淫羊藿苷对体外培养的人牙龈成纤维细胞的作用研究

目的 研究淫羊藿苷对体外培养的人牙龈成纤维细胞(human gingival fibroblasts,HGF)的作用.方法 体外培养HGF,用MTT法检测不同浓度的淫羊藿苷(0.001、0.01、0.1 μg/ml)、不同时间(24、48、72、96 h)作用下HGF的增殖水平.结果 淫羊藿苷(0.001～0.1 μg/ml)对HGF增殖具有促进作用(P<0.01),0.01 μg/ml 浓度作用最明显.结论 淫羊藿苷有促进HGF增殖的作用.     

三种中药提取物抑制牙周炎牙槽骨吸收的实验研究

目的:观察三种中药提取物淫羊藿苷、大黄素和黄芩苷联合应用对实验性牙周炎形成的影响。方法:将3 6只SD大鼠随机分为三组:正常对照组(N)、空白对照组(C)和实验组(E)。用大肠杆菌内毒素脂多糖(E LPS)牙龈局部注射法建立大鼠实验性牙周炎模型,E组造模期间在相同部位注射三种中药提取物混合液,C组注射生理盐水。光学显微镜下观察牙周组织病理学变化,并进行破骨细胞计数,测量釉牙本质界到牙槽嵴顶的距离。结果:E组大鼠牙周组织炎症反应明显轻于C组,破骨细胞数少(P <0 .0 1) ,牙槽骨吸收量小(P <0 .0 5 )。结论:淫羊藿苷、大黄素和黄芩苷联合应用可抑制实验性牙周炎牙槽骨吸收。     

淫羊藿苷对内毒素抑制牙周膜细胞ALP表达的影响

目的：探讨淫羊藿苷对人牙周膜细胞（periodontal ligament cells,PDLC）增殖及对内毒素（Lipopolysaccha-ride,LPS）干扰下碱性磷酸酶（alkaline phosphatase,ALP）的影响。方法：原代培养PDLC,MTT法检测不同浓度（0、10^-5～10^-9mol／L）淫羊藿苷对PDLC增殖的影响;RT—PCR、对硝基苯酚法测定淫羊藿苷对LPS抑制PDLC的ALPmRNA表达和分泌的影响。结果：淫羊藿苷在一定浓度下（10^-6～10^-7mol／L）可促进PDLC增殖;10υg／mLLPS可抑制PDLC的ALP活性;加入10^-6mol／L淫羊藿苷干扰后,对LPS抑制PDLC的ALP有拮抗作用,可提高其mRNA表达和活性。结论：淫羊藿苷在一定浓度时可促进PDLC增殖;可能通过拮抗LPS抑制其ALP的活性。     

淫羊藿苷、黄芩苷和大黄素联合作用对体外骨吸收的影响

目的研究淫羊藿苷、黄芩苷和大黄素单独及联合作用对体外骨吸收的影响。方法将从新生兔长骨分离的细胞与牙本质磨片共培养,建立体外骨吸收模型,并加入白细胞介素(IL)-1β及淫羊藿苷0.01μg/ml、黄芩苷0.01μg/ml、大黄素10μg/ml或其等比组合进行干预,动态观察并测量吸收陷窝的数量和面积。结果分离的细胞与牙本质片共同培养24 h后,牙本质片上出现少量吸收陷窝。体外共培养10 d,加入IL-1β的阴性对照组的陷窝数量和面积大于空白对照组(P<0.05)。同时加入提取物的各组陷窝数量和面积均小于阴性对照组(P<0.05)。其中,淫羊藿苷组、黄芩苷组和大黄素组3组之间无显著差异(P>0.05),而组合组陷窝数量和面积均小于上述3组(P<0.01)。结论淫羊藿苷0.01μg/ml、黄芩苷0.01μg/ml、大黄素10μg/ml等比组合可抑制IL-1β诱导的骨吸收。     

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.     

