促丝裂原激活蛋白激酶在牙髓干细胞向成牙本质细胞分化和牙髓损伤修复中的作用

通过诱导牙髓干细胞（DPSC）向成牙本质细胞方向分化,龋源性牙髓炎的治疗将不再局限于根管治疗这一临床选择,修复治疗也不再成为缺失牙治疗的唯一方案。促丝裂原激活蛋白激酶（MAPK）,尤其是P38MAPK通过直接或间接磷酸化特定的转录因子,将细胞外刺激信号转导至细胞及其核内,从而引起一系列细胞生物学反应,如细胞增殖、分化、转化和程序性死亡。骨形态发生蛋白-2、矿物三氧化物聚合体和Biodentine皆可诱导DPSC向成牙本质细胞分化,而三者正是通过MAPK信号转导通路发挥作用的。在组织工程支架诱导DPSC分化过程中,支架材料通过激活P38MAPK信号转导通路促进了DPSC的分化。此外,MAPK信号转导通路参与牙髓损伤修复中DPSC的迁移、黏附和分化,参与牙髓损伤修复中牙本质的形成。由于MAPK信号转导通路在细胞增殖、分化和生存等过程中都起着十分关键的作用,因此,深入研究其反应分子、作用底物和作用机制有着重要的理论和临床意义。     

Examination of the signal transduction pathways leading to activation of gelatinolytic activity by interleukin-1alpha and Porphyromonas gingivalis in human osteosarcoma cells

BACKGROUND: Recently, evidence show that matrix metalloproteinases (MMP) play an important role in the pathogenesis of periodontal diseases. However, the mechanisms and signal transduction pathways involved in the production of MMPs in human osteosarcoma cells are not fully understood. OBJECTIVES: The purpose of this study was to investigate the gelatinolytic activity in human osteosarcoma cells stimulated with interleukin-1alpha (IL-1alpha) or Porphyromonas gingivalis in the absence or presence of SB203580 (p38 inhibitor), U0126 [mitogen-activated protein kinase kinase (MEK) inhibitor], and LY294002 [phosphatidylinositaol 3-kinase (PI3K) inhibitor]. METHODS: IL-1alpha and the supernatants of P. gingivalis were used to evaluate gelatinolytic activity in human osteosarcoma cells using gelatin zymography. Furthermore, to search possible signal transduction pathways, SB203580, U0126, and LY294002 were added to test how they modulated the gelatinolytic activity. Results: Gelatin zymography demonstrated that the latent proforms of gelatinases MMP-2 and MMP-9 were released by human osteosarcoma cells. Secretion of MMP-9 was time-dependent by stimulating with IL-1alpha or P. gingivalis. In addition, SB203580, U0126, and LY294002 significantly reduced the IL-1alpha or P. gingivalis-stimulated MMP-9 production, respectively (p < 0.05). However, none of the kinase inhibitors affected the MMP-2 level compared with the control during the 4-day culture period (p > 0.05). CONCLUSIONS: Our findings demonstrated that IL-1alpha and P. gingivalis enhance MMP-9 production in human osteosarcoma cells, and the signal transduction pathways p38, MEK, and PI3K are involved in the inhibition of MMP-9. SB203580, U0126, and LY294002 suppress MMP-9 production and/or activity and may therefore be valuable therapeutics in MMP-mediated periodontal destruction, and might be proved clinically useful agents, in combination with standard treatment modalities, in the treatment of periodontitis.     

Signal transduction pathways involved in the stimulation of tissue type plasminogen activator by interleukin-1alpha and Porphyromonas gingivalis in human osteosarcoma cells

BACKGROUND: Recently, evidences have shown that tissue type plasminogen activator (t-PA) may play an important role in the pathogenesis of periodontal diseases. However, the mechanisms and signal transduction pathways involved in the production of t-PA in human osteosarcoma cells are not fully understood. OBJECTIVES: The purpose of this study was to investigate the caseinolytic activity in human osteosarcoma cell line U2OS cells stimulated with interleukin-1alpha (IL-1alpha) or Porphyromonas gingivalis in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. METHODS: IL-1alpha and the supernatants of P. gingivalis were used to evaluate the caseinolytic activity in U2OS cells by using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search possible signal transduction pathways, SB203580, U0126, and LY294002 were added to test how they modulated the caseinolytic activity. RESULTS: Casein zymography exhibited a caseinolytic band with a molecular weight of approximately 70 kDa, suggestive of the presence of t-PA. Secretion of t-PA was found to be stimulated with IL-1alpha and P. gingivalis during a 2-day culture period (p < 0.05). From the results of casein zymography and ELISA, SB203580, U0126, and LY294002 significantly reduced the IL-1alpha or P. gingivalis-stimulated t-PA production, respectively (p < 0.05). CONCLUSIONS: Our findings demonstrated that IL-1alpha and P. gingivalis enhance t-PA production in human osteosarcoma cells, and that the signal transduction pathways p38, MEK, and PI3K are involved in the inhibition of t-PA. SB203580, U0126, and LY294002 suppress t-PA production and/or activity and may therefore be valuable therapeutics in t-PA-mediated periodontal destruction, and might be proved clinically useful agents, in combination with standard treatment modalities, in the treatment of periodontitis.     

