FNDC5过表达慢病毒载体的构建及稳定转染THP-1细胞系

Construction of lentiviral vectors of fibronectin type Ⅲ domain-containing protein 5 overexpression and stable transfection of THP-1 cell line

摘要:

目的：构建过表达Ⅲ型纤连蛋白结构域包含蛋白5（fibronectin type Ⅲ domain-containing protein 5, FNDC5）基因的慢病毒载体，获得稳定转染FNDC5的THP-1细胞系。方法：PCR扩增目的基因FNDC5，将目的基因构建入pLenti-EF1a-EGFP-P2A-Puro慢病毒载体中，在DH5α感受态细胞中进行扩增，测序及鉴定合格后利用四质粒包装系统转染293T细胞，包装成过表达慢病毒颗粒并测定病毒滴度。Western blot检测重组慢病毒中FNDC5的表达情况后，将获得的重组慢病毒转染THP-1细胞系，荧光显微镜观察并计算其转染效率。经嘌呤霉素筛选建立FNDC5过表达的稳转细胞系后，将细胞分为3组，分别为空白组、空载组和FNDC5组，用Western blot及RT-PCR检测其FNDC5的表达情况。通过油红O染色后观察FNDC5过表达对THP-1巨噬细胞源性泡沫细胞脂质蓄积的影响。结果：PCR产物鉴定及测序结果显示成功构建FNDC5过表达慢病毒载体；转染293T细胞后可检测到绿色荧光，Western blot检测FNDC5-Flag标签有表达，包装后病毒的滴度为1.32×109 TU/mL；Western blot及RT-PCR检测重组慢病毒转染THP-1细胞系，与空载组对比，FNDC5组中FNDC5蛋白表达明显增加[（238.0±24.9）％]，有统计学意义（P=0.000）；与空载组对比，FNDC5组中FNDC5 mRNA表达水平明显增加[（228.5±3.4）％]，有统计学意义（P=0.000）。FNDC5过表达转染THP-1巨噬细胞源性泡沫细胞中的脂质蓄积减少。结论：本实验成功构建了FNDC5基因的过表达慢病毒载体，通过FNDC5过表达慢病毒感染建立了FNDC5稳转的THP-1细胞系，证明FNDC5可明显减少THP-1巨噬细胞源性泡沫细胞的脂质蓄积，为后续研究奠定基础。

Abstract:

Objective：To construct the lentiviral vector of fibronectin type Ⅲ domain-containing protein 5 （FNDC5） overexpression，and to obtain the THP-1 cell line stably transfected with FNDC5. Methods：FNDC5 gene was amplified by PCR and was then inserted to pLenti-EF1a-EGFP-P2A-Puro lentiviral vector. The recombinant vector was amplified in DH5α competent cells，and after se－quencing and identification，the vectors were transfected into 293T cell with the 4-plasmid packaging system to produce overexpression lentiviral particles. Virus titer was then measured. Western blot was used to measure the expression of FNDC5 in recombinant lentivirus；the recombinant lentivirus was transfected into THP-1 cell line，and a fluorescence microscope was used to calculate trans－fection efficiency. After a stably transfected cell line with FNDC5 overexpression was screened out by puromycin，the cells were divid－ed into blank group，empty group，and FNDC5 group，and Western blot and RT-PCR were used to measure the protein and mRNA expression of FNDC5. Oil red O staining was used to observe the effect of FNDC5 overexpression on lipid accumulation in THP-1 macrophage-derived foam cells. Results：PCR product identi－fication and sequencing results showed that a lentiviral vector of FNDC5 overexpression was successfully constructed. Green flu－orescent protein was detected after transfection of 293T cells，and the Flag-tag expression of FNDC5 was detected by Western blot. The titer of the lentivirus after packaging was 1.32×109 TU/mL. Western blot and RT-PCR were used to analyze the THP-1 cell line transfected with the lentivirus；compared with the empty group，the FNDC5 overexpression group had a significant increase in the protein and mRNA expression of FNDC5（238.0±24.9）% and （228.5±3.4）%，respectively，both P=0.000）. There was a reduction in lipid accumulation in THP-1 macrophage-derived foam cells with FNDC5 overexpression. Conclusion：A lentiviral vector of FNDC5 overexpression is successfully constructed in this study and a THP-1 cell line stably transfected with FNDC5 is established by the lentiviral vector of FNDC5 overexpression，indi－cating that FNDC5 can significantly reduce lipid accumulation in THP-1 macrophage-derived foam cells，which lays a foundation for further research.