和炎症牙髓中的表达。结果:炎性牙髓中SDF-1、CXCR4主要分布于炎性细胞、成牙本质细胞和微血管内皮细胞。而正常组牙髓少见SDF-1、CXCR4阳性细胞。炎性牙髓中SDF-1mRNA的表达较健康牙髓显著增强。结论:与正常牙髓相比,炎性牙髓组织中SDF-1、CXCR4阳性细胞明显增多。炎性牙髓中SDF-1表达水平明显上调。SDF-1/CXCR4轴可能参与了牙髓炎症损伤和修复过程。     

Caries-related bacteria and cytokines induce CXCL10 in dental pulp

Marked infiltration of inflammatory cells, such as activated T-cells, is observed in the progression of pulpitis; however, little is known about the mechanism of their recruitment into pulpal lesions. It has been recently demonstrated that CXC chemokine ligand 10 (CXCL10) chemoattracts CXC chemokine receptor 3 (CXCR3)-positive activated T-cells. We therefore examined whether CXCL10 is involved in the pathogenesis of pulpitis. CXCL10 mRNA expression levels in clinically inflamed dental pulp were higher than those in healthy dental pulp. Immunostaining results revealed that CXCL10 was detected in macrophages, endothelial cells, and fibroblasts in inflamed dental pulp, and that CXCR3 expression was observed mainly on T-cells. Moreover, cultured dental pulp fibroblasts produced CXCL10 after stimulation with live caries-related bacteria, peptidoglycans, and pro-inflammatory cytokines. In contrast, heat-killed bacteria did not induce CXCL10 secretion. These findings suggest that CXCL10-CXCR3 may play an important role in the pulpal immune response to caries-related bacterial invasion. Abbreviations: CXCL10, CXC chemokine ligand 10; CXCR3, CXC chemokine receptor 3; IFN, interferon; FBS, fetal bovine serum; LTA, lipoteichoic acid; PGN, peptidoglycan; IL, interleukin; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; CCL, C-C chemokine ligand; TLR, Toll-like receptor; NOD, nucleotide oligomerization domain; HDPF, human dental pulp fibroblasts.     

基质细胞衍生因子-1及其受体CXCR4在成体干细胞迁移中的作用

基质细胞衍生因子-1是一种重要的造血与非造血系干细胞形态发生因子和趋化因子。基质细胞衍生因子-1与其受体CXCR4结合,所介导的成体干细胞迁移归巢在多种组织器官损伤后的再生修复中发挥重要作用。基质细胞衍生因子-1及其受体CXCR4组成的功能轴在成体干细胞尤其是骨髓源干细胞迁移方面的研究进展,为研究牙髓干细胞迁移提供了新的方向。     

Expression of macrophage inflammatory protein 3alpha in human inflamed dental pulp tissue

Severe pulpitis resulting from dental caries is characterized by marked inflammatory infiltrate such as lymphocytes. Little is known about the recruitment of these cells into the dental pulp lesions of carious teeth. Macrophage inflammatory protein-3alpha (MIP-3alpha), a CC chemokine attracts CC chemokine receptor 6 (CCR6)-expressing T cells. We examined the distribution of MIP-3alpha-positive and/or CCR6-positive cells in human inflamed and normal dental pulp by immunohistochemistry. MIP-3alpha was observed in all inflamed pulp sections, and was mostly distributed in macrophages that had accumulated in the area adjacent to carious lesions. Furthermore, CCR6 expression was also observed in the infiltrating lymphocytes. In contrast, MIP-3alpha and CCR6 were rarely detected in normal pulp. These findings suggest that MIP-3alpha plays a role in the advancement of pulpal inflammation via the recruitment of CCR6-expressing lymphocytes.     

Natural modifiers of the inflammatory process in the human dental pulp.

Concentrations of the protease inhibitors alpha 1-antitrypsin and alpha 2-macroglobulin were determined in normal and inflamed human dental pulps. Carious pulpal exposure which is associated with polymorphonuclear leukocyte infiltration and release of lysosomal enzymes was chosen as the point of verifiable inflammatory activity in the pulp. Normal samples were collected from nondiseased third molar teeth treatment planned for extraction and inflamed human pulps were collected from teeth with deep carious lesions. One half of each sample was assayed for concentration of protease inhibitors by enzyme-linked immunosorbent assay and the remaining half was examined histologically to verify the clinical diagnosis and categorize the extent of the inflammatory process. alpha 1-Antitrypsin and alpha 2-macroglobulin were detected in normal and inflamed human dental pulps in the nanogram per milliliter range. Statistically significant differences were found in the concentrations of alpha 2-macroglobulin (p less than 0.01) in moderate to severe inflammation versus normal pulp categories and between mildly inflamed pulps and moderate to severely inflamed pulps (p less than 0.05). Although differences in concentrations of alpha 1-antitrypsin were seen between inflamed and normal pulps, the differences were not statistically significant. The presence of these two protease inhibitors in the human dental pulp tissue and the increase in their concentration in acute inflammation indicates that these proteins play a role in the pathogenesis of pulpal inflammatory disease.     

