| 张辉 孟蕾 李娟 杜沿林 李永明.小鼠RNA干扰基因RelA慢病毒载体的构建与鉴定[J].,2016,16(17):3206-3211 |
| 小鼠RNA干扰基因RelA慢病毒载体的构建与鉴定 |
| Construction and Identification of a Lentiviral Vector of RNA InterferenceTargeting Mouse RelA Gene |
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| DOI： |
| 中文关键词: RelA基因 慢病毒载体 RNA 干扰 成骨细胞 |
| 英文关键词: RelA gene Lentiviral vector RNAi Osteoblast |
| 基金项目:国家自然科学基金项目（31370943） |
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| 中文摘要: |
| 目的：构建小鼠RelA 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RelA 基因序列，设计特异
性的shRNA 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DH5-alpha，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RelA 基因及成骨相关基因
ALP、OCN、RANKL的表达。结果：成功构建小鼠RelA 基因的RNA干扰慢病毒载体，感染MC3T3-E1 细胞后，RelA 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RelA 基因的
RNA 干扰慢病毒载体。当小鼠成骨细胞RelA基因表达被干扰，NF-资B 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 RelA gene, and identify the vector
through transfecting into MC3T3-E1 cell.Methods:Particular shRNA sequences aiming at mouse RelA gene were designed and inserted
into the lentiviral vector GV-248 by DNA recombination technology. The recombinant plasmids were transfected into competent
DH5琢bacteria. 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 293T cell to
produce the lentiviral particles. The lentiviral particles were transfected into MC3T3-E1 cell, and the RelA 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-RelA were constructed successfully. The RelA gene expression in the transfected cells was
significantly inhibited at the mRNA and protein levels. Gene RANKL expression levels descend, while gene ALP and OCN expression
levels ascend.Conclusion:The lentiviral RNAi vector targeting mouse gene RelA has been successfully constructed. When RelA gene
expressions were obstructed and NF-kB 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. |
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