持续性压力促进成骨细胞分泌VEGF的机制研究

发布时间：2018-05-25 04:25

本文选题：持续性压力 + ERK1/2　；参考：《南京医科大学》2011年硕士论文

【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
[Abstract]:[purpose] To investigate the mechanism of continuous stress promoting VEGF secretion by osteoblasts. [methods] 1. Primary culture of osteoblasts was carried out in the calvaria of 1 and 2 day old SD rats. Osteoblasts were detected and identified. 2. The cells of the third passage were divided into two groups: the pressurized group and the unpressurized group. Each group was divided into two groups: the group without PD98059 preconditioning and the group without preconditioning. In the pressurized group, compressed air was applied to the sealed vessel for 100 kPa, and the pressure was 0.5 ~ 2.0 ~ 6.0 h, respectively. 3. The concentration of VEGF was detected by ELISA method in each group, the level of phosphorylated ERK1/2(phosphorylation ERK1 / 2, pERK1 / 2 in osteoblasts was detected by Western blot, and the changes of VEGF mRNA were detected by RT-PCR. [results] 1. Culture and identification of rat osteoblasts: after 1 day of primary culture, the primary cells were adherent, irregular, large and clear nucleus, triangular or fusiform. After 5-7 days of culture, 80-90% of the cells were fused after 7-8 days, and the cells were subcultured and purified by "adherent method". The cells were inoculated for 1 h, and the growth rate was 3 to 4 days, and the bottle-bottom was covered. The alkaline phosphatase (ALP) was detected qualitatively by NBT-BCIP in the same shape as that of the original culture, and the dark blue to blue purple compounds were found. 2. Persistent pressure significantly increased the phosphorylation level of ERK1/2, but the total protein level of ERK1/2 did not change significantly. 3.PD98059 significantly inhibited the ERK1/2 phosphorylation level in osteoblasts induced by continuous compression, and inhibited the expression of mRNA in VEGF at the same time. [conclusion] Continuous pressure regulates the secretion of VEGF in osteoblasts through the activity of ERK1/2
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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