淫羊藿苷对人牙周膜细胞增殖及内毒素作用下IL-6表达的影响

目的初步研究淫羊藿苷对人牙周膜细胞(human periodontal ligament cells,h PDLC)增殖以及在内毒素(Lipopolysaccharide,LPS)作用下淫羊藿苷对h PDLC白细胞介素6(IL-6)表达的影响。方法组织块法原代培养并鉴定h PDLC,采用MTT方法检测淫羊藿苷(0、10-5、10-6、10-7、10-8、10-9、10-10mol/L)作用下h PDLC增殖的变化;利用PCR、ELISA方法检测淫羊藿苷对在LPS刺激下的人牙周膜细胞IL-6分泌的情况。结果一定浓度范围下(10-6~10-7mol/L)淫羊藿苷对h PDLC的增殖具有积极作用;在10μg/m L LPS作用下h PDLC的IL-6表达增多、活性增强,加入10-6mol/L淫羊藿苷处理后,对此加强效果有一定的抑制作用。结论在一定浓度范围作用下淫羊藿苷可促进h PDLC的增殖;同时,淫羊藿苷对LPS导致h PDLC的IL-6表达增强有一定的抑制作用。     

五倍子水提取物对LPS抑制人牙龈成纤维细胞DNA合成和对细胞周期改变的影响

目的：研究五倍子水提取物对内毒素(LPS)引起人牙龈成纤维细胞(HGF)DNA合成和细胞周期改变的影响。方法：采用健康人正常牙龈组织原代培养的牙龈成纤维细胞，以25μg／mL LPS作为刺激因子，用流式细胞仪技术观察20μg／mL五倍子水提取物对LPS引起HGF细胞周期改变的影响。结果：LPS作用后，DNA合成前期细胞比例(G1期)明显上升，而DNA合成期细胞比例(S期)及细胞增殖指数下降，五倍子可改善此现象。结论：五倍子水提取物可显著改善LPS抑制HGF增殖，提示五倍子对牙龈组织具有保护作用，有助于牙周病的防治。     

中药黄芩苷对脂多糖刺激人牙周膜成纤维细胞分泌肿瘤坏死因子-α的影响

目的研究中药黄芩苷对脂多糖（LPS）刺激人牙周膜成纤维细胞（HPLFs）分泌肿瘤坏死因子-α（TNF-α）的影响。方法采用原代培养HPLFs技术和酶联免疫检测方法（ELISA），检测培养上清液中TNF-α的水平。结果0．001～10μg／ml黄芩苷和2μg／ml地塞米松均可显著抑制LPS刺激HPLFs分泌TNF-α的活性（P〈0．01），而不同浓度组之间比较，抑制作用无显著性差异（P〉0．05）。结论黄芩苷对LPS刺激HPLFs合成和分泌TNF-α有显著抑制作用，与地塞米松抗炎效果相近，作用机制与抑制TNF-α等炎性细胞因子过度产生有关，揭示了黄芩苷可能的抗炎作用机制。     

中药黄芩苷对人牙周膜成纤维细胞保护作用的实验研究

目的研究中药黄芩有效成分黄芩苷对脂多糖（LPS）抑制人牙周膜成纤维细胞（HPLFs）活性及对LPS损伤HPLFs超微结构的影响，并初步探讨其可能的作用机理。方法采用原代培养人牙周膜成纤维细胞技术、四唑盐（MTT）比色试验和透射电子显微镜技术（TEM）。结果LPS浓度为100μg／ml时明显抑制细胞活性，同时加入LPS和黄芩苷后，细胞活性明显增强（P〈0．05）。100μg／ml LPS对HPLFs各超微结构均有不同程度的损伤，特别是线粒体损伤严重，同时加入LPS扣黄芩苷（10μg／ml）后，与LPS组比较，超微结构损伤减轻，细胞器结构表现也较正常。结论中药黄芩苷能显著促进成纤维细胞的增殖，对LPS抑制HPLFs活性和LPS损伤HPLFs超微结构具有保护作用，其作用机制可能与降解LPS及作用于细胞膜表面阻止LPS进入细胞内部有关。     

中药黄芩有效成分黄芩苷对IL-1β刺激人牙周膜细胞产生PGE2的影响

目的:探讨不同浓度黄芩苷对白细胞介素-1β(Interleukin-1β,IL-1β)刺激人牙周膜细胞(periodontal ligament cells,PDLC)产生前列腺素E2(prostaglandin E2,PGE2)的影响.方法:运用细胞培养技术和酶联免疫检测方法,观察IL-1β刺激PDLC分泌PGE2的量以及不同浓度的黄芩苷对其产生PGE2量的影响.结果:在1 ng/mL IL-1β的刺激下,PDLC产生PGE2 的量随时间的延长而增加.0.01～10 μg/mL的黄芩苷均能显著抑制1 ng/mL IL-1β对 PDLC的刺激作用(P<0.01),而不同浓度组之间比较,抑制作用无显著性差异.结论:①PDLC具有合成和分泌PGE2的功能,IL-1β能有效刺激 PDLC产生PGE2.②黄芩苷对IL-1β刺激PDLC合成和分泌PGE2有显著抑制作用.     

