MEK抑制剂提高博莱霉素A5诱导口腔癌细胞株表达细胞凋亡的信号通路机制

博莱霉素A5是一种葡萄糖肽抗生素类的肿瘤化疗药物。以往的研究证实,博莱霉素A5在促进肿瘤细胞凋亡发生中发挥着重要作用。本实验利用人口腔表皮样癌KB细胞,研究博莱霉素A5、博莱霉素A5联合MEK(MAP-ERK激酶)抑制剂(PD98059)对促有丝分裂蛋白激酶(MAPK)信号通路家族中的Jun癌基因N末端激酶(JNK)和细胞外信号调节激酶(ERK)信号通路对细胞凋亡的影响,阐明博莱霉素A5促进细胞凋亡的信号通路传导,以期降低博莱霉素A5在联合化疗中的药量,减少其毒副作用,从而为联合化疗开辟一种新的治疗方法。材料和方法①细胞培养:KB细胞于改良的Du…     

红细胞生成素及其受体对成骨细胞的作用机制

红细胞生成素(EPO)与其受体(EPOR)结合引发信号转导,调节红细胞的增殖和分化.EPO激活贾纳斯激酶(JAK)及其下游的丝裂原激活蛋白激酶(MAPK)、磷脂酰肌醇-3-激酶(PI3K)、信号转导子和转录激活子(STAT)等信号传导通路,参与调节原始干细胞发育、细胞完整性、血管再生和神经发育等.EPO具有促进骨髓间质干细胞向成骨细胞分化的作用,促进成骨细胞的骨形成和骨折的愈合,在牵张成骨过程中可加快骨的愈合速度.EPO和EPOR在骨髓间质干细胞或骨髓基质细胞等成骨向分化和成骨细胞中,主要通过JAK-STAT、PI3K-蛋白激酶B、MAPK和核因子-κB等信号转导通路发挥作用,进而促进成骨细胞的骨形,这对于口腔正畸牙移动和颌面外科的牵张成骨等皆有十分重要的临床意义.     

34．MEK抑制剂提高博莱霉素A5诱导口腔癌细胞株表达细胞凋亡的信号通路机制[英]

博莱霉素A5是一种葡萄糖肽抗生素类的肿瘤化疗药物。以往的研究证实，博莱霉素A5在促进肿瘤细胞凋亡发生中发挥着重要作用。本实验利用人口腔表皮样癌KB细胞，研究博莱霉素A5、博莱霉素A5联合MEK(MAP-ERK激酶)抑制剂(PD98059)对促有丝分裂蛋白激酶(MAPK)信号     

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。     

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.     

平阳霉素对人脐静脉内皮细胞系ECV304的抑制作用及其机制的实验研究

目的：探讨平阳霉素对人脐静脉内皮细胞系ECV304的抑制作用及其相关机制。方法：用不同浓度的平阳霉素作用ECV304细胞不同时间,以MTT法比较平阳霉素作用24、48及72h后的细胞抑制率,应用DNA电泳实验、流式细胞术分析细胞凋亡、坏死情况、细胞周期及Fas、Bcl-2蛋白的表达。结果：MTT法显示随平阳霉素浓度升高和作用时间的延长,细胞生长抑制越明显DNA电泳证实合适浓度平阳霉素作用下细胞发生凋亡,过高浓度细胞则发生坏死;流式细胞术显示细胞凋亡率和坏死率随药物浓度和作用时间增加而增高,过高浓度细胞坏死占主导;平阳霉素各浓度作用24h后,G2—M期百分率增高,S期百分率降低,G0—G1期百分率无明显变化;Fas表达随药物浓度增加而递减,Bcl-2表达随药物浓度增加而升高。结论：平阳霉素能明显抑制ECV304细胞生长,并具有浓度和时间依赖性;平阳霉素可能通过诱导凋亡和坏死作用以抑制ECV304细胞的体外生长和增殖;细胞凋亡发生在G2—M期,是通过上调Fas蛋白并下调Bcl-2蛋白的表达来实现的。     