Mesenchymal Stem Cells Derived from Human Inflamed Dental Pulp Exhibit Impaired Immunomodulatory Capacity In Vitro

IntroductionDental pulp stem cells (DPSC) are very attractive in regenerative medicine. In this study, we focused on the characterization of the functional properties of mesenchymal stem cells derived from DPSCs. Currently, it is unknown whether inflammatory conditions present in an inflamed dental pulp tissue could alter the immunomodulatory properties of DPSCs. This study aimed to evaluate the immunomodulatory capacity in vitro of DPSCs derived from healthy and inflamed dental pulp.MethodsDPSCs from 10 healthy and inflamed dental pulps (irreversible pulpitis) were characterized according to the minimal criteria of the International Society for Cell Therapy, proliferation, differential potential, and colony-forming units. Furthermore, the immunomodulatory capacity of DPSCs was tested on the proliferation of T lymphocytes by flow cytometry and the in vitro enzyme activity of indoleamine 2, 3-dioxygenase.ResultsThere were no significant differences in the DPSC characteristics and properties such as immunophenotype, tridifferentiation, colony-forming units, and proliferation of the DPSCs derived from normal and inflamed pulp tissue. Furthermore, there were significant differences in the immunomodulatory capacity of DPSCs obtained from human healthy dental pulp and with the diagnosis of irreversible pulpitis.ConclusionsOur results showed that DPSCs isolated from inflamed dental pulp showed typical characteristics of MSCs and diminished immunosuppressive capacity in vitro in comparison with MSCs derived from healthy dental pulp. Further investigation in vivo is needed to clarify the mechanism of this diminished immunosuppressive capacity.     

颌面部火器伤后间接损伤的牙髓组织中白介素-1的表达和意义

目的：研究白细胞介素—1(IL—1)在犬颌面部火器伤间接损伤牙髓中的表达情况。方法：采用直径(d)5．80mm制式枪弹致伤犬下颌部的模型，利用免疫组化染色及定量分析方法检测颌面部火器伤后不同时间、下颌不同牙齿的牙髓组织中IL—1的动态变化。结果：IL—1在伤后牙髓组织中有阳性表达，并随时间的改变，呈动态变化过程。伤侧高于对侧：结论：IL—1在颌面部火器伤牙髓间接损伤过程中起重要作用。     

Recruitment of exogenous mesenchymal stem cells in mandibular distraction osteogenesis by the stromal cell-derived factor-1/chemokine receptor-4 pathway in rats

Distraction osteogenesis is widely used in orthopaedic and craniofacial surgery. However, its exact mechanism is still poorly understood. The purpose of this study was to find out whether there is systemic recruitment of mesenchymal stem cells (MSC) to the neocallus in the distraction gap by the stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) axis during osteogenesis. We examined the migration of MSC towards a gradient of SDF-1 in vitro. We also transplanted MSC labelled with green fluorescent protein (GFP) intravenously, with or without treatment with CXCR4-blocking antibody, into rats that had had unilateral mandibular distraction osteogenesis, and investigated the distribution of cells labelled with GFP in the soft callus after 24 h. We found that SDF-1 facilitated the migration potency of MSC both in vitro and in vivo, and this migration could be inhibited by AMD3100, an antagonist of CXCR4, and promoted by local infusion of exogenous SDF-1 into the distraction gap. This study provides a new insight into the molecular basis of how new bone is regenerated during distraction osteogenesis.     

