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 PDE7A gene silencing THP-1 cell lines by lentivirus-mediated RNA interference and expression of ABCA1

作　　者：向乐[1] 唐菀泽[1] 张志珍[1] 马卫列[1] 

机构地区：[1]广东医学院生物化学与分子生物学教研室,广东东莞523808

出　　处：《中国现代医学杂志》2016年第3期1-8,共8页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:81170267);广东省高等学校人才引进专项[No:粤财教(2013)246号]

摘　　要：目的利用慢病毒介导构建磷酸二酯酶7A(PDE7A)基因沉默人单核巨噬细胞(THP-1)株,并分析沉默细胞株的三磷酸腺苷结合盒转运体A1(ABCA1)的表达变化。方法以PDE7A基因为靶点,设计合成3段sh RNA片段,与慢病毒载体Pmi Rzip连接,构建重组质粒。测序鉴定后进行病毒包装,感染THP-1细胞,实时荧光定量聚合酶链反应(q PCR)进行分析,确定最佳干扰片段。嘌呤霉素筛选得到PDE7A基因沉默稳转细胞株。q PCR和Western blot检测鉴定所构建的细胞株,将其诱导成为泡沫细胞后鉴定ABCA1基因的表达。结果转染sh RNA1、sh RNA2、sh RNA3细胞的PDE7A相对表达量分别为(0.480±0.028)、(0.561±0.016)和(0.377±0.013),故选择sh RNA3作为干扰PDE7A基因的最佳片段。采用1.4 g/L嘌呤霉素成功筛选出PDE7A沉默细胞株,q PCR和Western blot检测PDE7A基因的干扰效率,其抑制效率>70%。将其诱导成巨噬泡沫细胞后,Western blot检测显示,ABCA1的表达量增加40%。结论成功构建PDE7A sh RNA慢病毒干扰载体,并筛选出PDE7A基因沉默的THP-1细胞株,且ABCA1的表达量增加。Objective To construct phosphodiesterase 7A （PDETA） 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 PDETA 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 PDE7A shRNAs was ana- lyzed by qPCR. Subsequently, the THP-1 cells were screened with Puromycin to get stable PDE7A 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 PDE7A 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 PDE7A gene fragment. PDE7A silence cell lines were successfully screened with 1.4 g/L Puromycin. PDETA 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 PDE7A silencing lentivirus interference vector has been successfully constructed, and PDETA 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A1 

分 类 号：R341[医药卫生—基础医学]