马来酸噻吗洛尔对人脐静脉内皮细胞VEGF传导通路表达的影响

目的:研究马来酸噻吗洛尔在人脐静脉内皮细胞(HUVEC)中血管生成信号传导通路的表达,以探讨这些药物作为治疗婴幼儿血管瘤的作用机制。方法:采用RT-PCR和Western blot检测蛋白激酶B(Akt)、细胞外调节蛋白激酶(ERK)、局部黏着斑激酶(FAK)和哺乳动物雷帕霉素靶蛋白(mTOR)的表达。应用SPSS17.0统计包进行数据分析。结果:RT-PCR检测发现,马来酸噻吗洛尔以剂量依赖的方式明显抑制血管内皮生长因子(vascular endothelial growth factor,VEGF)诱导的ERK、Akt、mTOR及FAK的mRNA表达;Western blot结果显示:马来酸噻吗洛尔以剂量依赖的方式明显抑制VEGF对磷酸化的细胞外调节蛋白激酶(p-ERK)、磷酸化蛋白激酶B(p-Akt)、磷酸化的哺乳动物雷帕霉素靶蛋白(p-mTOR)的调控。结论:马来酸噻吗洛尔可以通过抑制ERK、Akt、mTOR、FAK信号通路的表达,抑制血管瘤内皮细胞增殖和促进细胞凋亡,从而达到治疗婴幼儿血管瘤的目的。     

表皮生长因子受体抑制剂AG1487对舌鳞状细胞癌细胞Tca8113细胞外信号调节激酶抑制作用的研究

目的了解表皮生长因子受体抑制荆AG1487对舌鳞状细胞癌细胞细胞外信号调节激酶（ERK）的抑制作用。方法采用免疫细胞化学以及Western blot方法研究Tca8113细胞在不同浓度AG1487状态下，细胞外信号调节激酶的表达情况。结果免疫细胞化学结果显示：AG1487可明显抑制细胞核内活化ERK的表达，但对细胞质内全部ERK的表达没有明显影响；Westernblot结果进一步证实，AG1487可抑制活化ERK的表达，并且。活化ERK的表达与AG1487存在剂量依赖关系。结论表皮生长因子受体抑制剂AG1487可明显抑制其MAPK信号通路，进而对细胞的分裂增殖产生抑制作用。     

核因子-κB传导通路与舌癌细胞平阳霉素化疗敏感性的关系

的 探讨核因子-κB（NF-κB）/p65信号传导通路与Tca8113细胞平阳霉素化疗敏感性的关系。方法 以脂质体为载体,转染2 mg/L的p65反义寡核苷酸于Tca8113细胞 后,8 mg/L的平阳霉素处理细胞,3 h、6 h后免疫组化、Western blot法检测细胞核内p65形态和含量变化,48 h后MTT法检测细胞抑制率。结果 平阳霉素能激活细胞内的NF-κB/p65信号传导通路,而p65反义寡核苷酸转染能明显抑制该信号通路,在6 h时胞核内p65含量显著减少（P<0.05）,48 h时细胞抑制率明显提高（P<0.05）。结论 抑制NF-κB/p65信号传导通路能够提高Tca8113细胞平阳霉素化疗敏感性。     

p38丝裂素活化蛋白激酶信号通路在应力调控成肌细胞分化中的作用

目的研究在C2C12细胞成肌分化过程中应力对丝裂原活化蛋白激酶（MAPKs）信号通路中p38丝裂素活化蛋白激酶（p38MAPK）信号通路的影响。方法将6孔Bio Flex培养板上贴壁培养的C2C12细胞,以0.5 Hz的加载频率和10%的细胞拉伸变形幅度,分别进行拉伸培养2、6、12、24 h,应用Western blot免疫印迹检测总p38和磷酸化p-p38（Thr180/Tyr182）蛋白的表达情况。结果周期性机械拉伸在调控C2C12成肌细胞分化过程中,p38MAPK信号通路被激活。p38MAPK信号通路蛋白磷酸化水平在较高水平;而p38MAPK通路总蛋白表达维持在一基线水平,各组之间差异无统计学意义。加入p38MAPK信号通路特异性抑制剂SB203580后再加力,Myogenin的表达明显降低。结论p38MAPK信号通路在应力介导的C2C12成肌细胞分化过程中发挥重要作用,但不是这一调控过程的唯一通路。     

