工程化益生菌攻克结肠炎｜Absin ELISA 解析炎症机制

近年来，炎症性肠病（IBD）发病率持续攀升，传统治疗手段存在局限性，合成工程益生菌凭借靶向调控肠道菌群、调节肠道免疫的优势，成为肠道炎症治疗的明星研究方向。近期发表于《Cellular & Molecular Immunology》的重磅研究，成功构建了一株定植能力更强、抗炎效果优异的工程化大肠杆菌Nissle 1917（EcN-CPM），系统阐明了其缓解结肠炎的完整分子机制。Absin多款炎症因子ELISA试剂盒在本研究中承担核心检测工作，为实验数据的精准输出、机制论证提供了坚实保障。本文将从研究思路、核心成果、实验工具应用三大维度，带大家深度解析这篇高分文献。

一、研究背景与整体研究思路

1. 研究痛点

大肠杆菌Nissle 1917（EcN）是应用广泛的安全益生菌底盘菌株，遗传操作体系成熟，可用于改造治疗肿瘤、结肠炎、神经类疾病等多种病症。但天然EcN肠道定植能力弱，难以耐受胃酸、胆汁盐的胁迫，且肠道黏附效果差，极大限制了其作为合成益生菌的临床转化潜力。

过往单一改造耐酸/耐胆盐能力的策略，无法应对胃肠道多重胁迫；包埋等物理改良手段又存在耗材操作繁琐等问题。因此，本研究确立多功能基因编辑联合改造的核心思路，从“抗胁迫、强黏附、稳定植"三个维度定向改造EcN，同时结合动物模型、微生物组学、代谢组学、免疫检测等多技术联用，验证工程菌株的抗炎功效与作用机制。

2. 完整研究技术路线

1. 菌株分步改造：通过CRISPR-Cas9基因编辑，分模块优化EcN性能

• 过表达PatE（耐酸激活因子）+ MdtM（胆盐外排转运蛋白），提升菌株胃酸、胆汁盐耐受能力；

• 替换curli纤维（卷曲纤维）操纵子强启动子、敲除csgD，增强胞外卷曲纤维合成，强化肠道上皮黏附能力；

• 整合双模块基因元件，最终获得多功能工程菌株EcN-CPM。

2. 体外性能验证：依次检测菌株耐酸、耐胆盐、细胞黏附能力、生长活性。

3. 体内定植验证：利用荧光/荧光素酶标记菌株，在小鼠体内追踪肠道定植时长与定植量。

4. 结肠炎动物药效评价：构建DSS诱导小鼠结肠炎模型，从体征、肠道组织、肠道屏障、炎症水平评估药效。

5. 机制深度解析：16S rRNA测序分析肠道菌群变化、靶向/非靶向代谢组学检测粪便代谢物、检测肠道免疫相关基因与炎症因子表达，串联“菌群-代谢-免疫"调控通路。

6. 生物安全性评价：长期灌胃菌株，检测小鼠血常规、肝肾功能、行为学、脏器系数等，验证菌株临床应用安全性。

二、核心研究成果

本研究层层递进，从菌株改造到机制解析，取得四大突破性成果，完整揭示EcN-CPM缓解结肠炎的作用逻辑。

成果1：成功构建高抗逆、强定植的工程菌株EcN-CPM

天然EcN在胃酸（pH 2.5）、高浓度胆盐环境中存活率极低，且肠道黏附能力不足。本研究通过双基因组合改造实现性能飞跃：

• 耐酸耐胆盐能力显著提升：过表达PatE可激活大肠杆菌AR1-5全套耐酸系统，大幅提升酸性环境下存活率；MdtM可高效转运胆酸盐与脱氧胆酸盐，相比另一转运蛋白DinF作用更广。双基因共表达菌株在模拟胃肠道环境中，90分钟存活率远高于野生型菌株。

• 肠道黏附能力翻倍：替换curli纤维启动子并敲除调控基因csgD后，菌株卷曲纤维合成量显著增加，对小鼠结肠MC38细胞的黏附能力提升约2倍。

• 体内定植能力：整合两大改造模块的EcN-CPM生长活性不受影响；活体成像、粪便菌落计数结果证实，小鼠灌胃后，EcN-CPM在肠道内定植时长、定植数量均远超野生EcN，可在肠道稳定定植5天以上。

