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,36 h开始观察荧光标记,72 h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
Objective To construct a recombinant lentiviral vector carrying miR-146b and validate its infection efficiency in human pre-adipocytes. Methods The minigene fragment of miR-146b was amplified from human genomic DNA by PCR,then cloned into a lentivirus expression vector.After sequencing validated exactly,the expression plasmid and packaging plasmids Pcgvp,Rev,Vsvg were co-transfected into HEK-293T cells to produce lentivirus; the supernatant containing lentiviral particles was used to infect human preadipocytes,and its infection efficiency and miR-146b overexpression level were respectively tested by fluorescent observation and real-time quantitative PCR. Results MiR-46b lentiviral vector was constructed successfully,and infection efficiency in human pre-adipocytes reached over 85%,the expression level increased nearly four folds. Conclusion We have successfully constructed the lentiviral vector containing miR-146b with high overexpression in infected human pre-adipocytes,which will be useful to the future studying the function of miR-146b in human pre-adipocytes.
关键词
miR-146b /
慢病毒 /
人脂肪前体细胞
Key words
miR-146b /
lentivirus /
human preadipocytes
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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)
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