中国现代医学杂志Issue(3)：1-8,8.DOI:10.3969/j.issn.1005-8982.2016.03.001
慢病毒介导磷酸二酯酶7A基因沉默细胞株的构建及三磷酸腺苷结合盒转运体A1的表达变化*
Construction of PDF7A gene silencing THP-1 cell lines by lentivirus-mediated RNA interference and expression of ABCA1
摘要
Abstract
Objective To construct phosphodiesterase 7A (PDF7A) gene silence human acute monocytic leukemia (THP-1) cell lines by lentivirus-mediated RNA interference technique, and to analyze the expression of ATP-binding cassette transporter A1 (ABCA1) in THP-1 cell lines. Methods Three human gene targeted shRNA fragments (shRNA1, shRNA2 and shRNA3) and shRNA-NC fragment were designed and syn-thesized, then connected with lentiviral vector PmiRzip to construct recombinant plasmids. After DNA sequencing, the lentivirus was packaged, and infected the THP-1 cells. The inhibitory effect of shRNAs was ana-lyzed by qPCR. Subsequently, the THP-1 cells were screened with Puromycin to get stable gene silencing cell lines which were then identified by qPCR and Western blot. The expression of ABCA1 was de-termined after THP-1 cells were induced to develop macrophage foam cells. Results The relative expression of in the cells transfected with shRNA1, shRNA2 or shRNA3 was (0.480±0.028), (0.561±0.016) and (0.377 ± 0.013) respectively; then shRNA3 was chosen as interferent gene fragment. silence cell lines were successfully screened with 1.4 g/L Puromycin. gene interference efficiency was identi-fied by qPCR and Western blot, and inhibition efficiency was more than 70%. The expression of ABCA1 was increased by more than 40% after THP-1 cells were induced into macrophage foam cells. Conclusions silencing lentivirus interference vector has been successfully constructed, and gene silencing THP-1 cell lines have been screened out in which ABCA1 expression is increased.关键词
磷酸二酯酶7A/RNA干扰/慢病毒/三磷酸腺苷结合盒转运体A1Key words
phosphodiesterase 7A/RNA interference/lentivirus/ATP-binding cassette transporter A1分类
医药卫生引用本文复制引用
向乐,唐菀泽,张志珍,马卫列..慢病毒介导磷酸二酯酶7A基因沉默细胞株的构建及三磷酸腺苷结合盒转运体A1的表达变化*[J].中国现代医学杂志,2016,(3):1-8,8.基金项目
国家自然科学基金（No81170267）；广东省高等学校人才引进专项[No粤财教（2013）246号] （）
