基于Arg1/Nos2启动子的巨噬细胞极化实时报告系统构建与优化

Construction and optimization of two real-time macrophage polarization reporter systems based on Arg1/Nos2 promoters

摘要:

为满足活细胞条件下对巨噬细胞极化方向与强度进行非破坏、可重复、定量监测的需求，本研究分别构建并优化了以Arg1 (M2相关)与Nos2 (M1相关)启动子为核心的2套报告系统。采用分泌型NanoLuc (secreted NanoLuc, secNluc)报告系统作为检测工具，以脂多糖(lipopolysaccharide, LPS)与干扰素-γ (interferon-γ, IFN-γ)联合刺激，以及受照肿瘤细胞条件培养基分别作为M1样与肿瘤相关巨噬细胞样(M2偏向)诱导剂，在RAW264.7细胞中系统筛选Arg1启动子(P_{Arg1-0/-1/-2/-3})和Nos2启动子截短片段(P_{Nos2-0/-1/-2/-3/-4})，并比较增强子-启动子相对位置，建立稳转株并验证。结果显示，P_Arg1-2与P_Nos2-1在基础活性与诱导响应性之间表现出最佳平衡；增强子位置效应则与基因相关：在Arg1体系中，增强子位于启动子下游时的增强作用优于上游(7.8倍vs. 2.0倍)；而在Nos2体系中，增强子位于启动子上游时的增强作用优于下游(20.7倍vs. 2.4倍)。稳转株经条件培养基或LPS/IFN-γ刺激后，Nluc活力分别提升约10.0倍与8.5倍。通过有限稀释法进一步获得了RAW264.7-PNos2和RAW264.7-PArg1单克隆细胞株，并利用该系统验证了转化生长因子-β1对Nos2和Arg1表达的差异性调控及其时间依赖性。本研究所建立的报告系统可在体外活细胞条件下实现巨噬细胞极化趋势的连续、定量监测，适用于在体外模拟的肿瘤微环境中巨噬细胞极化趋势评估，并具备用于板式多条件比较、剂量-反应评估及高通量筛选的应用潜力，为巨噬细胞极化相关机制研究和干预因素筛选提供了实用工具。

Abstract:

To enable non-destructive, repeatable, and quantitative monitoring of macrophage polarization direction and intensity under live-cell conditions, we constructed and optimized two reporter systems centered on the Arg1 (M2-associated) and Nos2 (M1-associated) promoters, respectively. Using a secreted NanoLuc (secNluc) reporter system as the detection tool, we applied combined stimulation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ), and conditioned medium from irradiated tumor cells as M1-like and tumor-associated macrophage-like (M2-skewed) inducers, respectively. In RAW264.7 cells, systematic screening was performed for truncated Arg1 promoter fragments (P_{Arg1-0/-1/-2/-3}) and Nos2 promoter fragments (P_{Nos2-0/-1/-2/-3/-4}). The relative positions of enhancers and promoters were compared, followed by the establishment and validation of stable cell lines. The results showed that P_Arg1-2 and P_Nos2-1 achieved the best balance between basal activity and inducible responsiveness. The positional effect of enhancers was gene-dependent: in the Arg1 system, downstream enhancer placement was more effective than upstream placement (7.8-fold vs. 2.0-fold), whereas in the Nos2 system, upstream enhancer placement was more effective than downstream placement (20.7-fold vs. 2.4-fold). Upon conditioned-medium or LPS/IFN-γ stimulation, Nluc activity in the stable cell lines increased by approximately 10.0-fold and 8.5-fold, respectively. Monoclonal RAW264.7-PNos2 and RAW264.7-PArg1 cell lines were further obtained by limiting dilution, and the system was then used to verify the differential regulation of Nos2 and Arg1 expression by transforming growth factor-β1 and its time-dependent pattern. In summary, the established reporter systems enable continuous, quantitative monitoring of macrophage polarization in live cells under in vitro conditions. They are suitable for evaluating polarization trends in tumor-mimicking in vitro microenvironments and hold promise for plate-based multi-condition comparison, dose-response evaluation, and high-throughput screening, thereby providing a practical tool for mechanistic studies of macrophage polarization and for screening modulatory factors.