Cytotoxicity of metal ions to human oligodendroglial cells and human gingival fibroblasts assessed by mitochondrial dehydrogenase activity.

OBJECTIVES: To assess the variable concentrations of several metal salts on human oligodendrocyte MO3.13 and human gingival fibroblasts HGF and to enable any difference in cell type sensitivity to be examined. METHODS: Cytotoxicity was measured as mitochondrial dehydrogenase activity assessed by MTT assay. The mean of the 50% response (TC50) was calculated by using equation-fitting software (TableCurve 2D). RESULTS: The results of the MTT assay showed that metal ions induce reproducible cytotoxic effects in MO3.13 oligodendroglia and human gingival fibroblasts, that is dose dependent on the tested agent. Cadmium relatively showed the highest cytotoxic effects on MO3.13 cells (TC50 9.8 microM) whereas mercury showed the highest cytotoxic effects on HGF (TC50 74 microM) comparing with other tested metals. The two cell types responded differently. MO3.13 cells were more sensitive than the HGF to most of the metals. CONCLUSION: Metals have a wide range of toxicity to human oligodendroglial cells (MO3.13) and human gingival fibroblasts. Fortunately, however, in vivo the normal levels of these metals are much lower than those determined as toxic in vitro.     

Mechanical force augments the anti-osteoclastogenic potential of human gingival fibroblasts in vitro

Background and Objective: The cellular response of human gingival fibroblasts to a mechanical force is considered to be primarily anti‐osteoclastic because they produce relatively high levels of osteoprotegerin. However, there is little information available on the effects of compression force on the production of osteoprotegerin and osteoclastic differentiation by these cells. In this study, we examined how mechanical force affects the nature of human gingival fibroblasts to produce osteoprotegerin and inhibit osteoclastogenesis. Material and Methods: Human gingival fibroblasts were exposed to mechanical force by centrifugation for 90 min at a magnitude of approximately 50 g/cm². The levels of osteoprotegerin, receptor activator of nuclear factor‐κB ligand (RANKL), interleukin‐1β and tumor necrosis factor‐α were measured at various time‐points after applying the force. The effect of the centrifugal force on the formation of osteoclast‐like cells was also determined using a co‐culture system of human gingival fibroblasts and bone marrow cells. Results: Centrifugal force stimulated the expression of osteoprotegerin, RANKL, interleukin‐1β and tumor necrosis factor‐α by the cells, and produced a relatively high osteoprotegerin to RANKL ratio at the protein level. Both interleukin‐1β and tumor necrosis factor‐α accelerated the force‐induced production of osteoprotegerin, which was inhibited significantly by the addition of anti‐(interleukin‐1β) immunoglobulin Ig isotype; IgG (rabbit polyclonal). However, the addition of anti‐(tumor necrosis factor‐α) immunoglobulin Ig isotype; IgG1 (mouse monoclonal) had no effect. Centrifugal force also had an inhibitory effect on osteoclast formation. Conclusion: Application of centrifugal force to human gingival fibroblasts accelerates osteoprotegerin production by these cells, which stimulates the potential of human gingival fibroblasts to suppress osteoclastogenesis. Overall, human gingival fibroblasts might have natural defensive mechanisms to inhibit bone resorption induced by a mechanical stress.     

Effect of triclosan on interferon-gamma production and major histocompatibility complex class II expression in human gingival fibroblasts

BACKGROUND, AIMS: The effect of triclosan (2,4,4'-trichloro-2'-hydroxyl-diphenyl ether) on the production of interferon-gamma (IFN-gamma) and the expression of major histocompatibility complex (MHC) class II antigen was studied in human gingival fibroblasts isolated from 4 individuals. METHODS/RESULTS: All cell lines demonstrated high IFN-gamma production in 24-h cultures of human gingival fibroblasts stimulated by phytohemagglutinin (PHA) (5 microg/ml). Human gingival fibroblasts showed a high expression of MHC class II when stimulated with 500 and 1,000 pg/ml rIFN-gamma in 7-day cultures. Treatment of the cells with triclosan (0.5 microg/ml) reduced both IFN-gamma production and MHC class II expression in human gingival fibroblast cultures. Similar inhibitory effects on IFN-gamma production and MHC class II expression were observed when the anti-inflammatory agent dexamethazone (1 microM) was used. CONCLUSION: The present study further supports the view that the agent has an anti-inflammatory effect in addition to its antibacterial capacity.     