Dental stem cells: Progress and perspectives

Dental pulp stem cells (DPSCs) are thought to contribute to reparative dentin formation, and that they may correspond to heterogenous populations of precursor cells or represent distinct differentiation stages along the odontoblastic lineage. DPSCs share many similarities with mesenchymal stem cells of the bone marrow (BMSCs). It appears that the distribution of tissue stem cells is not random and, within the dental pulp, there are potentially several distinct niches of stem/progenitor cells. In addition to DPSCs, other dental stem cell populations have been isolated. As for DPSCs, further studies are still needed to evaluate their potential of differentiation and their regenerative activity. Up today, (1) the formal demonstration that pulpal resident stem cells are actually the reparative dentin-forming cells recruited in response to injury is still lacking; and (2) the origin, localization and precise identity of odontogenic stem cells remain largely unknown. Dental clonal cell lines may represent valuable tool to answer some fontamental questions concerning the dental stem cell biology. Altogether, the presence of dental cell populations displaying stem cell properties has opened new paths for considering regenerative therapies. This might be a prerequisite to design alternative strategies for capping and endodontic treatment, using stem cells.     

An immunohistological study on cyclooxygenase-2 in human dental pulp

Characteristics of cyclooxygenase-2 (COX-2) expressing cells in human dental pulp were immunohistologically studied. Extirpated pulpal tissues from extracted teeth were examined to elucidate the localization and distribution of COX-2. Pulpal tissues were examined by the labeled streptavidin biotin method using specific mouse monoclonal antibodies for COX-2. Cell types of the COX-2 expressing cells were also investigated by the double stain technique using both monoclonal antibodies for CD68/macrophage and anti-COX-2. COX-2 expressing cells could be found in all of the inflamed pulps, and these cells were mostly distributed close to the area of accumulation of inflammatory cells. COX-2 was mainly expressed in fibroblasts rather than macrophages. In contrast, COX-2 expressing cells were scarcely found in the normal pulps. These findings indicate that pulpal fibroblasts, as well as macrophages, may participate in the production of prostaglandin through COX-2 expression in pulpal inflammation, and might be involved in the pathogenesis of irreversible pulpitis.     

Pulp biology: progress during the past 25 years

During the past 25 years there has been a rapid expansion in our knowledge of the dentine and pulp complex. This paper provides representative examples of important advances that researchers have made in this field. Topics to be considered include: differentiation of odontoblasts, dentine matrix proteins, extent of odontoblast processes, pulpal stem cells, apoptosis, interstitial fluid pressure in normal and inflamed pulps, class II antigen-presenting cells of the pulp, cytokines, antibodies, pulpal calcifications, tertiary dentine and pulpal inflammation associated with bacterial contamination of exposed dentine beneath restorations.     

基质细胞衍生因子1α及其受体CXCR4在人牙髓细胞中的表达

目的 研究体外培养人牙髓细胞(human dental pulp cells,HDPC)上CXCR4的表达情况,检测大肠杆菌脂多糖(lipopolysaccharide,LPS)和肿瘤坏死因子α(TNF-α)刺激后HDPC培养上清液中基质细胞衍生因子1α(stromal cell-derived factor-1α,SDF-1α)的表达水平,探讨人工重组SDF-1α(recombinant human SDF-1α,rhSDF-1α)对HDPC增殖及迁移的影响.方法 采用免疫细胞化学及间接免疫荧光技术检测HDPC上CXCR4的表达.用不同浓度LPS(0.1、1、10、100 mg/L)和TNF-α(1、10、100μg/L)刺激HDPC 48 h后,ELISA法检测HDPC培养上清液中SDF-1α含量的变化.同时甲基噻唑基四唑(MTT)法及体外趋化实验观察不同浓度rhSDF-1α对HDPC增殖及迁移的影响.结果 正常HDPC胞膜表达CXCR4且其培养上清液分泌SDF-1α,浓度约为(4513.55±962.92)ng/L.在用LPS和TNF-α刺激HDPC后,SDF-1α的表达水平均显著降低(P＜0.05).50、100和200μg/L的rhSDF-1α可促进HDPC的增殖(P＜0.05),50和100μg/L rhSDF-1α作用9 h可显著趋化HDPC的迁移(P＜0.01).结论 CXCR4在HDPC上表达且SDF-1α能促进HDPC的增殖及迁移;SDF-1-CXCR4轴可能在牙髓组织损伤修复中发挥重要作用.     

