本文選題：持續(xù)性壓力 + ERK1/2　； 參考：《南京醫(yī)科大學(xué)》2011年碩士論文

【摘要】：[目的] 探討持續(xù)性壓力促進(jìn)成骨細(xì)胞分泌VEGF的機(jī)制。 [方法] 1.取1～2日齡SD乳大鼠顱蓋骨進(jìn)行成骨細(xì)胞原代培養(yǎng),后檢測并鑒定成骨細(xì)胞。 2.將傳代并培養(yǎng)至第三代的細(xì)胞分加壓組和不加壓組,每組再分成PD98059不預(yù)處理組和預(yù)處理組。加壓組在密閉容器內(nèi)采用壓縮空氣施以100 kPa的靜壓力,分別加壓0.5、2.0、6.0 h。 3.各組培養(yǎng)基ELISA法檢測VEGF濃度,提取各組細(xì)胞總蛋白,Western blot檢測各組成骨細(xì)胞內(nèi)磷酸化ERK1/2(phosphorylation ERK1/2, pERK1/2)的水平;提取各組細(xì)胞總mRNA,RT-PCR檢測VEGF mRNA的變化。 [結(jié)果] 1.大鼠成骨細(xì)胞的培養(yǎng)和鑒定:原代細(xì)胞培養(yǎng)1d后,貼壁展開,不規(guī)則,胞核大而清楚,呈三角形或梭形,培養(yǎng)5-7d后細(xì)胞半融合,7-8d后,80-90%融合,“貼壁法”傳代并純化細(xì)胞后,細(xì)胞接種1h即可貼壁,生長快,3-4d即可鋪滿瓶底,形態(tài)與原代相同,NBT-BCIP法定性檢測堿性磷酸酶發(fā)現(xiàn)深藍(lán)色至藍(lán)紫色化合物。 2.持續(xù)性壓力明顯增加ERK1/2的磷酸化水平;而ERK1/2總蛋白的量卻無明顯變化。 3.PD98059在顯著抑制持續(xù)性加壓所誘導(dǎo)的成骨細(xì)內(nèi)ERK1/2磷酸化水平的同時(shí),抑制了VEGF的mRNA的表達(dá)。 [結(jié)論] 持續(xù)性壓力通過ERK1/2的活性調(diào)節(jié)成骨細(xì)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
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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