【作者】 芮泽；

【导师】 殷国勇；

【作者基本信息】 南京医科大学 ， 外科学， 2011， 硕士

【摘要】 [目的]探讨持续性压力促进成骨细胞分泌VEGF的机制。[方法]1.取1～2日龄SD乳大鼠颅盖骨进行成骨细胞原代培养,后检测并鉴定成骨细胞。2.将传代并培养至第三代的细胞分加压组和不加压组,每组再分成PD98059不预处理组和预处理组。加压组在密闭容器内采用压缩空气施以100 kPa的静压力,分别加压0.5、2.0、6.0 h。3.各组培养基ELISA法检测VEGF浓度,提取各组细胞总蛋白,Western blot检测各组成骨细胞内磷酸化ERK1/2(phosphorylation ERK1/2, pERK1/2)的水平;提取各组细胞总mRNA,RT-PCR检测VEGF mRNA的变化。[结果]1.大鼠成骨细胞的培养和鉴定:原代细胞培养1d后,贴壁展开,不规则,胞核大而清楚,呈三角形或梭形,培养5-7d后细胞半融合,7-8d后,80-90%融合,“贴壁法”传代并纯化细胞后,细胞接种1h即可贴壁,生长快,3-4d即可铺满瓶底,形态与原代相同,NBT-BCIP法定性检测碱性磷酸酶发现深蓝色至蓝紫色化合物。2.持续性压力明显增加ERK1/2的磷酸化水平;而ERK1/2总蛋白的量却无明显变化。3.PD98059在显著抑制持续性加压所诱导的成骨细内ERK1/2磷酸化水平的同时,抑制了VEGF的mRNA的表达。[结论]持续性压力通过ERK1/2的活性调节成骨细胞VEGF的分泌更多还原

【Abstract】 Objective:To investigate the mechanism of VEGF expression in osteoblasts induced by durative pressure.Methods:1、osteoblast were cultured and accredited, from 1-2d SD rats to take primary cell2、The osteoblasts were mechanically stimulated on coverslips.The phosphorylation of ERK1/2 of osteoblasts was measured by Western blot.The mRNA of VEGF was measured by RT-PCR.Results:1、Osteoblasts in culture and identification: Osteoblasts were isolated from the calvariae of neonatal (one day old) Sprague-Dawley rats and cultured according to published protocols.Cells be expanded and adherented, anomaly,cells’s nucleus are big and clear,and form be triangle or fusiform shape,half- coalesce after cultured 5-7d,80%-90% coalesce after cultured 7-8d, passage. NBT-BCIP method qualitation to detect AKP and acquire dark blue and amethyst compound.2、The phosphorylation of ERK1/2 in osteoblasts treated with durative pressure significantly increased.3、The VEGF mRNA level of osteoblasts treated with durative pressure significantly increased and this increase was inhibited after treating with PD98059. Conclusions:These data suggests that durative pressure induce VEGF expression in osteoblasts, which was dependent the ERK1/2 activation.更多还原