發(fā)布時(shí)間：2018-04-05 13:18

  本文選題：MafB　切入點(diǎn)：破骨細(xì)胞　出處：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的：構(gòu)建小鼠MafB (v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B)基因重組腺病毒表達(dá)載體,闡明MafB 對(duì)小鼠破骨細(xì)胞分化的影響。并對(duì)破骨細(xì)胞的分化如何被MafB影響機(jī)制作一研究。方法：1.提取小鼠RAW264.7細(xì)胞總RNA逆轉(zhuǎn)錄為cDNA,以cDNA為模板RT-PCR獲得含Sal I /EcoR V雙酶切位點(diǎn)的目的基因MafB,經(jīng)限制性內(nèi)切酶作用后與穿梭質(zhì)粒pAdTrack-CMV鏈接,經(jīng)酶切鑒定正確的穿梭質(zhì)粒用Pme I線性化后轉(zhuǎn)化到含pAdEasy-1的大腸桿菌BJ5183菌株中同源重組成腺病毒載體pAd-MafB,經(jīng)Pac Ⅰ線性化后,在HEK 293細(xì)胞中包裝腺病毒Ad-MafB,感染小鼠RAW264.7細(xì)胞,實(shí)驗(yàn)分為空白對(duì)照組、空載體組(Ad-GFP組)和過表達(dá)組(Ad-MafB組),經(jīng)RT-PCR和Western blotting檢測(cè)MafB的表達(dá)水平,證明細(xì)胞內(nèi)有無MafB的過表達(dá)。2.RT-PCR和Western blotting法檢測(cè)細(xì)胞內(nèi)過表達(dá)MafB對(duì)成熟破骨細(xì)胞標(biāo)志物抗酒石酸酸性磷酸酶(Tartrate Resistant Acid Phosphatase, TRAP)與組織蛋白酶K(cathepsin K,CTSK)的影響。3.RT-PCR檢測(cè)過表達(dá)MafB時(shí),破骨細(xì)胞分化關(guān)鍵因子活化T細(xì)胞核因子c1(nuclear factor of activated T cells c1,NFAT c 1)和破骨細(xì)胞相關(guān)受體(osteoclast-associated receptor,OSCAR)的表達(dá)水平。4.經(jīng)抗酒石酸酸性磷酸酶(TRAP)染色法觀察MafB對(duì)小鼠可溶性核因子K B受體活化因子配體(receptor activator of NF-κB ligand, sRANKL)誘導(dǎo)破骨細(xì)胞分化的影響。5.通過構(gòu)建小鼠顱骨骨片體外溶骨模型,經(jīng)電鏡掃描分析破骨細(xì)胞溶骨特性的變化情況。結(jié)果：1.成功構(gòu)建了重組腺病毒Ad-MafB,可感染小鼠單核/巨噬細(xì)胞RAW264.7, RT-PCR和Western blotting結(jié)果均顯示與空白對(duì)照組和Ad-GFP組比較,Ad-MafB組RAW264.7細(xì)胞MafB mRNA表達(dá)顯著增強(qiáng)且蛋白相對(duì)表達(dá)明顯增加(P0.05)。2.RT-PCR和Western blotting結(jié)果表明,與空白對(duì)照組和Ad-GFP組比較,Ad-MafB組TRAP及CTSK mRNA表達(dá)減弱且蛋白相對(duì)轉(zhuǎn)錄及翻譯水平顯著降低(P0.05)3.通過RT-PCR對(duì)破骨細(xì)胞分化關(guān)鍵因子分析發(fā)現(xiàn),Ad-MafB組破骨細(xì)胞分化的最主要的轉(zhuǎn)錄因子NFAT c1及OSCAR表達(dá)明顯受抑制(P0.05)。4.TRAP染色,Ad-MafB組TRAP陽(yáng)性的多核巨細(xì)胞明顯少于Control組和Ad-GFP組。5.成功構(gòu)建小鼠顱骨骨片體外溶骨模型,電鏡掃描發(fā)現(xiàn)在RAW264.7細(xì)胞內(nèi)過表達(dá)MafB可抑制其在骨面形成溶骨陷窩。結(jié)論：1.成功構(gòu)建可在小鼠RAW264.7細(xì)胞中過表達(dá)MafB的重組腺病毒Ad-MafB,并且證實(shí)其可抑制破骨細(xì)胞標(biāo)志物TRAP及CTSK的生成。2.破骨細(xì)胞的分化程度下降是通過MafB降低關(guān)鍵轉(zhuǎn)錄因子NFAT cl活性及抑制OSCAR的激活來實(shí)現(xiàn)的。3.通過TRAP染色和電鏡掃描發(fā)現(xiàn)MafB可降低破骨細(xì)胞分化程度并降低其溶骨能力。
[Abstract]:Aim: to construct the recombinant adenovirus expression vector of mouse MafB musculoaponeurotic fibrosarcoma oncogene family (protein B) gene and elucidate the effect of MafB on the differentiation of mouse osteoclasts.How the differentiation of osteoclasts is affected by MafB is studied.Method 1: 1.The total RNA of mouse RAW264.7 cells was extracted by reverse transcription to cDNA. the target gene MafBcontaining Sal I / Ecor V double restriction site was obtained by using cDNA as template RT-PCR. Mafb was linked to the shuttle plasmid pAdTrack-CMV after restriction endonuclease treatment.By restriction endonuclease digestion, the correct shuttle plasmid was linearized with Pme I and transformed into the homologous recombinant adenovirus vector pAd-MafB in E. coli BJ5183 strain containing pAdEasy-1. After linearized with Pac 鈪，

本文編號(hào)：1714918