五倍子水提取物对牙龈卟啉菌膜泡抑制牙周膜成纤维细胞生物活性的影响

目的:探讨五倍子水提取物对牙龈卟啉菌(Porphyromonas gingivalisPg)膜泡(extracellular vesicles ECV)抑制牙周膜成纤维细胞生物活性的影响。方法:采用体外培养的人牙周膜成纤维细胞,用3H-TdR掺入方法,检测ECV对人牙周膜成纤维细胞DNA合成的影响,以及中药五倍子提取物对ECV这一生物活性的阻断作用。结果:细胞培养物中加入50μg/mLECV时,人牙周膜成纤维细胞DNA的合成受到明显抑制,与空白对照组相比P<0.01。当同时加入各浓度五倍子水提取物时其DNA合成量明显增加,与ECV组相比均(P<0.05),且随五倍子水提取物浓度升高,DNA的合成量也随之增高,并呈一定浓度依赖性。结论:适宜浓度的五倍子提取物可有效地阻断ECV的这种生物活性作用,从而可推测其对牙周病的发生、发展能起到一定的阻断作用。     

牙龈卟啉单胞菌脂多糖对人牙周膜成纤维细胞胶原吞噬作用的影响

目的研究牙周病致病菌牙龈卟啉单胞菌脂多糖（LPS）对体外培养的人牙周膜成纤维细胞（hPDLF）胶原吞噬作用的影响。方法将不同质量浓度的LPS加入体外培养的hPDLF 48 h后,采用荧光定位术和流式细胞技术检测hPDLF胶原吞噬率的变化。结果LPS导致hPDLF胶原吞噬率显著增加（P<0.05）。结论牙龈卟啉单胞菌脂多糖具有促进hPDLF吞噬胶原的作用,可能是牙周组织破坏机制之一。     

碱性成纤维细胞生长因子对成骨细胞和牙周膜成纤维细胞迁移、增殖的影响

目的　明确碱性成纤维细胞生长因子(bFGF)对体外成骨细胞和牙周膜成纤维细胞迁移、增殖的影响,以 探讨在牙种植体组织界面局部应用bFGF诱导类牙周膜形成的可行性。方法　同一只SD大鼠来源的成骨细胞和 牙周膜成纤维细胞经传代培养至第4代,建立体外创伤模型,分别在普通培养基和含bFGF的培养基中培养,观察 细胞迁移情况,四唑盐比色实验(MTT)测定细胞的增殖速度。结果　普通培养基中,成骨细胞迁移速度快于成纤维 细胞。加bFGF培养基中牙周膜成纤维细胞迁移速度明显快于其他各组,同时MTT结果显示加入bFGF能明显促进 两种细胞的增殖。结论　bFGF能明显促进牙周膜成纤维细胞的增殖、移行。     

牙龈卟啉单胞菌超声提取物对人牙周膜细胞活性的影响

目的探讨牙龈卟啉单胞菌超声提取物对人牙周膜细胞活性的影响。方法组织块法体外培养人牙周膜细胞（hPDLC）,以6．25、12．5、25、50mg·L^-1质量浓度的超声提取物作用于hPDLC24h,阴性对照组为无超声提取物作用的hPDLC;用12．5mg·L^-1质量浓度的超声提取物分别作用于hPDLC24、48、72、96h,阴性对照组亦培养24、48、72、96h。以甲噻唑四唑氮比色比较试验组与对照组hPDLC的活性变化,用SPSS13．0软件包对数据行单因素方差分析和独立样本t检验。结果当作用于hPDLC的超声提取物质量浓度为6．25～50mg·L^-1时,经过24h,试验组hPDLC的活性低于阴性对照组;当超声提取物质量浓度为12．5mg·L^-1时,在24-96h内,试验组hPDLC的活性低于阴性对照组。结论牙龈卟啉单胞菌可导致体外培养的hPDLC的活性降低,且作用呈质量浓度和时间依赖性。     

