本文關(guān)鍵詞： 骨硬化蛋白　出處：《山東大學(xué)學(xué)報(醫(yī)學(xué)版)》2017年03期 　論文類型：期刊論文

【摘要】：目的構(gòu)建含有小鼠骨硬化蛋白(Sost)基因3'非編碼區(qū)(UTR)的熒光素酶報告基因載體(pMIR-REPORTSOST UTR),利用原代培養(yǎng)的小鼠顱骨成骨細(xì)胞及M C3T3-E1小鼠前成骨細(xì)胞系,探究在成骨分化過程中Sost基因表達(dá)所受到的轉(zhuǎn)錄后調(diào)控作用。方法成骨誘導(dǎo)C57BL/6小鼠原代顱骨成骨細(xì)胞及MC3T3-E1細(xì)胞,分別檢測各細(xì)胞中Sost基因mRNA和蛋白的相對表達(dá)水平。采用PCR克隆Sost基因3'UTR區(qū),連接到熒光素酶報告基因載體pMIR-REPORT上,命名構(gòu)建重組載體為pMIR-REPORT-SOST UTR。將pMIR-REPORT-SOST UTR和pMIR-REPORT轉(zhuǎn)染至顱骨成骨細(xì)胞、M C3T3-E1細(xì)胞及NIH3T3小鼠胚胎成纖維細(xì)胞中,根據(jù)轉(zhuǎn)染質(zhì)粒將細(xì)胞分為pMIR-REPORT-SOST UTR組和pMIR-REPORT組,檢測各組細(xì)胞中熒光素酶的相對表達(dá)水平。結(jié)果成骨誘導(dǎo)小鼠顱骨成骨細(xì)胞及MC3T3-E1細(xì)胞Sost的mRNA水平較對照組均明顯升高(P0.05),而Sost的蛋白水平與對照組相比變化不大(P0.05)。在小鼠顱骨成骨細(xì)胞及MC3T3-E1細(xì)胞中,pMIR-REPORT-SOST UTR轉(zhuǎn)染組較pMIR-REPORT轉(zhuǎn)染組的熒光素酶相對表達(dá)水平均有所下降(P0.05),而在NIH3T3細(xì)胞中pMIR-REPORT轉(zhuǎn)染組和pMIR-REPORT-SOST UTR轉(zhuǎn)染組的熒光素酶相對表達(dá)水平差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論成功構(gòu)建了pMIR-REPORT-SOST UTR,且Sost基因的3'UTR區(qū)參與了具有細(xì)胞特異性的轉(zhuǎn)錄后調(diào)控。
[Abstract]:Objective to construct a luciferase reporter gene vector pMIR-REPORTSOST UTR-containing mouse osteosclerotic protein Sost3 'noncoding region (UTR3). The primary cultured osteoblasts of mouse cranial bone and MC3T3-E1 preosteoblast were used. To explore the posttranscriptional regulation of Sost gene expression during osteogenic differentiation. Methods Osteogenic C57BL / 6 mouse primary calvarial osteoblasts and MC3T3-E1 cells were induced by osteogenesis. The relative expression levels of mRNA and protein of Sost gene were detected respectively. PCR was used to clone the 3UTR region of Sost gene. Linked to luciferase reporter gene vector pMIR-REPORT. Name the build recombinant vector pMIR-REPORT-SOST UTR. change the pMIR-REPORT-SOST. UTR and pMIR-REPORT were transfected into osteoblasts. MC3T3-E1 cells and NIH3T3 mouse embryonic fibroblasts. According to the transfection plasmid, the cells were divided into pMIR-REPORT-SOST UTR group and pMIR-REPORT group. Results the mRNA levels of Sost in osteoblasts and MC3T3-E1 cells of osteogenic mice were significantly higher than those of the control group (P < 0.05). P0.05). Compared with the control group, the protein level of Sost did not change significantly (P 0.05). It was found in the osteoblasts and MC3T3-E1 cells of the mouse skull. The relative level of luciferase expression in pMIR-REPORT-SOST UTR transfection group was lower than that in pMIR-REPORT transfection group (P 0.05). However, there was no significant difference in luciferase expression between pMIR-REPORT transfection group and pMIR-REPORT-SOST UTR transfection group in NIH3T3 cells. Conclusion pMIR-REPORT-SOST UTR was successfully constructed. Moreover, the 3 Sost region of Sost gene is involved in cell-specific post-transcriptional regulation.
【作者單位】： 山東大學(xué)口腔醫(yī)學(xué)院;山東省口腔組織再生重點實驗室;山東大學(xué)口腔醫(yī)院牙體牙髓科;
【基金】：國家自然科學(xué)基金(81100757)
【分類號】：R68
【正文快照】： 網(wǎng)絡(luò)出版地址:http://www.cnki.net/kcms/detail/37.1390.R.20170302.1110.014.html骨硬化蛋白(sclerostin,SOST)是由Sost基因編碼的含有213個氨基酸的糖蛋白,它可以通過與LRP5/6受體結(jié)合而抑制經(jīng)典的Wnt信號通路,從而抑制成骨細(xì)胞的增殖和分化[1-2]。Sost基因敲除小鼠的骨量，

本文編號：1444102