【摘要】：目的Rap1b是一種Ras樣GTP酶,可參與氧化應激,通過調控多種信號通路如MAPK、B-Raf/MEK/ERK和Akt信號通路調節(jié)細胞的增殖和存活,在增殖、分化、形態(tài)形成及凋亡中起分子開關的作用,還能調控腫瘤細胞的增殖和轉移。還有研究表明Rap1b可以促進內皮遷移與血管生成,以及可以選擇性地抑制成熟破骨細胞,進而阻斷病理性骨吸收。另外,本課題組在前期研究中發(fā)現(xiàn),Rap1b還參與斑馬魚早期胚胎發(fā)育。然而,Rap1b基因對成骨分化過程的調節(jié)作用,尚無明確的文獻報導。因此,了解Rap1b的生物學功能具有重要意義。.MC3T3-E1細胞是從新生C57BL/6小鼠顱頂骨中分離得到的一種前成骨細胞,已被證實其具有成骨細胞的表型分化特征。因此,本研究以鼠前成骨細胞(Mc3T3E1)為研究對象,探討Rap1b對鼠前成骨(Mc3T3E1)細胞成骨分化早期的影響,在進一步闡明其在成骨分化中的調節(jié)作用的理論基礎的研究中有重要意義。方法利用成骨誘導液誘導Mc3T3E1成骨向分化,利用實時定量PCR分別檢測誘導后0、3、7d的Rap1bmRNA的相對表達量。同時向實驗組細胞分別轉染Rap1b-mus-462 和 Rap1b-mus-703,4 d 后進行 ALP 染色,同時測定 Rap1b 的mRNA相對表達量,并對轉染后細胞中成骨指標ALP、Runx2和Osterix0、3、7d時的mRNA相對表達量進行測定。再利用劃痕實驗觀察siRNA轉染對Mc3T3E1細胞遷移能力的影響。結果Real-time PCR結果顯示在成骨誘導早期的不同時間點,Rap1b的表達量隨誘導時間的增加而增加。當Rap1b的表達降低時,各成骨指標ALP、Runx2和Osterix的表達量也隨之降低,Mc3T3E1細胞遷移能力也有所減弱。結論Rap1b對Mc3T3E1細胞成骨分化早期以及細胞遷移有一定的促進作用。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R781

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