Fig. 4 Construction of an engineered EcN strain with high intestinal colonization ability. A Schematic illustration of the construction of the EcN-CPM and EcN-PM. B qRT‒PCR analysis of the expression of patE, mdtM, csgE, csgB, and csgD in EcN and EcN-CPM. C Growth curves of EcN, EcN-PM, EcN-Csg, and EcN-CPM. D Electron microscopy-based observation of curli fibers in the EcN, EcN-PM, EcN-Csg, and EcN-CPM (scale bar = 1 μm). E Normalized amyloid production of the EcN, EcN-PM, EcN-Csg, and EcN-CPM groups. F Comparison of the adhesion ability of the EcN, EcN-PM, EcN-Csg, and EcN-CPM groups to MC38 cells. The data are presented as the mean ± SEM. The statistical significance between the two groups was analyzed using a t-test. Statistical comparisons among groups (more than or equal to three groups) were performed using one-way analysis of variance (ANOVA) (*p < 0.05, **p < 0.01, ***p < 0.001)

成果2：EcN-CPM显著缓解DSS诱导的小鼠结肠炎，保护肠道屏障

在DSS结肠炎模型中，相比空白组、野生EcN处理组，EcN-CPM治疗组展现出优异的抗炎表型：

• 小鼠体重下降幅度显著减少，盲肠、结肠长度恢复正常；

• 结肠组织HE染色显示，肠道上皮结构完整、隐泡排列规则，组织病理评分大幅降低；

• FITC-葡聚糖实验证明，EcN-CPM可修复受损肠道屏障，降低肠道通透性；

• 核心炎症因子水平得到精准调控：结肠组织中IL-1β、CXCL1、TNF-α、IL-6、IL-17A、IFN-γ等促炎因子大幅下调，抗炎因子IL-10显著回升，恢复至健康小鼠水平。

Fig. 6 Therapeutic efficacy of EcN-CPM against DSS-induced colitis. A Schematic illustration of the animal experiment. B Changes in body weight and weight change of the mice. C The photographs depict representative cecal and colonic specimens collected from mice on day 10. The lengths of the colon (D) and cecum (E) in the different groups. F HE staining analysis of colon tissues collected from the mice in different groups on day 10. These HE-stained images are representative cross-sections of colon tissues. Scale bars represent 100 μm (main images) and 20 μm (high-magnification views). G Comparative analysis of histopathological scores in colon tissues among the four groups of mice. H Serum concentrations of FITC-dextran in different groups. I Concentrations of seven cytokine markers in the colon tissues of different groups. The data are presented as the mean ± SEM. Statistical comparisons among groups (more than or equal to three groups) were performed using one-way analysis of variance (ANOVA) (*p < 0.05, **p < 0.01, ***p < 0.001)

成果3：重塑肠道菌群结构，驱动有益菌群增殖

16S rRNA测序结果证实，EcN-CPM可逆转结肠炎引发的肠道菌群紊乱：

• 显著降低致病菌Escherichia-Shigella（志贺菌属）丰度，该菌会破坏肠道屏障、加剧炎症反应；

• 显著富集norank_f_Muribaculaceae（穆里杆菌科），这类菌群是肠道核心有益菌，可合成短链脂肪酸（SCFAs）；

• 菌群差异与结肠炎表型关联分析表明，菌群结构的改变与炎症缓解、肠道修复高度相关。

Fig. 7 EcN-CPM administration affects the abundance of cecal and colonic microbial genera and their correlations with colitis-associated phenotypes. Relative and inferred absolute abundances of the cecal (A) and colonic (B) microbiomes at the genus level. C Correlations between the cecal and colonic microbes and colitis-associated phenotypes. The association between the colitis phenotype and microbes was determined by Spearman’s rank correlation. The data are presented as the mean ± SEM. Statistical comparisons among groups (more than or equal to three groups) were performed using one-way analysis of variance (ANOVA) (*p < 0.05, **p < 0.01)

成果4：解析“菌群-代谢-免疫"三重抗炎机制

本研究串联代谢组与免疫检测，完整阐明EcN-CPM的抗炎通路，也是全文最核心的机制亮点：

• 促进短链脂肪酸（SCFAs）恢复：EcN-CPM上清液可直接促进norank_f_Muribaculaceae增殖，逆转DSS诱导的SCFAs耗竭；SCFAs含量与志贺菌属负相关、与穆里杆菌科正相关。

• 调控Th17/Treg免疫平衡：SCFAs是调控肠道Th17/Treg稳态的关键代谢物。EcN-CPM处理后，结肠组织中Treg细胞标志物（Foxp3、Ctla4、Cd25）表达上调，Th17细胞相关基因（IL-17A、Roryt）表达下调，失衡的IL-17A/Foxp3比值恢复稳态，从细胞层面抑制过度炎症。