Raf激酶抑制蛋白对促分裂原活化蛋白激酶通路的调控作用

Raf激酶抑制蛋白是一种高度保守的表达蛋白,它作为促分裂原活化蛋白激酶信号级联反应的内源性调控者,在生物体的生长和分化中扮演着重要的作用。Raf激酶抑制蛋白位于促分裂原活化蛋白激酶级联反应的交叉口,能调控多条信号传导通路,主要通过调节Raf-1的活性来介导其调控机制。因此,Raf激酶抑制蛋白可能是促分裂原活化蛋白激酶级联通路中一个新的关键调控位点。     

舌鳞癌细胞Tca8113中蛋白激酶C和丝裂原活化蛋白激酶激酶/细胞外调节蛋白激酶通路对诱导型一氧化氮合酶表达的影响

目的 在舌鳞癌细胞Tca8113中,探索与细胞增殖密切相关的诱导型一氧化氮合酶（NOS-2）的表达调控机制。方法 采用RNAi技术沉默Tca8113细胞中NOS-2、蛋白激酶C（PKC）-α、PKC-β和PKC-δ的基因;Griess Reagent法检测NOS-2基因沉默后一氧化氮（NO）生成量;CCK8法测定细胞增殖活性;实时定量荧光聚合酶链反应（q-PCR）技术检测各种方法处理后NOS-2、PKC-α、PKC-β和PKC-δ的基因表达;Western blotting技术测定丙二醇甲醚醋酸酯（PMA）作用细胞后的细胞外调节蛋白激酶（ERK）1/2磷酸化程度。结果 利用NOS-2的siRNA处理后,Tca8113细胞的增殖能力明显降低（P<0.01）;PKC的活性与NOS-2的基因表达成负相关（P<0.05）;PKC亚型PKC-α、PKC-β和PKC-δ共同参与NOS-2的基因调控（P<0.01）;丝裂原活化蛋白激酶激酶（MEK）/ERK通路负调控NOS-2的基因表达（P<0.05）;PKC通过MEK/ERK通路负调控NOS-2的基因表达（P<0.05）。结论 在Tca8113细胞中,PKC通过MEK/ERK通路负调控与细胞增殖相关的NOS-2的基因表达。     

生物力刺激和促丝裂原激活蛋白激酶对骨改建的影响

生物力信号转导是骨生物学研究的热点之一.通过研究流体剪切力、细胞外基质形变等生物力刺激下成骨细胞系的应答发现,生物力信号转导涉及促丝裂原激活蛋白激酶(MAPK)信号转导通路在内的多种信号系统.生物力刺激作用于整联蛋白、钙离子通道和脂筏等感受器,激活MAPK信号转导通路并通过级联反应调节下游分子的活性,如核心结合因子-α1和激活蛋白1等转录因子,进而调控成骨细胞的功能.同时生物力刺激诱导的MAPK信号转导通路与雌激素受体、甲状旁腺素受体和1,25-二羟胆骨化醇受体等信号转导通路存在交联作用,是生物力信号转导的重要途径.     

Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation of cultured human dental pulp cells by low-power gallium-aluminium-arsenic laser irradiation

AIM: To examine whether low-power laser irradiation (LPLI) promotes cellular proliferation of human dental pulp-derived fibroblast-like cells (dental pulp cells). METHODOLOGY: Dental pulp cells were obtained by primary culture of human dental pulp tissues from extracted third molar teeth. The phosphorylation of the mitogen-activated protein kinase (MAPK) family after LPLI of these cells was investigated by Western blotting. By using a specific MAPK/ERK kinase (MEK) inhibitor (PD098059), the specific effect of LPLI on the MAPK pathway was also investigated by Western blotting as described above. The incorporation of [3H]thymidine into the cells after LPLI was determined, and statistical analysis was performed by Wilcoxon signed-ranks test. RESULTS: Extracellular signal-regulated protein kinase (ERK) 1/2 was phosphorylated between 5 and 30 min after LPLI. Moreover, PD098059 inhibited LPLI-mediated ERK1/2 activation. LPLI did not affect p38 MAPK or c-Jun N-terminal kinase (JNK) phosphorylation. But LPLI did not stimulate [3H]thymidine incorporation into these cells. CONCLUSIONS: These results indicated that LPLI activated MAPK/ERK, a signal for proliferation, differentiation and survival, but did not activate the stress signals p38 MAPK and JNK in human dental pulp cells.     

