Rap1b对鼠前成骨细胞（Mc3T3-E1）成骨分化早期的影响
发布时间：2018-07-20 21:27
【摘要】：目的Rap1b是一种Ras样GTP酶,可参与氧化应激,通过调控多种信号通路如MAPK、B-Raf/MEK/ERK和Akt信号通路调节细胞的增殖和存活,在增殖、分化、形态形成及凋亡中起分子开关的作用,还能调控肿瘤细胞的增殖和转移。还有研究表明Rap1b可以促进内皮迁移与血管生成,以及可以选择性地抑制成熟破骨细胞,进而阻断病理性骨吸收。另外,本课题组在前期研究中发现,Rap1b还参与斑马鱼早期胚胎发育。然而,Rap1b基因对成骨分化过程的调节作用,尚无明确的文献报导。因此,了解Rap1b的生物学功能具有重要意义。.MC3T3-E1细胞是从新生C57BL/6小鼠颅顶骨中分离得到的一种前成骨细胞,已被证实其具有成骨细胞的表型分化特征。因此,本研究以鼠前成骨细胞(Mc3T3E1)为研究对象,探讨Rap1b对鼠前成骨(Mc3T3E1)细胞成骨分化早期的影响,在进一步阐明其在成骨分化中的调节作用的理论基础的研究中有重要意义。方法利用成骨诱导液诱导Mc3T3E1成骨向分化,利用实时定量PCR分别检测诱导后0、3、7d的Rap1bmRNA的相对表达量。同时向实验组细胞分别转染Rap1b-mus-462 和 Rap1b-mus-703,4 d 后进行 ALP 染色,同时测定 Rap1b 的mRNA相对表达量,并对转染后细胞中成骨指标ALP、Runx2和Osterix0、3、7d时的mRNA相对表达量进行测定。再利用划痕实验观察siRNA转染对Mc3T3E1细胞迁移能力的影响。结果Real-time PCR结果显示在成骨诱导早期的不同时间点,Rap1b的表达量随诱导时间的增加而增加。当Rap1b的表达降低时,各成骨指标ALP、Runx2和Osterix的表达量也随之降低,Mc3T3E1细胞迁移能力也有所减弱。结论Rap1b对Mc3T3E1细胞成骨分化早期以及细胞迁移有一定的促进作用。
[Abstract]:Objective Rap1b is a Ras-like GTP-like enzyme involved in oxidative stress. Rap1b regulates cell proliferation and survival by regulating many signal pathways such as MAPK- Raf- Rafr / MEK / ERK and Akt signaling pathways, and plays a molecular switch role in proliferation, differentiation, morphogenesis and apoptosis. It can also regulate the proliferation and metastasis of tumor cells. Other studies have shown that Rap1b can promote endothelial migration and angiogenesis, and selectively inhibit mature osteoclasts, thus blocking pathological bone resorption. In addition, Rap1b was also found to be involved in the early embryonic development of zebrafish. However, the regulation of Rap1b gene on osteogenic differentiation has not been clearly reported. Therefore, it is important to understand the biological function of Rap1b. MC3T3-E1 cells are a kind of preosteoblasts isolated from the cranioparietal bone of C57BL / 6 mice. It has been proved that Rap1b has the phenotypic differentiation characteristics of osteoblasts. Therefore, in this study, the effect of Rap1b on the early stage of osteogenic differentiation of rat preosteoblast (Mc3T3E1) was studied. It is of great significance to further elucidate the theoretical basis of its regulatory role in osteogenic differentiation of rat preosteoblast (Mc3T3E1). Methods the osteogenic differentiation of Mc3T3E1 was induced by osteogenic inducer, and the relative expression of Rap1b mRNA on the 7th day after induction was detected by real-time quantitative PCR. At the same time, the experimental cells were transfected with Rap1b-mus-462 and Rap1b-mus-703 respectively for ALP staining for 4 days, and the relative expression of Rap1b mRNA was measured. The relative expression of ALPnRunx2 and Osterix0 were measured at the 7th day after transfection. The effect of siRNA transfection on the migration of Mc3T3E1 cells was observed by scratch assay. Results Real-time PCR results showed that the expression of Rap1b increased with the increase of induction time at different time points in the early stage of osteogenesis. When the expression of Rap1b was decreased, the expression of ALPnRunx2 and Osterix also decreased, and the migration ability of Mc3T3E1 cells was also decreased. Conclusion Rap1b can promote early osteogenic differentiation and cell migration of Mc3T 3E1 cells.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781
[Abstract]:Objective Rap1b is a Ras-like GTP-like enzyme involved in oxidative stress. Rap1b regulates cell proliferation and survival by regulating many signal pathways such as MAPK- Raf- Rafr / MEK / ERK and Akt signaling pathways, and plays a molecular switch role in proliferation, differentiation, morphogenesis and apoptosis. It can also regulate the proliferation and metastasis of tumor cells. Other studies have shown that Rap1b can promote endothelial migration and angiogenesis, and selectively inhibit mature osteoclasts, thus blocking pathological bone resorption. In addition, Rap1b was also found to be involved in the early embryonic development of zebrafish. However, the regulation of Rap1b gene on osteogenic differentiation has not been clearly reported. Therefore, it is important to understand the biological function of Rap1b. MC3T3-E1 cells are a kind of preosteoblasts isolated from the cranioparietal bone of C57BL / 6 mice. It has been proved that Rap1b has the phenotypic differentiation characteristics of osteoblasts. Therefore, in this study, the effect of Rap1b on the early stage of osteogenic differentiation of rat preosteoblast (Mc3T3E1) was studied. It is of great significance to further elucidate the theoretical basis of its regulatory role in osteogenic differentiation of rat preosteoblast (Mc3T3E1). Methods the osteogenic differentiation of Mc3T3E1 was induced by osteogenic inducer, and the relative expression of Rap1b mRNA on the 7th day after induction was detected by real-time quantitative PCR. At the same time, the experimental cells were transfected with Rap1b-mus-462 and Rap1b-mus-703 respectively for ALP staining for 4 days, and the relative expression of Rap1b mRNA was measured. The relative expression of ALPnRunx2 and Osterix0 were measured at the 7th day after transfection. The effect of siRNA transfection on the migration of Mc3T3E1 cells was observed by scratch assay. Results Real-time PCR results showed that the expression of Rap1b increased with the increase of induction time at different time points in the early stage of osteogenesis. When the expression of Rap1b was decreased, the expression of ALPnRunx2 and Osterix also decreased, and the migration ability of Mc3T3E1 cells was also decreased. Conclusion Rap1b can promote early osteogenic differentiation and cell migration of Mc3T 3E1 cells.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781
【参考文献】
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1 杨小燕;何志旭;舒莉萍;金皎;黄t，
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