小鼠MafB基因重组腺病毒载体的构建及其对破骨细胞分化的影响

Construction of recombinant adenovirus of mouse MafB gene and its effect on osteoclast differentiation

目的:构建小鼠肌腱膜纤维肉瘤癌基因B型(MafB)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。方法:提取小鼠RAW264.7细胞总RNA,逆转录为cDNA,以cDNA为模板PCR获得目的基因MafB,连接入穿梭质粒pAdTrack-CMV,经酶切鉴定正确的穿梭质粒用PmeⅠ线性化后转化到含pAdEasy-1的大肠杆菌BJ5183菌株中,经PacⅠ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法检测MafB的表达水平,经抗酒石酸酸性磷酸酶(TRAP)染色、RT-PCR和Western blotting法观察MafB对小鼠可溶性核因子κB受体活化因子配体诱导的破骨细胞分化的影响。结果:与空白对照组和Ad-GFP组比较,Ad-MafB组RAW264.7细胞MafB mRNA表达水平明显升高且蛋白相对表达水平明显增加(P<0.05);TRAP染色,Ad-MafB组TRAP阳性细胞明显少于空白对照组和Ad-GFP组;与空白对照组和Ad-GFP组比较,Ad-MafB组TRAP和组织蛋白酶K(CTSK)mRNA表达降低且蛋白相对表达水平显著降低(P<0.05)。结论: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,证实MafB可抑制破骨细胞的分化。

Objective To construct the recombinant adenovirus vector of mouse MafB gene,and to elucidate the influence of MafB in osteoclast differentiation.Methods The total RNA of RAW264.7 cells in mouse was extracted and reversedly transcripted to cDNA,and the target gene MafB was obtained by RT-PCR with cDNA as template.The MafB gene was connected to shuttle vector pAdTrack-CMV and linearized with PmeⅠ and transfected to competent E.coli BJ5183 containing pADEasy-1.The adenovirus Ad-MafB in HEK 293 cells was amplified after linearization with PacⅠ and the RAW264.7cells were infected.The RAW264.7cells were devided into control group,empty-vector group（Ad-GFP group）and over-expression group（Ad-MafB group）.The expression levels of MafB were detected by RT-PCR and Western blotting method.TRAP staining was applied to observe the osteoclast differentiation and maturation.RT-PCR and Western blotting method were applied to detect the expression of TRAP and cathepsin K（CTSK）.Results Compared with control group and Ad-GFP group,the expression levels of MafB mRNA and protein in Ad-MafB group were increased（P〈0.05）.The TRAP staining results showed that the number of TRAP-positive cells in Ad-MafB group was less than those in control group and Ad-GFP group.Compared with control group and Ad-GFP group,the expression levels of TRAP and CTSK mRNA and proteins in Ad-MafB group were decreased（P〈0.05）.Conclusion The recombinant adenovirus Ad-MafB over expressing MafB in mouse RAW264.7cells is successfully constructed,and it can inhibit the differentiation of osteoclasts.