Aloin Inhibits Interleukin (IL)‐1β−Stimulated IL‐8 Production in KB Cells

Background: Interleukin (IL)‐1β, which is elevated in oral diseases including gingivitis, stimulates epithelial cells to produce IL‐8 and perpetuate inflammatory responses. This study investigates stimulatory effects of salivary IL‐1β in IL‐8 production and determines if aloin inhibits IL‐1β?stimulated IL‐8 production in epithelial cells. Methods: Saliva was collected from volunteers to determine IL‐1β and IL‐8 levels. Samples from volunteers were divided into two groups: those with low and those with high IL‐1β levels. KB cells were stimulated with IL‐1β or saliva with or without IL‐1 receptor agonist or specific mitogen‐activated protein kinase (MAPK) inhibitors. IL‐8 production was measured by enzyme‐linked immunosorbent assay (ELISA). MAPK protein expression involved in IL‐1β?induced IL‐8 secretion was detected by Western blot. KB cells were pretreated with aloin, and its effect on IL‐1β?induced IL‐8 production was examined by ELISA and Western blot analysis. Results: Saliva with high IL‐1β strongly stimulated IL‐8 production in KB cells, and IL‐1 receptor agonist significantly inhibited IL‐8 production. Low IL‐1β–containing saliva did not increase IL‐8 production. IL‐1β treatment of KB cells induced activation of MAPK signaling molecules as well as nuclear factor‐kappa B. IL‐1β?induced IL‐8 production was decreased by p38 and extracellular signal‐regulated kinase (ERK) inhibitor treatment. Aloin pretreatment inhibited IL‐1β?induced IL‐8 production in a dose‐dependent manner and inhibited activation of the p38 and ERK signaling pathway. Finally, aloin pretreatment also inhibited saliva‐induced IL‐8 production. Conclusions: Results indicated that IL‐1β in saliva stimulates epithelial cells to produce IL‐8 and that aloin effectively inhibits salivary IL‐1β–induced IL‐8 production by mitigating the p38 and ERK pathway. Therefore, aloin may be a good candidate for modulating oral inflammatory diseases.     

Synergistic effects of verapamil on pingyangmycin-induced cytotoxicity and apoptosis in KB cells

OBJECTIVES: Previous studies have shown that pingyangmycin (PYM; bleomycin A5) can induce two distinct modes of cell death (necrosis, apoptosis). At high concentrations, PYM can be considered as an apoptosis mimetic drug. In this study, we explored the possibility that the membrane-modifying agent verapamil might affect the transport function of PYM through the plasma membrane, resulting in inducing apoptosis of tumor cells at low concentration of PYM. METHODS: Cytotoxicity, flow cytometry and DNA fragmentation assays were used to detect the interaction of verapamil and PYM in human oral carcinoma cell line KB cells. RESULTS: Our results indicated that verapamil can enhance the cytotoxicity of PYM against KB cells with the non-toxic doses (P<0.05). The cell viability at a concentration of 500 microg ml-1 of PYM was 35+/-2% compared with control and 10 microg ml-1 verapamil decreased the cell viability lower to 28+/-1%. In addition, because of the synergistic effect of verapamil, KB cells apoptosis was found to be induced when treated with a lower concentration of PYM (50 microg ml-1) for 24 h by flow cytometry and DNA fragmentation assays. CONCLUSIONS: Verapamil was found to enhance PYM-induced cytotoxicity and apoptosis in KB cells. The responsiveness of PYM might be explained by the effective accumulation of PYM by verapamil in KB cells mediated by the inhibition of PYM efflux function of the cells.     

