CAJ | 학술논문

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。
목적：위탐토인급성단핵세포백혈병세포계( THP-1)중담고순전운상관적ABCG1기인적표체대동맥죽양경화발생발전적작용,구건ABCG1만병독간우재체,병험증간우효과、진행공능검측。방법침대인ABCG1적cDNA서렬,설계량개RNA간우적과핵감산단련DNA。장기삽입도만병독표체재체상；장구건적재체화포장질립공전염293T세포；통과실시형광정량PCR급단백질면역인적검측간우재체대 ABCG1표체수평적영향；통과검측외류솔진행공능험증。결과구건적ABCG1기인적소간우RNA만병독표체재체감염THP-1세포가이유효지억제ABCG1 mRNA화단백표체수평,차감소 ABCG1개도적담고순외류,감염조외류솔소우대조조(23.87%±0.45%비30.57%±1.00%,P﹤0.001)。결론만병독표체재체성공고저THP-1세포적ABCG1,병위후속적ABCG1재거서세포중생물학작용적연구창조조건。
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-1 cell model for further detection of ABCG1 function.