格列美脲对大鼠下颌骨成骨细胞PI3K／Akt通路的影响

目的研究格列美脲（glimepiride）对大鼠下颌骨成骨细胞PI3K／Akt通路的影响。方法分离培养原代下颌骨成骨细胞，进行成骨细胞鉴定，将成骨细胞以1×10^5接种于25cm^2培养瓶中，细胞融合后加入10μmol／L格列美脲分别干预0、15、30、60、120min。提取蛋白，取等量蛋白，采用凝胶电泳，分别加入兔抗大鼠PI3KP85、Akt／P-Akt抗体、IRS-1／P-IRS-1抗体和G肌动蛋白，37℃孵育4h，洗膜后，经辣根过氧化物酶标记的羊抗兔IgG孵育50min，化学发光法显示抗原抗体复合物，X线片显影并定影，所有杂交信号在Labworks4．5成像分析仪系统测定条带密度。蛋白目的条带水平用与β肌动蛋白的比值表示。以上实验重复3次。结果在格列美脲诱导下，PIKP85、Akt／P—Akt、IRS-1／P—IRS-1表达显著提高。结论格列美脲在成骨细胞中也能激活PI3K／Akt通路。

Effect of glimepiride on PDK/Akt pathway in rat osteoblasts

Objective To investigate the effect of glimepiride on PI3K/Akt pathway in rat mandible osteoblasts.Methods Primary osteoblasts were isolated and cultured.Osteoblasts were seeded onto 25 cm2 plates at a density of 1× 105 cells/plate.All the groups were cultured for 15 min,30min,60 min and 120 min,respectively.The cell lyses were collected equally to ensure each sample containing equal protein.The electrophoresis was performed and detected with antibodies against PI3- kinase P85,IRS-1,P-IRS-1,Akt,and P-Akt (Ser-473),which were diluted in TBS-T for 4 hours at room temperature.After three washes by TBS-T,the blots were subsequently incubated with a sheep anti-rabbit peroxidase-conjugated secondary antibody for 50 min at room temperature.The blots were visualized by enhanced chemiluminescence using Labworks 4.5 Image Acquisition System.For normalization purposes,the same blot was also probed with rabbit anti-rat β-action antibody.Results The expression of PI3K P85,Akt/P Akt and IRS-1/P-IRS-1 protein expression was significantly increased by glimepiride in the cultured osteoblasts.Conclusions Glimepiride activates the PI3K/Akt pathway in osteoblasts.