机械压力对牙周膜成纤维细胞P38蛋白激酶激活及转位的影响

目的：研究P38蛋白激酶是否参与机械压力在牙周膜成纤维细胞的信号转导过程,初步探讨咬合创伤对牙周组织损伤机制。方法：本实验通过免疫组化法观察机械压力刺激后,牙周膜细胞内磷酸化P38蛋白激酶不同时间定位、强度的变化。结果：当压力大于200Kpa时,细胞内P38蛋白激酶磷酸化程度增强,且在30分钟时染色颗粒由脑浆转移至胞核,约60分钟后染色降至刺激前水平。结论：初步证明机械力信号可通过细胞路膜转导,并激活脑浆内P38蛋白激酶转移至胞核。     

机械力诱导牙周膜成纤维细胞p38 MAPK磷酸化反应的实验研究

目的：研究p38蛋白激酶(p38 MAPF)是否参与机械压力在牙周膜成纤维细胞的信号转导过程，初步探讨咬合创伤对牙周组织的损伤机制。方法：本实验通过免疫印迹法观察机械压力刺激后，牙周膜细胞内磷酸化p38 MAPK不同时段强度的变化。结果：当压力值为200kPa时，细胞内p38 MAPK磷酸化程度明显增强，约60min后磷酸化水平降至刺激前水平。结论：初步证明机械力信号可通过跨膜转导激活p38 MAPK，引起牙周膜细胞功能改变，导致牙周组织改建。     

周期性张应变对人牙周膜细胞迁移的影响

目的 探讨周期性张应变对人牙周膜细胞(human periodontal ligament cells,hPDLC)迁移的影响及其相关机制,为进一步了解机械力对hPDLC功能的影响提供资料.方法 应用FX-4000T细胞应变加载系统,对体外培养的hPDLC施加周期性张应变,牵张幅度分别为10%和20%,加载时间为6 h和24 h,频率均为0.1 Hz,以未加载的静态细胞作为对照组,应用划痕法观察hPDLC的迁移,蛋白质印迹法检测hPDLC的基质金属蛋白酶9(matrix metalloproteinases-9,MMP-9)的表达变化;用细胞外信号调节蛋白激酶(extracellular signal-regulated kinase,ERK)的特异性抑制剂PD98059预处理hPDLC后,在0.1 Hz、10%幅度条件下,加载6 h,检测细胞p-ERK1/2和MMP-9表达的变化.用细胞迁移划痕法观察加载10%和20%幅度张应变,观察24 h后PD98059和MMP家族的抑制剂强力霉素(doxycycline,DOX)对hPDLC迁移的影响.结果 细胞迁移划痕实验结果显示,牵张幅度和加载时间均可显著促进细胞迁移.与静态组相比,施加牵张幅度分别为10%和20%的张应变6 h后,hPDLC迁移变化不明显,但加载24 h后,10%和20%幅度的张应变均显著促进了hPDLC迁移(P<0.05),细胞迁移率分别为(45 ±8)%和(66±14)%,且20%比10%幅度牵张对细胞迁移的促进作用更显著(P<0.05).蛋白印迹法结果显示,周期性张应变促进了hPDLC的MMP-9表达.牵张幅度对细胞MMP-9的表达有显著影响(P<0.05),但加载时间对细胞MMP-9的表达无显著影响.ERK1/2的抑制剂PD98059不仅可以明显抑制张应变诱导的p-ERK1/2活化,而且可通过ERK信号通路明显抑制张应变诱导的细胞MMP-9表达.细胞迁移划痕实验还证实,加载幅度分别10%和20%的张应变24 h后,抑制剂PD98059和DOX均能有效抑制周期性张应变诱导的hPDLC迁移.结论 周期性张应变可通过激活hPDLC的ERK信号通路,促进细胞的MMP-9表达,从而诱导体外培养的hPDLC迁移.     

