钙调神经磷酸酶-T细胞核因子信号通路在应力诱导成肌细胞凋亡中的作用

目的 探讨钙调神经磷酸酶（CaN）-T细胞核因子（NFAT）信号通路在应力介导的成肌细胞凋亡中的作用。方法 构建成肌细胞体外培养-力学刺激模型,利用多通道应力加载系统对细胞加载不同时间的周期性张应力,加入CaN的特异性抑制剂环孢素（CsA）作为对比。采用Hoechst 33258染色法和流式细胞术检测成肌细胞凋亡情况,实时聚合酶链式反应检测CaN和NFAT mRNA的表达情况,蛋白质印迹法检测NFAT3的蛋白含量。结果 随加力时间的延长,细胞凋亡逐渐增加,CaN亚基CnA、CnB及NFAT3的mRNA表达及NFAT3蛋白含量逐渐升高;加入 CsA后,细胞凋亡减少,CnA、NFAT3的mRNA表达及NFAT3的蛋白含量明显减少。结论 周期性张应力可以诱导成肌细胞发生凋亡;CaN-NFAT信号通路可能参与了周期性张应力诱导的成肌细胞凋亡。     

人脐静脉内皮细胞的分离培养和鉴定及酪氨酸激酶受体-2在其中的表达

目的分离和培养人脐静脉内皮细胞（HUVECs）,对其进行鉴定,并探讨酪氨酸激酶受体-2（Tie-2）基因在HUVECs中的表达情况。方法用胰蛋白酶消化、分离HUVECs并对其进行培养,根据细胞生长特点、形态特征和Ⅷ因子免疫荧光组化技术对细胞进行鉴定。分别采用逆转录-聚合酶链反应（RT-PCR）和SABC免疫细胞化学染色对HUVECs中Tie-2 mRNA和蛋白表达情况进行检测。结果原代培养的HUVECs约于24 h完全贴壁,4～5 d后融合成单层铺路石样结构。Ⅷ因子免疫荧光组化法证实细胞是HUVECs。RT-PCR检测到HUVECs中Tie-2 mRNA条带明显,SABC免疫细胞组化法检测到Tie-2蛋白在HUVECs胞浆、核膜中呈强阳性表达,阳性率为85％以上。结论胰蛋白酶灌流消化法可获得高纯度的HUVECs,Tie-2在HUVECs中呈强阳性表达。     

Alpha1-adrenergic receptor stimulation induces the expression of receptor activator of nuclear factor kappaB ligand gene via protein kinase C and extracellular signal-regulated kinase pathways in MC3T3-E1 osteoblast-like cells

The receptor activator of nuclear factor kappaB ligand (RANKL) produced by bone marrow stromal/osteoblast cells is a crucial regulator of osteoclastgenesis and bone resorption. Osteoblastic cells have been demonstrated to express alpha(1)-adrenergic receptors. OBJECTIVE: The purpose of this study was to test the hypothesis that alpha(1)-adrenergic receptor stimulation induces the expression of RANKL gene via protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways in osteoblastic cells. DESIGN: The steady-state mRNA levels of RANKL and activation of ERK in mouse MC3T3-E1 osteoblast-like cells were analyzed by semi-quantitative RT-PCR and Western blotting, respectively. RESULTS: In three alpha(1)-adrenergic receptor subtype mRNAs, alpha(1b)- and alpha(1d)-subtypes were expressed in MC3T3-E1 cells. The mRNA levels of RANKL were increased by phenylephrine (alpha(1)-agonist) in time- and dose-dependent manners. Prazosin (alpha(1)-antagonist) suppressed the phenylephrine-induced RANKL mRNA expression, but yohimbine (alpha(2)-antagonist) and propranolol (beta-antagonist) did not. Phorbol 12-myristate 13-acetate (PMA, PKC activator) increased RANKL mRNA expression and GF109203X (PKC inhibitor) suppressed the phenylephrine-induced RANKL mRNA expression. Both phenylephrine and PMA stimulated the phosphorylation of ERK, while both prazosin and GF109203X inhibited phenylephrine-induced ERK activation. Pretreatment with PD98059 (ERK kinase inhibitor) inhibited both the phosphorylation of ERK and the expression of RANKL gene induced by phenylephrine in MC3T3-E1 cells. CONCLUSION: These results show that alpha(1b)- and alpha(1d)-adrenergic receptor subtype genes are expressed and the expression of RANKL mRNA may be regulated by alpha(1)-adrenergic receptor stimulation in osteoblastic cells. The induction of RANKL mRNA by activating the alpha(1)-adrenergic receptor is probably mediated via PKC and ERK signalling pathways in osteoblastic cells.     

Up-regulation of vascular endothelial growth factor expression by adenosine through adenosine A2 receptors in the rat tongue treated with endotoxin

The main focus of the present investigation is to evaluate a differential effect of adenosine on the up-regulation of vascular endothelial growth factor (VEGF) expression through adenosine A₂ receptors in the rat tongue treated with endotoxin (lipopolysaccharide: LPS). Angiogenesis in the rat tongue treated with LPS/incomplete Freund's adjuvant (IFA) or endotoxin/IFA/adenosine A₂ receptor (A₂R) antagonists was examined using immunohistochemistry for LYVE-1, ED1, ED2, OX6, langerin and VEGF, and real-time polymerase chain reaction (PCR) for VEGF. The distributional density of both blood vessels and OX6⁺ cells was significantly increased at day 8 after injection of LPS/IFA. The immunoreactive products of VEGF were intensely labelled in the cytoplasm of various antigen presenting cells (APCs) including dendritic cells (DCs) with double-immunofluorescence technique. Increase in VEGF mRNA expression level, the occupancy ratio of blood vessels, and the number of ED1⁺, ED2⁺, OX6⁺, and langerin⁺ cells was inhibited in the injured tongue of rats as a consequence of the treatment with A₂R antagonists. The present results indicate that the LPS-induced adenosine might promote angiogenesis by the up-regulation of VEGF expression in macrophages/DCs through A₂ receptors. This suggests that the synergistic interaction between toll-like receptor (TLR) and A₂ receptor signalling observed in vivo plays an important role in oral mucosal wound healing.