Resin composite monomers alter MTT and LDH activity of human gingival fibroblasts in vitro.

OBJECTIVES: Substances such as monomers may be released from composite resin systems and may induce adverse effects in biological tissues. The aim of this study is to investigate the cytotoxic concentrations of resin composite monomers on cultures of human gingival fibroblasts. METHODS: A range of dilutions of five resin composite monomers (HEMA, HPMA, DMAEMA, TEGDMA, and Bis-GMA) were added to the culture medium of human gingival fibroblasts for 24 h. Their cytotoxic effects were measured by using two colorimetric functional assays, mitochondrial dehydrogenase activity (MTT) and lactate dehydrogenase activity (LDH) assay. The logP values (water/octanol partition) of test monomers were also calculated computationally. RESULTS: Mitochondrial reducing activity assessed with the MTT test was inhibited by all monomers and all the monomers increased the LDH release in a reproducible dose dependent manner. A wide range of TC 50 values (concentrations altering MTT and LDH activity by 50%) (0.32-5.8 mM by MTT assay and 0.36-6.7 mM by LDH assay) was observed. Ranking of composite resin monomer cytotoxicities (TC 50) were similar for both the MTT and LDH assays, (Bis-GMA>TEGDMA>DMAEMA>HPMA >HEMA). However, the MTT assay was found to be more sensitive than the LDH assay, particularly when lower doses of the tested monomers were determined. The ranking of TC 50 concentrations correlated with the calculated logP values. SIGNIFICANCE: Monomers used in dental restorative materials show a variety of toxic effects on gingival fibroblasts. A combination approach using MTT and LDH assays provides valuable information about their toxic effects.     

Cytokines differentially regulate CXCL10 production by interferon-gamma-stimulated or tumor necrosis factor-alpha-stimulated human gingival fibroblasts

Background and Objective: CXC chemokine 10 (CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T‐helper 1 cells. In this study we examined the effects of cytokines on CXCL10 production by human gingival fibroblasts. Material and Methods: Human gingival fibroblasts were exposed to pro‐inflammatory cytokines (interleukin‐1β, tumor necrosis factor‐α), a T‐helper 1 cytokine (interferon‐γ), T‐helper 2 cytokines (interleukin‐4, interleukin‐13), T‐helper 17 cytokines (interleukin‐17A, interleukin‐22) and regulatory T‐cell cytokines (interleukin‐10, transforming growth factor‐β1) for 24 h. CXCL10 production by human gingival fibroblasts was examined by enzyme‐linked immunosorbent assay. Results: Human gingival fibroblasts produced CXCL10 protein upon stimulation with interleukin‐1β, tumor necrosis factor‐α and interferon‐γ. Treatment of human gingival fibroblasts with interferon‐γ in combination with tumor necrosis factor‐α or interleukin‐1β resulted in a synergistic production of CXCL10. However, interleukin‐4 and interleukin‐13 inhibited CXCL10 production by interferon‐γ‐stimulated or tumor necrosis factor‐α‐stimulated‐human gingival fibroblasts. On the other hand, interleukin‐17A and interleukin‐22 enhanced CXCL10 production by human gingival fibroblasts treated with interferon‐γ and inhibited CXCL10 production by tumor necrosis factor‐α‐stimulated human gingival fibroblasts. Furthermore, the anti‐inflammatory cytokine, interleukin‐10, inhibited CXCL10 production by both interferon‐γ‐ and tumor necrosis factor‐α‐stimulated human gingival fibroblasts, but transforming growth factor‐β1 enhanced interferon‐γ‐mediated CXCL10 production by human gingival fibroblasts. Conclusion: These results mean that the balance of cytokines in periodontally diseased tissue may be essential for the control of CXCL10 production by human gingival fibroblasts, and the production of CXCL10 might be important for the regulation of T‐helper 1 cell infiltration in periodontally diseased tissue.