尼古丁调控人牙周膜细胞自噬水平的实验研究

目的 探讨尼古丁对人牙周膜细胞（hPDLCs）自噬水平的影响。方法 选取因正畸治疗而拔除的前磨牙,采用组织块法分离培养hPDLCs。通过Western blot法筛选尼古丁影响hPDLCs自噬的最佳作用时间及浓度,使用透射电子显微镜（TEM）和免疫荧光染色法检测该作用时间及浓度下hPDLCs自噬体形成情况和自噬标志蛋白LC3的表达情况。结果 LC3Ⅱ蛋白表达在尼古丁作用的12 h内持续升高,从而确定12 h为最佳作用时间;LC3Ⅱ蛋白表达上调具有尼古丁浓度依赖性,1×10^-5 mol·L^-1为尼古丁最佳作用浓度。TEM和免疫荧光染色证实尼古丁在此浓度及作用时间下hPDLCs细胞质内自噬体的数量增加,自噬标志蛋白LC3表达升高。结论 在一定条件下,尼古丁能够上调hPDLCs自噬水平,为进一步研究细胞自噬与吸烟相关牙周炎的关系奠定了基础。     

The up-regulation of heme oxygenase-1 expression in human gingival fibroblasts stimulated with nicotine

BACKGROUND: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. Heme oxygenase-1 (HO-1) is known as a stress-inducible protein and functions as an antioxidant enzyme. There is limited information on the expression of HO-1 in smoking-associated periodontal disease. OBJECTIVES: The aim of the present study was to investigate the effects of nicotine on the expression of HO-1 protein in cultured human gingival fibroblasts in vitro and further to compare HO-1 expression in gingival tissues obtained from cigarette smokers and non-smokers in vivo. METHODS: Western blot assay was used to investigate the effects on human gingival fibroblasts exposed to nicotine. In addition, antioxidants catalase, superoxide dismutase (SOD), and N-acetyl-l-cysteine (NAC) were added to test how they modulated the effects on nicotine-induced HO-1 expression. Gingival biopsies taken from the flap surgery of 20 male patients with periodontal disease (10 cigarette smokers and 10 non-smokers) were examined by immunohistochemistry. RESULTS: The exposure of quiescent human gingival fibroblasts to 10 mm nicotine resulted in the induction of HO-1 protein expression in a time-dependent manner (p < 0.05). The addition of glutathione (GSH) precursor NAC inhibited the nicotine-induced HO-1 protein expression (p < 0.05). However, SOD and catalase did not decrease the nicotine-induced HO-1 protein expression (p > 0.05). The results from immunohistochemistry demonstrated that HO-1 expression was significantly higher in cigarette smokers (p < 0.05). HO-1 was noted in the basal layers of epithelium, inflammatory cells, and fibroblasts in specimens from cigarette smoking. CONCLUSIONS: Taken together, these results suggest that HO-1 expression is significantly up-regulated in gingival tissues from cigarette smokers, and nicotine may, among other constituents, be responsible for the enhanced HO-1 expression in vivo. The regulation of HO-1 expression induced by nicotine is critically dependent on the intracellular GSH concentration.     

口腔癌相关成纤维细胞对舌癌细胞株细胞外信号调节激酶通路的影响

目的研究口腔癌相关成纤维细胞（CAFs）对舌癌细胞株细胞外信号调节激酶（ERK）通路的影响。方法以口腔CAFs条件培养基刺激舌癌细胞株Tca8113和将Tca8113细胞与口腔CAFs共同培养,采用Western杂交检测特定时间Tca8113细胞总ERK和总pERK的表达。结果Tca8113细胞经口腔CAFs条件培养基刺激或与口腔CAFs共同培养后,总pERK的表达迅速增加,总pERK与总ERK的比值增加。结论口腔CAFs对癌细胞ERK通路有活化作用。     

Mitogen-activated protein kinases mediate interleukin-1beta-induced receptor activator of nuclear factor-kappaB ligand expression in human periodontal ligament cells

BACKGROUND AND OBJECTIVE: Interleukin-1beta-stimulated receptor activator of nuclear factor-kappaB ligand (RANKL) expression in human periodontal ligament cells is partially mediated by endogenous prostaglandin E2, whereas mitogen-activated protein kinases (MAPKs) are implicated in regulating various interleukin-1-responsive genes. We investigated herein the involvement of MAPKs in interleukin-1beta-stimulated RANKL expression in human periodontal ligament cells. MATERIAL AND METHODS: Human periodontal ligament cells were pretreated separately with specific inhibitors of MAPKs, including extracellular signal-regulated kinase, p38 MAPK and c-Jun N-terminal kinase, and subsequently treated with interleukin-1beta. Following each treatment, the phosphorylation of each MAPK, the expression of RANKL, and the production of prostaglandin E2 were determined. RANKL activity was evaluated using an assay to determine the survival of prefusion osteoclasts. RESULTS: Interleukin-1beta induced RANKL expression at the mRNA and protein levels, as well as RANKL activity in human periodontal ligament cells. Interleukin-1beta also activated extracellular signal-regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Pretreatment with each MAPK inhibitor partially, but significantly, suppressed interleukin-1beta-induced RANKL expression and its activity, as well as prostaglandin E2 production. CONCLUSION: In human periodontal ligament cells, three types of MAPK inhibitor may abrogate RANKL expression and activity induced by interleukin-1beta, directly or indirectly through partial suppression of prostaglandin E2 synthesis. In addition, extracellular signal-regulated kinase, p38 MAPK, and c-Jun N-terminal kinase signals may co-operatively mediate interleukin-1beta-stimulated RANKL expression and its activity in those cells.     