五倍子、黄芩提取物对牙龈卟啉菌超微结构的影响

目的:观察五倍子、黄芩提取物对牙龈卟啉菌(Porphyromonas gingivalis Pg)超微结构的影响.方法:采用透射电镜进行观察.结果:黑色的细菌经20 g/L的五倍子提取物液处理后变为淡褐色,可使Pg大部分胞壁不完整,破裂呈不连续状态或消失,胞浆内可见较多空泡,有的胞浆内可见黑色沉淀颗粒.20g/L的黄芩液处理后细菌仍为黑色,电镜下可见其细胞变形,胞壁变薄或破裂,胞浆消化呈空泡,有的胞浆内可见黑色沉淀颗粒.结论:20 g/L的五倍子、黄芩提取物可明显破坏Pg的细胞结构.     

静磁场对人牙龈成纤维细胞碱性磷酸酶活性的影响

目的:研究不同磁路设计的磁性附着体所产生静磁场对人牙龈成纤维细胞酶学的相关作用.方法:使用自主设计的磁场加载系统,产生不同强度的静磁场,对体外培养的人牙龈成纤维细胞进行不同时间的磁场加载.通过与对照组的比较,探讨磁场对该类细胞碱性磷酸酶活性的影响.结果:改变加载强度(120 mT、10 mT、0mT)或加载时间(1、3、5个加载周期),静磁场对人牙龈成纤维细胞碱性磷酸酶活性均无显著性影响(P＞0.05).结论:静磁场对牙龈成纤维细胞碱性磷酸酶活性没有影响,初步提示开放及闭合磁路系统磁性附着体所产生静磁场,对牙龈成纤维细胞的相关酶不存在生物学效应.     

牙周膜成纤维细胞对成骨细胞细胞数量和碱磷酶活性的影响

目的:观察人牙周膜成纤维细胞(human periodontal ligament fibroblast cells,HPLFs)对成骨细胞(Osteoblast cells,OBs)细胞数量和碱磷酶活性的影响,为进一步探讨正畸牙齿移动的生物学机制奠定基础。方法:建立人OBs与HPLFs共培养系统,通过细胞计数及生化检测法观察人牙周膜成纤维细胞对成骨细胞细胞数量和碱性磷酸酶(alkalinephosphatase,ALP)活性的影响。结果:3d和5d时,共培养组OBs细胞计数分别为4.5×104及8.5×104,明显高于对照组,且两组分别与对照组OBs细胞计数有显著性差异(P<0.05)。transwell共培养组与对照组相比,在3d时,两组OBsALP活性有显著性差异(P<0.05),5d及7d时差异尤其显著(P<0.01),transw ell共培养组OBsALP活性低于对照组。结论:人HPLFs能增加OBs的细胞数量,但抑制OBsALP活性。     

不同fimA基因型牙龈卟啉单胞菌对牙龈成纤维细胞表达白细胞介素-8的影响

目的:研究不同fimA基因型牙龈卟啉单胞菌(Porphyromonas gingivalis, Pg)对人牙龈成纤维细胞表达IL- 8的影响,探讨fimA基因型与Pg致病力差异之间的关系.方法: Pg ATCC 33277(Ⅰ型),WCSP115(Ⅱ型),WCSP1.5(Ⅲ型),W83(Ⅳ型)分别与牙龈成纤维细胞在标准条件下共同孵育,于孵育后1、3、6、12 h收集细胞和上清,应用实时荧光定量RT- PCR和ELISA法分别检测牙龈成纤维细胞IL- 8 mRNA和蛋白的动态表达.结果:与对照组比较,各fimA型Pg对牙龈成纤维细胞IL- 8 mRNA和蛋白的表达均有明显的促进作用(P<0.01);其中ⅡfimA型组IL- 8 mRNA和蛋白的表达量明显高于其它fimA型组,不同时间点IL- 8 mRNA相对表达量分别为20.42±2.21～103.01±12.50,蛋白分泌水平为(137.46±4.97～323.24±13.74) pg/ml;而Ⅲ fimA型Pg组的表达水平弱于其它fimA型组,IL- 8 mRNA相对表达量为3.61±0.39～12.88±1.56,蛋白分泌水平为(44.83±3.68～189.19±87.34) pg/ml, 差异有统计学意义(P<0.05).结论:Pg可促进牙龈成纤维细胞IL- 8 mRNA和蛋白的表达,且各fimA型Pg的促进作用有所差异.提示: fimA基因型的不同可能是Pg的致病力差异的基因基础.     