• 恢复7种抗炎代谢物：非靶向代谢组学发现，EcN-CPM可特异性恢复DSS处理后耗竭的马尿酸、松萝酸等7种经典抗炎代谢物，多途径协同抑制肠道炎症。

Fig. 8 EcN-CPM alleviates inflammation by remodeling the gut microbiota and enhancing the levels of fecal metabolites to shift Th17/Treg transcriptional signatures. A Schematic of the experiment assessing the effects of EcN and EcN-CPM supernatants on the relative abundance of norank_f_Muribaculaceae in the fecal microbiota. B The relative abundance of norank_f_Muribaculaceae in groups treated with EcN or EcNCPM supernatants compared with the LB-treated control (Con). C EcN-CPM administration reversed the DSS-induced reduction in the abundance of six short-chain fatty acids (SCFAs). D Correlation analysis between the abundance of the six SCFAs and the relative abundance of Escherichia-Shigella and norank_f_Muribaculaceae. E mRNA expression levels of Foxp3, Ctla4, and Cd25 in colon tissues from the four groups of mice. F Expression levels of IL-17A and Roryt mRNA in colon tissues from the four groups of mice. G The IL-17A/Foxp3 ratio in the colons of the four groups of mice. H Venn diagrams of fecal metabolites identified via nontargeted metabolomics across the four experimental groups. I Principal component analysis (PCA) of gut metabolite profiles among the four groups of mice. J EcN-CPM administration increased the abundance of seven fecal metabolites that were reduced in the DSS group, as determined by nontargeted metabolomics analysis. K Schematic of the mechanism by which EcN-CPM alleviates inflammation through gut microbiome remodeling, which enhances the production of functional fecal metabolites to shift Th17/Treg transcriptional signatures. The data are presented as the mean ± SEM. Statistical comparisons among groups (more than or equal to three groups) were performed using one-way analysis of variance (ANOVA) (*p < 0.05, **p < 0.01, ***p < 0.001)

成果5：EcN-CPM具备良好生物安全性

为期30天的长期灌胃实验证实，EcN-CPM不会影响小鼠体重、进食饮水、神经行为；血常规、肝肾功能、脏器系数均无异常，无明显毒副作用，具备临床转化的安全基础。

三、Absin产品应用详解

在本研究结肠炎药效评价、免疫机制验证的关键环节，研究人员采用Absin品牌ELISA试剂盒完成小鼠结肠组织中多种炎症因子的定量检测，该部分对应原文图6I（7种细胞因子含量检测），是判断菌株抗炎效果、支撑免疫机制结论的核心实验工具。

Fig. 6 Therapeutic efficacy of EcN-CPM against DSS-induced colitis.  I Concentrations of seven cytokine markers in the colon tissues of different groups. The data are presented as the mean ± SEM. Statistical comparisons among groups (more than or equal to three groups) were performed using one-way analysis of variance (ANOVA) (*p < 0.05, **p < 0.01, ***p < 0.001)

1. 文中使用的Absin产品清单及对应检测指标

2. Absin ELISA试剂盒在本研究中的核心作用

精准量化炎症水平，支撑药效结论
DSS诱导结肠炎后，肠道会爆发剧烈炎症，促炎因子大量分泌，抗炎因子表达被抑制。研究人员利用Absin ELISA试剂盒对各组小鼠结肠组织匀浆进行定量检测，直观证明EcN-CPM可显著下调促炎因子、改善肠道炎症状态，为工程菌株缓解结肠炎的药效结论提供直接生化数据支撑。

串联免疫通路，机制逻辑
肠道炎症与Th17/Treg免疫失衡密切相关，细胞因子是该通路的核心标志物。Absin ELISA试剂盒精准完成因子定量检测，结合qRT-PCR基因表达结果，印证了EcN-CPM通过调节肠道免疫稳态抑制炎症的分子机制，让菌群、代谢、免疫三大研究模块形成完整逻辑闭环。

适配复杂样本，保障实验稳定性
本实验检测样本为小鼠结肠组织匀浆，基质复杂、干扰成分多。Absin ELISA试剂盒采用成熟双抗体夹心技术，具备高特异性、高灵敏度、低非特异性结合的特点，可有效规避组织样本带来的干扰，检测结果重复性佳、数据误差小，很好满足高分期刊对实验数据严谨性的要求。

3. 产品核心优势

• 物种高度匹配：全系小鼠炎症因子ELISA试剂盒，针对小鼠样本优化体系，适配结肠炎、肠道菌群、免疫调控类动物实验；

• 检测灵敏度优异：可精准识别组织、细胞上清、血清等样本中的低丰度细胞因子，有效捕捉炎症早期的微弱表达变化；

• 操作简单高效：预包被板式设计，简化实验流程、缩短操作时长，适合大批量动物样本的批量筛查；

• 数据认可度高：检测结果可与病理切片、qRT-PCR等主流实验结果相互佐证，已被多篇高分SCI期刊引用。

产品信息