构建LILRA2过表达慢病毒载体及建立THP-1-LILRA2稳定转染细胞株OA

Objective To construct a recombinant lentiviral vector overexpressing leukocyte immunoglobulin-like receptor subfamily A member 2(LILRA2),establish a stable LILRA2-overexpressing human myeloid leukemia mononuclear cell strain(THP-1),and preliminarily investigate the impact of LILRA2 on the expression of pro-in-flammatory cytokines.Methods The pLV-SFFV-MCS-EF1-ZsGreen1-T2A-Puro backbone vector was double-diges-ted with BamH Ⅰ/Xba Ⅰ to construct the LILRA2-overexpressing recombinant lentiviral vector.After lentiviral packaging,concentration,and titer determination,the optimal multiplicity of infection(MOI=30)and puromycin screening concentration(2 μg/mL)for THP-1 cells were determined.Following lentiviral infection,puromycin se-lection was performed to obtain stable transfectants.LILRA2 expression was verified by RT-qPCR and Western blot.TNF-α and IL-1β expression was determined by RT-qPCR.Results Sequencing confirmed the successful construc-tion of the LILRA2-overexpressing recombinant lentiviral vector.The fluorescence-positive rate in stably transfected THP-1 cells exceeded 90％.LILRA2 mRNA(P<0.001)and protein(P<0.05)expression levels in the THP-1-LILRA2 group were significantly higher than those in the THP-1-NC negative control group.TNF-α(P<0.05)and IL-1β(P<0.05)mRNA levels in the THP-1-LILRA2 group were significantly higher than those in the THP-1-NC negative control group.Conclusions The LILRA2-overexpressing lentiviral vector was successfully constructed and a stable LILRA2-overexpressing THP-1 cell strain was established.LILRA2 overexpression upregulates the expres-sion of pro-inflammatory cytokines in monocytes.