黄芩苷对牙龈成纤维细胞和牙周膜细胞上ICAM-1表达调节的研究

目的 :观察黄芩苷对IL - 1β诱导的牙龈成纤维细胞 (humangingivalfibrobl -asts ,HGF)和牙周膜细胞 (peri odontalligamentcells ,PDLcells)上细胞间粘附分子 - 1表达的影响。方法 :应用体外细胞培养和细胞免疫组化及图像分析方法。结果 :正常牙龈成纤维细胞和牙周膜细胞上细胞间粘附分子 - 1表达阴性或弱阳性 ;1μg/mLIL - 1β能诱导细胞间粘附分子 - 1在牙龈成纤维细胞和牙周膜细胞上强阳性表达 ;0 .1μg/mL黄芩苷对IL - 1β诱导的牙龈成纤维细胞和牙周膜细胞上细胞间粘附分子 - 1的表达有显著抑制作用 (P <0 .0 1)。结论 :黄芩苷可抑制IL -1β诱导的细胞间粘附分子 - 1在牙龈成纤维细胞和牙周膜细胞上表达 ,提示黄芩苷具有抗细胞粘附的作用     

骨形成蛋白-2和碱性成纤维细胞生长因子对牙周膜细胞碱性磷酸酶活性的联合效应

目的 :了解重组人骨形成蛋白 - 2 (rhBMP - 2 )和碱性成纤维细胞生长因子 (bFGF)单独和联合作用对人牙周膜细胞 (PDLC)碱性磷酸酶 (ALP)活性的影响。方法 :体外培养人PDLC ,分别用不同浓度的rhBMP- 2和bFGF单独或联合作用 ,用酶动力学方法检测PDLC的ALP活性。结果 :5 0～ 2 0 0 μg/L浓度的rhBMP - 2可显著增强人PDLC的ALP活性 (P <0 .0 1) ,而 10 μg/L浓度的bFGF可显著抑制人PDLC的ALP活性(P <0 .0 1) ,rhBMP - 2和bFGF联合作用仍可较明显地增强人PDLC的ALP活性 (P <0 .0 5 )。结论 :rhBMP - 2和bFGF联合应用可增强人PDLC的ALP活性     

Porphyromonas gingivalis lipopolysaccharide modulates the responsiveness of human periodontal ligament fibroblasts to platelet-derived growth factor

Lipopolysaccharides (LPS) prepared from periodontopathic bacteria have been known to induce various biological responses which may lead to periodontal tissue breakdown. The purpose of this study was to determine if Porphyromonas gingivalis LPS could affect cellular functions of human periodontal ligament fibroblasts (HPLF). We showed here the responsiveness of cultured HPLF to platelet-derived growth factor (PDGF)-BB, a growth factor for mesenchymal cells, in the presence of P. gingivalis LPS. DNA synthesis of HPLF was enhanced in a dose-dependent manner when LPS were co-incubated for 48 h; thereafter, it decreased to the baseline level within 24 h incubation. The stimulating effect of PDGF-BB was further enhanced by the pretreatment of HPLF with LPS (10 μg/ml) for 48 h. The binding assay of [¹²⁵I] PDGF-BB and the flow cytometric assay using rabbit antiserum to human PDGF receptor (PDGF-R) β-type indicated that this enhancement was due to the increase of the number of PDGF-R β-type on HPLF. Immunoprecipitation using antiserum to human PDGF-R β-type also showed that the synthesis of PDGF-R β-type was augmented in the LPS-treated HPLF. These results indicate that P. gingivalis LPS stimulate cellular proliferation and responsiveness to PDGF-BB of cultured HPLF. These cellular reactions may be mediated by PDGF-BB binding, followed by increased synthesis of the receptor protein.     

