红藻氨酸致痫后大鼠海马ERK、P38 MAPK和JNK的活性变化

目的研究红藻氨酸(KA)诱导大鼠癫痫发作后海马组织细胞外调节蛋白激酶(ERK)、p38MAPK和c-jun氨基末端激酶(JNK)的活性(磷酸化状态)的变化情况。方法立体定向大鼠侧脑室内注射KA引起大鼠癫痫发作,采用Western-blot方法观察KA致痫后大鼠海马中活性ERK、p38MAPK和JNK的变化。结果KA诱导大鼠癫痫发作后,海马组织ERK、p38MAPK和JNK的磷酸化水平开始增高,分别于30min、1h和30min后达高峰,呈对照组的4.76倍、2.16倍和3.95倍,两组比较差异具有显著性(P<0.01),之后逐渐下降。结论KA致痫大鼠癫痫发作后,海马组织MAPKs的活性产生变化,其信号通路可能参与癫痫发作后海马组织的病理生理反应过程。     

G蛋白耦联受体激酶结合蛋白1通过调节细胞外调节激酶1/2的活性促进成骨细胞迁移

目的探索G蛋白耦联受体激酶结合蛋白1(GITI)在成骨细胞迁移中的作用,并分析其机理。方法通过Western blot方法检测GIT1蛋白在鼠的成骨细胞内的表达；用免疫荧光染色方法确定：在血小板衍生生长因子(PDGF)不刺激和刺激的条件下,GIT1和细胞外调节激酶1/2(ERK1/2)在成骨细胞内的位置；用共同免疫沉淀的方法测定GIT1和ERK1/2相互结合,并且用免疫荧光双染的方法确定这两种蛋白相互结合的位置；用包含GIT1-RNA发夹结构的腺病毒感染成骨细胞后,用免疫荧光染色方法确定磷酸化ERK1/2(pERK1/2)在成骨细胞内的位置,用划痕愈合法检测在PDGF刺激下的迁移能力。结果在成骨细胞内,PDGF刺激导致了GIT1和ERK1/2的相互结合,并且这种结合发生在成骨细胞的局部粘附内。包含GIT1-RNA发夹结构的腺病毒明显抑制了pERK1/2招募至成骨细胞局部粘附内以及PDGF所刺激的成骨细胞的迁移。结论在PDGF刺激下,GIT1招募pERK1/2至成骨细胞的局部粘附内,从而促进成骨细胞的迁移。     

东菱迪芙对大鼠局灶性脑缺血再灌注后JNK和ERK活性的影响

目的探讨东菱迪芙对局灶性脑缺血再灌注大鼠脑内c-Jun氨基末端激酶(JNK)和胞外信号调节酶(ERK)活性的影响。方法采用大脑中动脉线栓法(MCAO)建立大鼠局灶性脑缺血再灌注模型,用免疫组织化学法和免疫印迹法检测ERK和JNK的活性,同时观察缺血侧脑组织形态学变化、脑梗死体积比、凋亡细胞数。结果东菱迪芙可下调脑缺血再灌注大鼠脑组织JNK蛋白的活性,上调ERK蛋白的活性,并降低梗死体积、坏死和凋亡细胞数。结论东菱迪芙对大鼠局灶性脑缺血再灌注损伤有保护作用,抑制JNK凋亡通路、促进ERK生存通路, 从而减轻细胞凋亡是其脑保护机制之一。     

K-Ras gene status and expression of Ras/mitogen-activated protein kinase (MAPK) signaling molecules in ameloblastomas

