NOD1与增强型绿色荧光蛋白标记的活化T细胞核因子2共表达细胞株的构建  

Establishment of cell line with NOD1 receptor and enhanced green fluorescent protein labeled nucleus factor of activated T cells 2

作　　者：王佳琪 李玉蕾 雍政 周培岚 苏瑞斌 WANG Jia-qi;LI Yu-lei;YONG Zheng;ZHOU Pei-lan;SU Rui-bin(Nanjing University of Chinese Medicine,Nanjing 210046,China;Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]南京中医药大学,江苏南京210046 [2]军事医学研究院,北京100850

出　　处：《药学学报》2025年第4期1012-1018,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(82273909)。

摘　　要：建立NOD1和增强型绿色荧光蛋白(EGFP)标记的活化T细胞核因子2(NFAT2)(EGFP-NFAT2)共表达细胞株。将pcDNA3.1-NOD1-3×Flag-hygro重组质粒转染至U2OS-EGFP-NFAT2细胞,经潮霉素压力筛选后加入脂多糖(lipopolysaccharides,LPS)孵育30 min,通过高内涵筛选系统检测细胞核内绿色荧光强度,验证EGFP-NFAT2核转位,筛选得到46株稳定表达NOD1的细胞株U2OS-EGFP-NFAT2-NOD1,其中4号细胞株细胞核内绿色荧光强度最强,故选定其为U2OS-EGFP-NFAT2-NOD1细胞株进行功能验证。采用实时定量PCR(RT-qPCR)检测该细胞株中NOD1 mRNA水平和Western blot法检测NOD1蛋白的表达水平,结果显示在传代5~20代内NOD1 mRNA均稳定表达,并且该细胞株中可明显表达NOD1蛋白,而对照细胞U2OS-EGFP-NFAT2中未见NOD1蛋白表达。将U2OS-EGFP-NFAT2-NOD1细胞接种于96孔板,加入组蛋白(histone)孵育30 min,通过高内涵筛选系统检测EGFPNFAT2核转位,验证该细胞株NOD1功能的特异性,发现组蛋白在不同浓度范围内使U2OS-EGFP-NFAT2-NOD1稳转细胞中EGFP-NFAT2核转位明显增加。将U2OS-EGFP-NFAT2-NOD1稳转细胞株分为溶媒对照组、NOD1拮抗剂nodinitib-1+组蛋白组、组蛋白组。药物孵育时间均为30 min。通过高内涵筛选系统观测EGFP-NFAT2核转位,结果表明与组蛋白组比较,nodinitib-1+组蛋白组EGFP-NFAT2核转位较明显减弱(P<0.05)。以上结论表明成功构建了共表达NOD1和EGFP-NFAT2的稳转细胞U2OS-EGFP-NFAT2-NOD1,可用于靶向NOD1的病原微生物的抗性化合物筛选及其分子机制研究。To establish the cells stably co-expressing NOD1 receptor and enhanced green fluorescent protein(EGFP)-tagged nuclear factor of activated T cells 2 nuclear factor (NFAT2)(EGFP-NFAT2) in U2OS cell,the NOD1 (NM_006092) pcDNA3.1-3×Flag-hygro recombinant plasmid was transfected into U2OS-EGFP-NFAT2cells,which were screened by pressure of hygromycin B and then incubated with NOD1 agonist lipopolysaccharides (LPS) for 30 min,and the green fluorescence intensity in the nucleus of the cells was detected by the high content screening assay.There were 46 cell strains expressing NOD1 in U2OS-EGFP-NFAT2 cells by EGFP-NFAT2 nuclear translocation assay.Among these cells,cells No 4 had the highest nuclear translocation function.Therefore,it was selected as the U2OS-EGFP-NFAT2-NOD1 cell for functional validation.The expression levels of NOD1 mRNA and protein in the selected U2OS-EGFP-NFAT2-NOD1 cells and the control cell U2OS-EGFP-NFAT2 were examined by real-time quantitative PCR (RT-qPCR) and Western blot.The results showed that NOD1 mRNA was stably expressed in this stably transfected cell line for 5-20 generations,and NOD1 protein was expressed in U2OS-EGFP-NFAT2-NOD1 stably transfected cell line,whereas no NOD1 protein was expressed in the control cell U2OS-EGFP-NFAT2.U2OS-EGFP-NFAT2-NOD1 cells were treated with histones or LPS for 30 min,and the EGFP-NFAT2 nuclear translocation was detected by the high content screening assay.Histones were found to significantly increase the EGFP-NFAT2 nuclear translocation in U2OS-EGFP-NFAT2-NOD1 stably transfected cells over a range of concentrations.The U2OS-EGFP-NFAT2-NOD1 cells were divided into the solvent control group,NOD1 receptor antagonist nodinitib-1+histone group,and histone group.The drug incubation time was 30 min,and the specificity of the NOD1 cells was verified by observing the EGFP-NFAT2 nuclear translocation through the high content screening assay.Compared with the histones group,the nodinitib-1+histones group significantly decreased EGFP-NFAT2 nuclear translocation in

关 键 词：病原微生物 NOD1受体 NFAT转录因子类 核转位实验 组蛋白 

分 类 号：R966[医药卫生—药理学]