Cell-bound and extracellular matrix-associated alkaline phosphatase activity in rat periodontal ligament

In previous studies it was noted that alkaline phosphatase (ALP) activity in periodontal ligament does not only seem to be related to cells but may also be associated with the extracellular matrix. In an attempt to clarify this we studied the distribution of the enzyme at the electron microscopic level. In addition, ALPactivity was assessed biochemically following extraction of the ligament with (i) agents dissolving the membrane or splitting the phosphatidylinositol anchor (Triton X-100 or phosphatidylinositol-phospholipase C, respectively), and (ii) a matrix-degrading enzyme cocktail (collagenase, hyaluronidase and elastase). Histochemical observations revealed (a) a heterogeneous distribution of ALP-activity, with highest activity adjacent to the alveolar bone and (b) two pools of activity; one bound to cells and one associated with the collagenous extracelluJar matrix. In line with this were the biochemical data indicating that approximately 10% of the enzyme activity was firmly bound to the extracellular matrix and 90% to plasma membranes. Isoelectric focusing did not reveal differences between the two fractions, both samples yielding a single broad band corresponding with an isoelectric point of about 4.4.     

Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis

Scheres N, Laine ML, de Vries TJ, Everts V, van Winkelhoff AJ. Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01229.x © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth‐supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis. This study aimed to investigate the effects of viable P. gingivalis on the expression of genes associated with inflammation and bone degradation by these fibroblast subsets. Material and Methods: Primary human GF and PDLF from six healthy donors were challenged in vitro with viable P. gingivalis W83 for 6 h. Gene expression of inflammatory cytokines in GF and PDLF was analyzed using real‐time PCR, and protein expression was analyzed using ELISA. Results: Viable P. gingivalis induced a strong in vitro inflammatory response in both GF and PDLF. We found increased gene expression of interleukin (IL)‐1β, IL‐6, IL‐8, tumor necrosis factor‐α, monocyte chemotactic protein‐1 and regulated upon activation, normal T‐cell expressed and secreted (RANTES). Macrophage colony‐stimulating factor was induced and the expression of osteoprotegerin was decreased in GF, but not in PDLF. In nonchallenged cells, a higher level of expression of IL‐6 was observed in GF than in PDLF. Between individual donors there was large heterogeneity in responsiveness to P. gingivalis. Also, in each individual, either GF or PDLF was more responsive to P. gingivalis. Conclusion: Considerable heterogeneity in responsiveness to P. gingivalis exists both between GF and PDLF and between individuals, which may be crucial determinants for the susceptibility to develop periodontitis.     

淫羊藿苷抑制牙龈卟啉单胞菌超声提取物对人牙周膜细胞增殖及骨保护素表达的影响

目的 探讨不同浓度淫羊藿苷抑制牙龈卟啉单胞菌超声提取物对人牙周膜细胞增殖及骨保护素（OPG）表达的影响.方法 体外培养人牙周膜细胞,用四唑盐（MTT）法检测不同浓度淫羊藿苷（0、0.001、0.01、0.1、1 μg/ml）及50μg/ml牙龈卟啉单胞菌超声提取物,不同时间（24、48、72h）作用下人牙周膜细胞的增殖水平.用RT-PCR及Western blot检测48h人牙周膜细胞骨保护素mRNA和蛋白的表达.结果 淫羊藿苷从0.01～1μg/ml对人牙周膜细胞增殖及骨保护素mRNA和蛋白的表达有促进作用（P＜0.01）,浓度为0.1μg/ml作用最显著.结论 淫羊藿苷可抑制牙龈卟啉单胞菌超声提取物对人牙周膜细胞增殖及骨保护素mRNA和蛋白表达的影响,促进人牙周膜细胞增殖及骨保护素mRNA和蛋白表达.