PP-1蛋白稳定高表达MC3T3-E1成骨样细胞系的建立

目的建立稳定高表达蛋白质磷酸酶-1(protein phasphatase-1,PP-1)的MC3T3-E1成骨样细胞系。方法采用RT-PCR技术从MC3T3-E1细胞中扩增蛋白质磷酸酶-1(PP-1)基因,将获得的基因定向插入pCI-neo真核表达质粒中,PCR及双酶切鉴定后用脂质体将表达载体转染入MC3T3-E1细胞,经G418加压有限稀释筛选,建立稳定高表达PP-1的MC3T3-E1成骨样细胞系,采用Western blot法检测PP-1的蛋白表达情况。结果 PCR及双酶切鉴定表明表达载体pCI-neo-PP-1构建正确;Western blot检测结果显示PP-1蛋白能在转染pCI-neo-PP-1的MC3T3-E1细胞中稳定高表达。结论成功建立了稳定高表达PP-1的MC3T3-E1成骨样细胞系,为进一步深入研究PP-1在细胞力学信号转导通路中的功能及作用机制奠定了实验基础。     

Role of p38 mitogen-activated protein kinase pathway in Porphyromonas gingivalis lipopolysaccharide-induced VCAM-1 expression in human aortic endothelial cells

Background: Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) has been reported to induce the expression of vascular cell adhesion molecule‐1 (VCAM‐1) in vascular endothelial cells. This finding suggests the potential roles for Pg in the pathogenesis of atherosclerosis. However, the mechanism involved in Pg LPS‐induced VCAM‐1 production in endothelial cells remains unclear. Methods: Quantitative real‐time polymerase chain reaction and Western blotting were used, respectively, to investigate the mRNA expression and protein production of VCAM‐1 in human aortic endothelial cells (HAECs) induced by Pg LPS. The involvement of the p38 mitogen‐activated protein kinase (p38 MAPK) cell signaling pathway in VCAM‐1 expression was investigated by assays with specific inhibitors. Results: Pg LPS–induced expression in HAECs of VCAM‐1 occurred in a dose‐ and time‐dependent manner. In addition, the p38 MAPK inhibitor (SB 203580) significantly attenuated Pg LPS–induced VCAM‐1 expression. Conclusion: Activation of p38 MAPK is at least partially involved in Pg LPS–induced VCAM‐1 expression in HAECs, which may contribute to the acceleration of atherosclerosis.     

Behaviour of human osteoblastic cells cultured on plasma-sprayed titanium implants in the presence of nicotine

Objectives: The aim of this work was to analyse the behaviour of human bone marrow osteoblastic cells cultured on the surface of routinely used plasma‐sprayed titanium implants in the presence of plasmatic and salivary nicotine levels reported in smokers. Material and methods: Human bone marrow cells (first subculture) were seeded on titanium implants and cultured for 35 days in α‐minimal essential medium supplemented with 10% foetal bovine serum, 50 μg/ml ascorbic acid, 10 mM β‐glycerophosphate and 10 nM dexamethasone. Seeded implants were exposed to nicotine, 10–1 mg/ml, from days 1 to 35, and characterized for cell morphology, viability/proliferation, alkaline phosphatase (ALP) activity and matrix mineralization. Results: Low levels of nicotine, 10 and 50 ng/ml, representative of the plasma concentrations reported in smokers, did not cause significant effects in the cell behaviour, although a small induction in cell growth and functional activity appeared to occur. Higher nicotine levels, 0.01–1 mg/ml, within those attained in saliva through tobacco use, caused evident dose‐dependent effects in osteoblastic cell behaviour, i.e., a stimulatory effect in cell growth, ALP activity and matrix mineralization, at concentrations up to 0.2 mg/ml, and a deleterious effect at higher levels. Conclusions: Considering the high tissue diffusion potential of nicotine, the results suggest the possibility of a direct modulation of the osteoblast activity as a contributing factor to the overall effect of nicotine in the bone microenvironment around dental implants.