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

doi:10.16352/j.issn.1001-6325.2026.04.0498

基础医学与临床 ›› 2026, Vol. 46 ›› Issue (4): 498-503.doi: 10.16352/j.issn.1001-6325.2026.04.0498

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

黄高翔¹, 魏冬梅^2,3, 杨耀¹, 郭夏宁², 周凯⁴, 栾晶^2*

1.联勤保障部队第九二四医院病理科,临床医学研究中心,广西桂林 541002;
2.陕西省脑疾病重点实验室,西安医学院基础与转化医学研究所,陕西西安 710021;
3.陕西科技大学化学与化工学院天然产物稳定同位素组学实验室,陕西西安 710021;
4.西北大学附属医院(西安市第三医院)耳鼻咽喉科,陕西西安 710018

收稿日期:2025-04-01 修回日期:2025-07-17 出版日期:2026-04-05 发布日期:2026-03-24
通讯作者: ^*15619239851@163.com
基金资助:
国家自然科学基金(8230060948,32260239);陕西省重点研发计划(2024SF-YBXM-347)

Construction of LILRA2-overexpressing lentiviral vector and establishment of THP-1-LILRA2 stable transfected cell strain

HUANG Gaoxiang¹, WEI Dongmei^2,3, YANG Yao¹, GUO Xianing², ZHOU Kai⁴, LUAN Jing^2*

1. Department of Pathology, Clinical Research Center, the 924th Hospital of the Chinese People′s Liberation Army Joint Logistic Support Force,Laboratory of Metabolic Diseases Research, Guilin 541002;
2. Shaanxi Key Laboratory of Brain Disorders Institute of Basic and Translational Medicine, Xi′an Medical University, Xi′an 710021;
3. College of Chemistry and Chemical Engineering, Isotopomics in Chemical Biology, Shaanxi University of Science and Technology, Xi′an 710021;
4. Department of Otolaryngology, Xi′an No. 3 Hospital, the Affiliated Hospital of Northwest University, Xi′an 710018, China

Received:2025-04-01 Revised:2025-07-17 Online:2026-04-05 Published:2026-03-24
Contact: ^*15619239851@163.com

摘要/Abstract

摘要： 目的通过构建过表达白细胞免疫球蛋白样受体亚家族A成员2 (LILRA2)重组慢病毒载体和建立过表达LILRA2的稳定转染人髓系白血病单核细胞THP-1细胞株,初步探讨LILRA2对单核细胞炎性因子表达的影响。方法采用BamH Ⅰ/Xba Ⅰ双酶切pLV-SFFV-MCS-EF1-ZsGreen1-T2A-Puro骨架载体,构建LILRA2过表达重组慢病毒载体。经慢病毒包装、浓缩、滴度测定后,通过病毒感染实验确定THP-1细胞株的最佳感染复数(MOI=30)和嘌呤霉素筛选浓度(2 μg/mL)。慢病毒载体感染THP-1细胞后经嘌呤霉素筛选获得稳定转染细胞株,采用RT-qPCR和Western blot验证LILRA2的表达,验证后采用RT-qPCR检测TNF-α和IL-1β的表达。结果测序证实过表达LILRA2的重组载体构建成功。稳定转染的THP-1中荧光阳性率达到90%以上。THP-1-LILRA2组中LILRA2 mRNA(P＜0.001)和蛋白(P＜0.05)的表达水平均显著高于THP-1-NC阴性对照组。THP-1-LILRA2组中TNF-α(P＜0.05)和IL-1β(P＜0.05)mRNA表达水平均显著高于阴性对照组。结论成功构建了LILRA2过表达慢病毒载体且建立了稳定过表达LILRA2的THP-1细胞株,LILRA2过表达能够上调单核细胞炎性因子的表达。

关键词: 白细胞免疫球蛋白样受体亚家族A成员2(LILRA2), THP-1细胞, 慢病毒载体, 促炎性因子

Abstract: 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-inflammatory cytokines. Methods The pLV-SFFV-MCS-EF1-ZsGreen1-T2A-Puro backbone vector was double-digested 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 selection 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 construction 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 expression of pro-inflammatory cytokines in monocytes.

Key words: leukocyte immunoglobulin like receptor subfamily A member 2(LILRA2), THP-1 cells, lentiviral vector, pro-inflammatory cytokines

中图分类号:

R392.12

黄高翔, 魏冬梅, 杨耀, 郭夏宁, 周凯, 栾晶. 构建LILRA2过表达慢病毒载体及建立THP-1-LILRA2稳定转染细胞株[J]. 基础医学与临床, 2026, 46(4): 498-503.

HUANG Gaoxiang, WEI Dongmei, YANG Yao, GUO Xianing, ZHOU Kai, LUAN Jing. Construction of LILRA2-overexpressing lentiviral vector and establishment of THP-1-LILRA2 stable transfected cell strain[J]. Basic & Clinical Medicine, 2026, 46(4): 498-503.

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

Construction of LILRA2-overexpressing lentiviral vector and establishment of THP-1-LILRA2 stable transfected cell strain

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