小鼠MafB过表达对单核细胞向破骨细胞分化的影响

发布时间：2018-04-05 13:18

  本文选题：MafB　切入点：破骨细胞　出处：《重庆医科大学》2015年硕士论文

【摘要】：目的：构建小鼠MafB (v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B)基因重组腺病毒表达载体,阐明MafB 对小鼠破骨细胞分化的影响。并对破骨细胞的分化如何被MafB影响机制作一研究。方法：1.提取小鼠RAW264.7细胞总RNA逆转录为cDNA,以cDNA为模板RT-PCR获得含Sal I /EcoR V双酶切位点的目的基因MafB,经限制性内切酶作用后与穿梭质粒pAdTrack-CMV链接,经酶切鉴定正确的穿梭质粒用Pme I线性化后转化到含pAdEasy-1的大肠杆菌BJ5183菌株中同源重组成腺病毒载体pAd-MafB,经Pac Ⅰ线性化后,在HEK 293细胞中包装腺病毒Ad-MafB,感染小鼠RAW264.7细胞,实验分为空白对照组、空载体组(Ad-GFP组)和过表达组(Ad-MafB组),经RT-PCR和Western blotting检测MafB的表达水平,证明细胞内有无MafB的过表达。2.RT-PCR和Western blotting法检测细胞内过表达MafB对成熟破骨细胞标志物抗酒石酸酸性磷酸酶(Tartrate Resistant Acid Phosphatase, TRAP)与组织蛋白酶K(cathepsin K,CTSK)的影响。3.RT-PCR检测过表达MafB时,破骨细胞分化关键因子活化T细胞核因子c1(nuclear factor of activated T cells c1,NFAT c 1)和破骨细胞相关受体(osteoclast-associated receptor,OSCAR)的表达水平。4.经抗酒石酸酸性磷酸酶(TRAP)染色法观察MafB对小鼠可溶性核因子K B受体活化因子配体(receptor activator of NF-κB ligand, sRANKL)诱导破骨细胞分化的影响。5.通过构建小鼠颅骨骨片体外溶骨模型,经电镜扫描分析破骨细胞溶骨特性的变化情况。结果：1.成功构建了重组腺病毒Ad-MafB,可感染小鼠单核/巨噬细胞RAW264.7, RT-PCR和Western blotting结果均显示与空白对照组和Ad-GFP组比较,Ad-MafB组RAW264.7细胞MafB mRNA表达显著增强且蛋白相对表达明显增加(P0.05)。2.RT-PCR和Western blotting结果表明,与空白对照组和Ad-GFP组比较,Ad-MafB组TRAP及CTSK mRNA表达减弱且蛋白相对转录及翻译水平显著降低(P0.05)3.通过RT-PCR对破骨细胞分化关键因子分析发现,Ad-MafB组破骨细胞分化的最主要的转录因子NFAT c1及OSCAR表达明显受抑制(P0.05)。4.TRAP染色,Ad-MafB组TRAP阳性的多核巨细胞明显少于Control组和Ad-GFP组。5.成功构建小鼠颅骨骨片体外溶骨模型,电镜扫描发现在RAW264.7细胞内过表达MafB可抑制其在骨面形成溶骨陷窝。结论：1.成功构建可在小鼠RAW264.7细胞中过表达MafB的重组腺病毒Ad-MafB,并且证实其可抑制破骨细胞标志物TRAP及CTSK的生成。2.破骨细胞的分化程度下降是通过MafB降低关键转录因子NFAT cl活性及抑制OSCAR的激活来实现的。3.通过TRAP染色和电镜扫描发现MafB可降低破骨细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，

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