敲低ABCG1慢病毒表达载体的构建及功能检测

目的为探讨人急性单核细胞白血病细胞系(THP-1)中胆固醇转运相关的ABCG1基因的表达对动脉粥样硬化发生发展的作用,构建ABCG1慢病毒干扰载体,并验证干扰效果、进行功能检测。方法针对人ABCG1的cDNA序列,设计两个RNA干扰的寡核苷酸单链DNA。将其插入到慢病毒表达载体上;将构建的载体和包装质粒共转染293T细胞;通过实时荧光定量PCR及蛋白质免疫印迹检测干扰载体对ABCG1表达水平的影响;通过检测外流率进行功能验证。结果构建的ABCG1基因的小干扰RNA慢病毒表达载体感染THP-1细胞可以有效地抑制ABCG1 mRNA和蛋白表达水平,且减少ABCG1介导的胆固醇外流,感染组外流率小于对照组(23.87%±0.45%比30.57%±1.00%,P<0.001)。结论慢病毒表达载体成功敲低THP-1细胞的ABCG1,并为后续的ABCG1在巨噬细胞中生物学作用的研究创造条件。

Construction of a lentiviral vector of ABCG1 short hairpin RNA and function detection

Objective To investigate the biological function of human lipid transporter gene ABCG1 in THP-1 cell line in the development and progression of atherosclerosis,the lentiviral vector of ABCG1 short hairpin RNA（ shRNA） was constructed and its knockdown effect and function was determined. Methods ABCG1 shRNA was designed and constructed based human ABCG1 cDNA sequence using a lentiviral vector.The expression of ABCG1 was measured by real-time PCR and Western blot,and function was examined by Cholesterol efflux assay. Results Construct of lentiviral vector effectively inhibited ABCG1 mRNA and protein levels and decreased the ABCG1-mediated cholesterol efflux from THP-1-derived macrophages.（ infection group： 23.87% ±0.45% vs. control group： 30.57% ±1.00%,P〈0.001）. Conclusions A lentivirus RNA inference vector targeting ABCG1 gene is successfully constructed,which provided THP-1cell model for further detection of ABCG1 function.