Expression and significance of stromal cell-derived factor-1alpha and its receptor CXCR4 in human dental pulp cells

OBJECTIVE: To investigate the expression of CXCR4 in cultured human dental pulp cells (HDPC) in vitro and the corresponding ligand SDF-1alpha level of HDPC supenatants stimulated by lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha), and to explore the role of SDF-1alpha on the proliferation and the migration of HDPC. METHODS: The expression of CXCR4 in HDPC was detected by immunocytochemistry technique and indirect immunofluorescence technique. The culture supernatants of HDPC were collected after HDPC had been simulated by LPS and TNF-alpha of different concentrations for 48h and then the SDF-1alpha level was assayed by quantitative sandwich ELISA. Meanwhile, the effects of recombinant human SDF-1alpha (rhSDF-1alpha) on the proliferation and the migration of HDPC at different concentrations were observed by MTT and Boyden Chamber Assay. RESULTS: CXCR4 was expressed in cytomembrane of HDPC and SDF-1alpha was secreted into their normal cell supernatants with a concentration of (4513.55 +/- 962.92) ng/L. The secretion of SDF-1alpha was both significantly decreased by stimulation with LPS and TNF-alpha (P < 0.05). In addition, rhSDF-1alpha stimulated the HDPC proliferation at the concentrations of 50, 100, 200 microg/L (P < 0.01) and increased the chemotactic migration of HDPC significantly after 9h's incubation with the concentrations of 50, 100 microg/L (P < 0.05). CONCLUSIONS: SDF-1alpha accelerated the proliferation and the migration of HDPC which expressed CXCR4. SDF-1-CXCR4 axis may play a role in repair of pulp injury.     

正常及炎症状态人牙髓中八聚体结合转录因子4B的表达

目的 研究人正常及炎症牙髓组织中八聚体结合转录因子4B(Oct-4B)的表达特点,检测大肠杆菌脂多糖刺激后人牙髓细胞( HDPCs)中Oct-4B的表达水平,以探讨Oct-4B在牙髓炎症中的可能作用.方法 采用免疫组织化学和反转录聚合酶链反应( RT-PCR)方法检测正常及炎症牙髓组织中Oct-4B的表达情况.RT-PCR检测1 mg/L脂多糖刺激HDPC 24、48、72 h后Oct-4B和热休克蛋白70(HSP70)表达水平的变化.结果 正常牙髓组织中未检测到Oct-4B的表达,炎症牙髓组织病灶处牙髓成纤维细胞和炎症细胞胞质中Oct-4B表达强阳性.炎症牙髓组织中Oct-4B mRNA水平显著高于正常牙髓组织(P<0.05).脂多糖刺激48.72 h后,HDPC中Oct-4B和HSP70 mRNA水平同步上调(P<0.05).结论 Oct-4B在炎症牙髓组织中高表达,且脂多糖刺激可上调牙髓细胞内Oct-4B表达,Oct-4B可能参与牙髓炎症修复过程.     

人牙龈干细胞的分离鉴定及基质细胞衍生因子-1对其趋化效应的研究

目的 研究基质细胞衍生因子-1（SDF-1）受体CXCR4在人牙龈干细胞（GMSCs）上的表达及SDF-1对人GMSCs的趋化效应。方法 通过有限稀释法分离并培养人GMSCs,检测其表面干细胞标志物的表达情况,测试其克隆形成率及多向分化能力,利用免疫荧光染色法检测人GMSCs上SDF-1受体CXCR4的表达,用Transwell细胞培养室检测不同质量浓度SDF-1对人GMSCs的趋化反应,光镜下计数迁移至滤膜下侧面的不同视野的细胞数。结果 人GMSCs具有较高的自我更新能力,在体外呈克隆状生长,表达间充质干细胞表面标志物CD44、CD73、CD90、CD105和CD166,而造血干细胞表面标志物CD14、CD34和CD45的表达为阴性。体外诱导培养的人GMSCs能够向成骨细胞及成脂细胞分化,其克隆形成率为21.4%±2.8%。免疫荧光染色显示,人GMSCs表达SDF-1受体CXCR4。SDF-1的质量浓度为100、200 ng·mL^-1时,Transwell细胞培养室中迁移的细胞数目（每高倍视野分别为189.3±4.4和164.6±4.9）显著多于空白对照组（每高倍视野47.8±2.5）（P<0.01）;使用CXCR4中和抗体处理后,人GMSCs的迁移效应明显受到抑制（每高倍视野降低为29.0±2.4,P<0.01）。结论 人GMSCs表达趋化因子SDF-1受体CXCR4,SDF-1对人GMSCs有趋化效应,这种趋化效应可能是通过其特异性受体CXCR4介导的。     

牙髓干细胞的研究和应用展望

成体干细胞的研究在组织和器官修复再生中具有重要的意义。近年来，在现代细胞生物学、分子生物学和分子遗传学研究推动下，有关牙髓干细胞的来源、体外培养、细胞表型和功能等研究积极展开，本文将对其研究进展及在牙髓疾病和牙齿再生修复中的应用前景作一综述。