人miR-146b慢病毒载体构建及人脂肪细胞中表达验证

发布时间：2018-05-03 14:44

本文选题：miR-b + 慢病毒　；参考：《中国儿童保健杂志》2013年11期

【摘要】：目的构建人miR-146b慢病毒表达载体,包被病毒并检测其在人脂肪细胞中过表达效果。方法以人基因组DNA为模板,PCR高保真扩增包含miR-146b区域上下游各100bp左右的基因组DNA,亚克隆入慢病毒表达载体,与包装质粒共转染HEK-293T细胞,包装病毒,收取病毒上清,纯化后感染人脂肪前体细胞,36h开始观察荧光标记,72h收取细胞,抽提RNA,Realtime PCR检测慢病毒感染下miR-146b的相应表达量。结果成功构建人miR-146b慢病毒表达载体,包装获得的病毒感染人脂肪细胞的效率可达到85%以上,miR-146b过表达水平可接近4倍。结论本研究成功构建了人miR-146b慢病毒表达载体,包被的慢病毒可以在人脂肪细胞中实现过表达效果,为后续功能研究奠定了基础。
[Abstract]:Aim to construct a human miR-146b lentivirus expression vector and to investigate its overexpression in human adipocytes. Methods Human genomic DNA was used as template to amplify the genomic DNA containing the upstream and downstream 100bp of the miR-146b region, and subcloned into the lentivirus expression vector, then co-transfected with the packaging plasmid into HEK-293T cells to package the virus and collect the virus supernatant. After being purified and infected with human adipocyte precursor cells for 36 h, the fluorescent labeled cells were observed for 72 hours, and the miR-146b expression was detected by extractable RNAs Realtime PCR under lentivirus infection. Results the expression vector of human miR-146b lentivirus was successfully constructed. The efficiency of infected human adipocytes by packaging could reach more than 85% and the overexpression level of miR-146b could be nearly 4 times. Conclusion the expression vector of human miR-146b lentivirus was successfully constructed in this study. The coated lentivirus could be over-expressed in human adipocytes, which laid a foundation for further functional study.
【作者单位】：南京医科大学儿科研究所;南京医科大学附属南京妇幼保健院;
【基金】：国家自然科学基金(81100619) 江苏省医学创新团队项目(LJ201108) 南京市科技发展计划(201104013)
【分类号】：R373

【参考文献】