中文摘要：

目的：构建小鼠Rel A基因的RNA干扰慢病毒载体,转染小鼠成骨样细胞并鉴定。方法：针对小鼠Rel A基因序列,设计特异性的sh RNA序列,应用基因重组技术插入慢病毒载体GV-248。得到的重组质粒转化感受态大肠杆菌DH5α,筛选得到阳性克隆并扩大培养。所得质粒进行测序分析确定载体构建成功。重组质粒载体及包装辅助质粒转染293T细胞,得到目的病毒并测定相应病毒滴度。慢病毒转染MC3T3-E1细胞后,Real-time PCR及Western blot检测MC3T3-E1细胞Rel A基因及成骨相关基因ALP、OCN、RANKL的表达。结果：成功构建小鼠Rel A基因的RNA干扰慢病毒载体,感染MC3T3-E1细胞后,Rel A基因的表达明显受到抑制,同时RANKL基因表达水平明显下降,ALP、OCN基因表达水平明显上升。结论：成功构建了小鼠Rel A基因的RNA干扰慢病毒载体。当小鼠成骨细胞Rel A基因表达被干扰,NF-κB通路被抑制后,小鼠成骨细胞成骨相关基因ALP、OCN的表达明显上升,成骨功能增强;同时RANKL的表达明显下降,其介导的破骨细胞骨吸收功能减弱。

英文摘要：

Objective： To construct a lentiviral vector of RNA interference targeting mouse Rel A gene, and identify the vector through transfecting into MC3T3-E1 cell. Methods： Particular sh RNA sequences aiming at mouse Rel A gene were designed and inserted into the lentiviral vector GV-248 by DNA recombination technology. The recombinant plasmids were transfected into competent DH5αbacteria. Then the bacteria were cultured in ampicillin medium, and the candidate clones were getted and amplified. The plasmids were identified to be correct by DNA sequencing. The recombinant and two packaging plasmids were co-transfected into 293 T cell to produce the lentiviral particles. The lentiviral particles were transfected into MC3T3-E1 cell, and the Rel A expression were assayed by Real-time PCR and Western blot analysis. The genes of osteogenesis including ALP, OCN, RANKL were assayed by the same way.Results： The lentiviral RNAi vector GV-248-Rel A were constructed successfully. The Rel A gene expression in the transfected cells was significantly inhibited at the m RNA and protein levels. Gene RANKL expression levels descend, while gene ALP and OCN expression levels ascend. Conclusions： The lentiviral RNAi vector targeting mouse Rel A gene has been successfully constructed. When Rel A gene expressions were obstructed and NF-κB signaling ways were inhibited, the expressions of ALP and OCN ascend, and this led to more bone formation. At the same time, the expressions of RANKL descend, and this led to less bone resorption by osteoclast.