丝氨酸/苏氨酸蛋白激酶通路介导的p53基因抑制口腔鳞状细胞癌增殖和侵袭的实验研究

目的 探讨人重组p53腺病毒（rAd-p53）注射液抑制口腔鳞状细胞癌增殖和侵袭的分子机制。方法 采用 rAd-p53注射液转染人舌鳞状细胞癌细胞系Tca8113,观察p53基因过表达对该细胞系增殖及侵袭能力的影响,并用蛋白免疫印迹的方法检测Ad-p53转染前后Tca8113细胞系内丝裂原激活的蛋白激酶（MAPK）、丝氨酸/苏氨酸蛋白激酶（AKT）信号通路相关蛋白,细胞周期及凋亡调控蛋白细胞周期素D1（Cydin D1）、P21、Bcl-2的表达。结果 Ad-p53 转染后显著抑制Tca8113细胞系增殖和侵袭（P<0.01）,并促进细胞凋亡（P<0.001）。蛋白免疫印迹结果显示,rAd-p53 转染后显著提高了Tca8113细胞P53和P21蛋白的表达,同时显著下调了Cydin D1、Bcl-2蛋白的表达及AKT蛋白的磷酸化（P<0.01）。结论 AKT信号通路可能是p53引起的口腔鳞癌细胞增殖和侵袭抑制的关键分子机制,Cyclin D1、P21和Bcl-2蛋白可能是AKT信号通路的下游调控基因,AKT信号通路及下游调控基因有望成为肿瘤基因治疗靶点。     

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.     

Midkine inhibits apoptosis via extracellular signal regulated kinase (ERK) activation in PC12 cells

Midkine (MK) is a new member of the family of heparin-binding neurotrophic factors. MK has several important biological effects and plays an important role in the development and survival of neurons. The mechanism by which MK exerts its neurotrophic actions, however has not been sufficiently clarified. To understand the intracellular pathway activated by MK, we established an apoptosis-induction system with the neuronal cell line PC12 and studied the involvement of the mitogen-activated protein kinase (MAPK) cascade in neuroprotective actions of MK. We demonstrate here that MK rescued PC12 cells from apoptosis induced by serum deprivation in a dose-dependent manner. MK also activated extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), which are well known as signal transducer acting downstream several receptors. PD98059, an inhibitor of MAPK kinase (MAPKK), inhibited ERK activation and also prevented the trophic effect of MK. These results indicate that MK exerts its neuroprotective actions mainly via ERK activation.     

The potential role of transient receptor potential type A1 as a mechanoreceptor in human periodontal ligament cells

Transient receptor potential type A1 (TRPA1) is reported to be a Ca²⁺‐permeable channel and is activated by cold temperatures and mechanical stimuli in the hair cells and in dorsal root ganglion. Using a DNA microarray, we found that TRPA1 was significantly up‐regulated in human periodontal ligament (hPDL) cells 2 d after intermittent mechanical stimulation (iMS) loading compared with unloaded cells. Although hPDL cells are known to respond to mechanical stimulation induced by occlusal force, little is known about the expression and functional role of TRPA1 in these cells. Therefore, we investigated the effects of iMS on TRPA1 expression and its signaling pathway in hPDL cells. Intermittent mechanical stimulation loading up‐regulated TRPA1 expression in hPDL cells in a time‐dependent manner, but had no effect on other mechanoreceptors. Furthermore, iMS significantly increased the phosphorylation of mitogen‐activated protein kinases (MAPKs), especially extracellular signal‐regulated kinase 1/2 (ERK1/2) and p38, and the expression of C‐C chemokine ligand 2 (CCL2). Transient receptor potential type A1 agonists also increased MAPK phosphorylation and the intracellular Ca²⁺ concentration. By contrast, inhibition or silencing of TRPA1 partially suppressed iMS‐induced MAPK phosphorylation. In summary, iMS during occlusion activates TRPA1 and MAPK signaling in periodontal ligament tissues, suggesting that TRPA1 regulates the mechanosensitivity of occlusal force via activation of MAPKs in hPDL cells.