BACKGROUND: To clarify the roles of rat sarcoma (Ras)/mitogen-activated protein kinase (MAPK) signaling pathway in oncogenesis and cytodifferentiation of odontogenic tumors, K-Ras gene status and expression of Ras, Raf1, MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)1, and ERK1/2 proteins were analyzed in ameloblastomas as well as in tooth germs. METHODS: Paraffin sections of 10 tooth germs and 46 benign and 6 malignant ameloblastomas were examined immunohistochemically for the expression of K-Ras, Raf1, MEK1, and ERK1/2. Frozen tissue samples of 22 benign ameloblastomas and 1 malignant (metastasizing) ameloblastoma were analyzed by direct DNA sequencing to detect K-Ras gene alteration. RESULTS: Immunohistochemical reactivity for K-Ras, Raf1, MEK1, and ERK1/2 was detected in both normal and neoplastic odontogenic epithelium, and these molecules were reactive chiefly with odontogenic epithelial cells neighboring the basement membrane. Plexiform ameloblastomas showed slightly stronger expression of these Ras/MAPK signaling molecules than follicular ameloblastomas. Keratinizing cells and granular cells showed decreased reactivity for the signaling molecules. Basal cell ameloblastomas showed slightly stronger reactivity for the signaling molecules than did the other subtypes. K-Ras immunoreactivity in malignant ameloblastomas was lower than that in dental lamina of tooth germs. Direct DNA sequencing showed a GGT to GCT point mutation at codon 12 of K-Ras gene in one ameloblastoma. Conclusion: Expression of K-Ras, Raf1, MEK1, and ERK1/2 in tooth germs and ameloblastomas suggests that Ras/MAPK signaling pathway functions to regulate cell proliferation and differentiation in both normal and neoplastic odontogenic epithelium. K-Ras gene status implied that K-Ras mutations might play a minor role in oncogenesis of odontogenic epithelium.     

Differential regulation of cytokine genes in gingival epithelial cells challenged by Fusobacterium nucleatum and Porphyromonas gingivalis

IL-8 mRNA in human gingival epithelial cells (HGECs) is up-regulated by Fusobacterium nucleatum, and up-/down-regulated by Porphyromonas gingivalis in a complex interaction in the early stages (< or = 4 h) after infection. The mechanisms involved in this regulation in response to F. nucleatum and/or P. gingivalis infection, and identification of co-regulated cytokine genes, are the focus of this investigation. Heat, formalin or protease treatment of F. nucleatum cells attenuated the IL-8 mRNA up-regulation. NF-kappaB, mitogen-activated protein kinase (MAPK) p38 and MAPK kinase/extracellular signal-regulated kinase (MEK/ERK) pathways were involved in IL-8 mRNA induction by F. nucleatum. Pretreatment of P. gingivalis with heat, formalin or protease enhanced IL-8 mRNA induction. NF-kappaB, MARK p38, and MEK/ERK pathways were also involved in this induction. In contrast, down-regulation of IL-8 mRNA by P. gingivalis involved MEK/ERK, but not NF-kappaB or MAPK p38 pathways. cDNA arrays analysis revealed that mRNA down-regulation by P. gingivalis is a specific reaction that only a number of genes, e.g. IL-1beta, IL-8, macrophage inflammatory protein-2alpha, and migration inhibitory factor-related protein-14, are affected based on examination of 278 cytokine/receptor genes. These data indicate that F. nucleatum and P. gingivalis trigger specific and differential gene regulation pathways in HGECs.     

磷酸化的细胞外信号调节蛋白激酶1/2在正常小鼠脑内的免疫细胞化学定位

细胞外信号调节蛋白激酶(ERK)1/2是重要的信号转导分子。现已知在正常动物的中枢神经系统内有其活化形式即磷酸化的ERK1/2(pERK1/2)分子的存在,但其在小鼠脑内的分布目前还没有全面的观察。本研究用免疫组织化学技术(ABC法)研究了pERK1/2样免疫反应阳性产物在脱臼处死的正常小鼠全脑内的分布。结果发现pERK1/2在正常小鼠脑内有广泛的表达,阳性产物主要存在于神经元,亦见于个别白质内的胶质细胞,脑膜及室管膜细胞也有表达。强阳性表达的核团主要有:岛皮质、视听皮质、边缘前皮质、扣带前皮质、海马垂直部、弓状核、蓝斑和小脑Purkinje细胞;中等阳性表达的核团主要有:感觉运动皮质、外侧隔区、内侧杏仁核、皮质杏仁核和外侧杏仁核、丘脑室旁核前部、视交叉上核、穹隆下器、终板血管器、前腹侧视前核和下丘脑背内侧核;弱阳性表达的核团主要有:视上核、下丘脑室旁核大细胞部、下丘脑后区、顶盖前区、室周灰质腹外侧柱、A5区、孤束核和延髓腹外侧网状结构等。本文结果观察到pERK1/2在正常小鼠脑内广泛存在,提示pERK1/2作为重要的信号转导分子,可能参与许多脑区在正常状态下的功能活动,揭示其分布特点为了解其在脑内的多样性功能提供了形态学依据。     

