【摘要】：背景:細(xì)胞外基質(zhì)磷酸化糖蛋白基因在骨的礦化和吸收、成骨細(xì)胞與破骨細(xì)胞的平衡中起重要的作用,研究細(xì)胞外基質(zhì)磷酸化糖蛋白的功能及其調(diào)控機(jī)制可為骨質(zhì)疏松癥的治療提供新的思路。目的:分析轉(zhuǎn)錄因子Runx2在小鼠前成骨細(xì)胞中對細(xì)胞外基質(zhì)磷酸化糖蛋白基因啟動子的調(diào)控作用,進(jìn)而初步研究轉(zhuǎn)錄因子Runx2在骨形成發(fā)育過程中的作用。方法:首先根據(jù)Genbank中Runx2的基因序列構(gòu)建Runx2真核表達(dá)載體;然后利用雙熒光素酶基因檢測報告系統(tǒng)分析Runx2對不同長度的細(xì)胞外基質(zhì)磷酸化糖蛋白基因啟動子轉(zhuǎn)錄活性的影響,以確定Runx2有明顯作用的啟動子區(qū)段,分析3種MAPK信號通路抑制劑調(diào)控Runx2對細(xì)胞外基質(zhì)磷酸化糖蛋白基因啟動子轉(zhuǎn)錄活性的影響;最后利用定時定量RT-PCR法分析Runx2對細(xì)胞外基質(zhì)磷酸化糖蛋白基因啟動子表達(dá)活性的影響。結(jié)果與結(jié)論:成功構(gòu)建Runx2真核表達(dá)載體;雙熒光素酶基因檢測報告系統(tǒng)分析顯示Runx2能夠上調(diào)細(xì)胞外基質(zhì)磷酸化糖蛋白基因啟動子在前成骨細(xì)胞中的轉(zhuǎn)錄活性,在(-300-+66)366 bp片段區(qū)域內(nèi)上調(diào)效果較為顯著,Runx2可通過激活MEK激酶MAPK通路上調(diào)細(xì)胞外基質(zhì)磷酸化糖蛋白啟動子活性;定時定量RT-PCR法檢測再次驗證Runx2上調(diào)細(xì)胞外基質(zhì)磷酸化糖蛋白基因啟動子的表達(dá)水平。提示轉(zhuǎn)錄因子Runx2可以通過MEK激酶MAPK信號通路對細(xì)胞外基質(zhì)磷酸化糖蛋白基因表達(dá)進(jìn)行調(diào)節(jié),為探討細(xì)胞外基質(zhì)磷酸化糖蛋白在骨形成發(fā)育過程中的意義奠定基礎(chǔ)。
[Abstract]:Background: extracellular matrix phosphorylated glycoprotein gene plays an important role in the mineralization and absorption of bone and the balance between osteoblasts and osteoclasts. Studying the function of extracellular matrix phosphorylated glycoprotein and its regulatory mechanism may provide new ideas for the treatment of osteoporosis. Aim: to investigate the role of transcription factor Runx2 in the regulation of extracellular matrix phosphorylated glycoprotein gene promoter in mouse preosteoblasts, and to investigate the role of transcription factor Runx2 in bone formation and development. Methods: firstly, the eukaryotic expression vector of Runx2 was constructed according to the gene sequence of Runx2 in Genbank. Then the effects of Runx2 on the transcriptional activity of different length extracellular matrix phosphorylated glycoprotein gene promoter were analyzed by double luciferase gene detection report system to determine the promoter region of Runx2. The effects of three MAPK signal pathway inhibitors on the transcriptional activity of extracellular matrix phosphorylated glycoprotein gene promoter were analyzed. Finally, the effect of Runx2 on the expression of extracellular matrix phosphorylated glycoprotein gene promoter was analyzed by timed quantitative RT-PCR. Results & conclusion: Runx2 eukaryotic expression vector was successfully constructed. Double luciferase gene detection system analysis showed that Runx2 could up-regulate the transcriptional activity of extracellular matrix phosphorylated glycoprotein gene promoter in preosteoblasts, and significantly increased the activity in (-300-66) 366 bp region. Runx2 upregulated the activity of extracellular matrix phosphorylated glycoprotein promoter by activating MEK kinase MAPK pathway. The expression of extracellular matrix phosphorylated glycoprotein gene promoter was up-regulated by Runx2. It is suggested that transcription factor Runx2 can regulate the expression of extracellular matrix phosphorylated glycoprotein gene through MEK kinase MAPK signaling pathway, which lays a foundation for the study of the significance of extracellular matrix phosphorylated glycoprotein in bone formation and development.
【作者單位】： 濰坊醫(yī)學(xué)院口腔醫(yī)學(xué)院;濰坊醫(yī)學(xué)院附屬醫(yī)院;濰坊醫(yī)學(xué)院;
【基金】：國家自然科學(xué)基金(81441107) 山東省自然科學(xué)基金(ZR2012HQ036) 濰坊醫(yī)學(xué)院大學(xué)生科技創(chuàng)新基金項目(KX2013011)~~
【分類號】：R318.08

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