降钙素基因相关肽通过ERK1/2信号通路对角质形成细胞增殖的影响

目的： 研究降钙素基因相关肽（CGRP）对角质形成细胞增殖活性的影响，并探讨其可能涉及的信号转导通路。方法： ①胸腺嘧啶掺入法（［3H］-TdR）观察CGRP诱导的角质形成细胞株HaCaT细胞增殖，及CGRP受体拮抗剂CGRP8-37、细胞外信号调节激酶ERK1/2特异性抑制剂PD98059对CGRP诱导的增殖活性的影响；②免疫印迹技术观察CGRP诱导后ERK1/2的磷酸化，及CGRP8-37、PD98059对ERK1/2磷酸化的影响。结果： ①CGRP在一定范围内可剂量依赖性地诱导HaCaT细胞增殖，该作用可被CGRP8-37和 PD98059阻断；②CGRP可时间依赖性地诱导HaCaT细胞ERK1/2的磷酸化，CGRP8-37和PD98059可减弱其作用。结论： CGRP可诱导HaCaT细胞增殖，CGRP受体及其相关的ERK1/2信号通路参与其调控机制。     

Cooperation between IL-7 and the pre-B cell receptor: a key to B cell selection

Hematopoiesis is regulated by a variety of signals that either originate within a developing cell or are supplied by the surrounding environment in secreted- or contact-dependent forms. This review discusses the effects of one secreted factor, interleukin-7, on the development of B lymphocytes. We describe a molecular mechanism for a crucial checkpoint during B lineage maturation, based on the integration of signals mediated by the pre-B cell receptor, the interleukin-7 receptor, and the environment in which these signals are received.     

Study of platelet-rich fibrin promoting endothelial cell differentiation and angiogenesis induced by transplantation of adipose-derived stem cells

Diabetic patients are characterized by long wound healing time, and adipose stem cells (ADSCs) can secrete growth factors to promote angiogenesis and improve diabetic wound healing. In this research, we attempted to interrogate the impact of platelet-rich fibrin (PRF) on ADSCs in diabetic wound healing. ADSCs were harvested from human adipose tissues and identified through flow cytometry. After pretreatment with cultured medium supplemented with different concentrations of PRF (2.5%, 5%, and 7.5%), proliferation and differentiation capacity of ADSCs were assessed by CCK-8 assay, qRT-PCR and immunofluorescence (IF), respectively. Tube formation assay measured angiogenesis. Western blot analysis analyzed expression of endothelial markers and the extracellular signal-regulated kinase (ERK) and serine/threonine kinase (Akt) pathways in PRF-induced ADSCs. The CCK-8 experiment indicated that PRF enhanced proliferation of ADSCs in dose-dependent manner, relative to normal control group. The expression of endothelial markers and the capacity of tube formation were significantly promoted by 7.5% PRF. The release of growth factors containing vascular endothelial grow factor (VEGF) and insulin-like growth factor-1 (IGF-1) from PRF was increased with the extension of detection time. When the receptors of VEGF or/and IGF-1 were neutralized, ADSCs differentiation into endothelial cells were obviously inhibited. Additionally, PRF stimulated ERK and Akt pathways, and the inhibitors of ERK and Akt attenuated PRF-induced differentiation of ADSCs into endothelial cells. In conclusion, PRF promoted endothelial cell differentiation and angiogenesis induced by ADSCs in diabetic wound healing, which appears to give guidance for treating patients.     

金粉蕨素拮抗氧化损伤所抑制的内皮细胞增殖的作用及其机制

目的　探讨金粉蕨素拮抗Menadione氧化损伤所抑制的内皮细胞增殖的作用及其机制。方法　以Menadione(O- 2 )损伤人脐静脉内皮细胞作为氧化损伤模型 ,采用MTT法和细胞计数法 ,观察不同浓度金粉蕨素对Menadione损伤内皮细胞生长抑制率的影响 ;利用硝酸还原酶法测定培养液中NO含量 ;以Westernblot检测细胞eNOS活性及磷酸化ERK1 / 2的表达。结果　金粉蕨素保护组与损伤组相比 ,内皮细胞的生长抑制率明显降低 ,培养液中NO含量增高 ,eNOS活性增强 ,磷酸化ERK1 / 2表达上调。结论　NO和ERK1 / 2通路可能介导了金粉蕨素拮抗Menadione氧化损伤所抑制内皮细胞增殖的保护作用