基于类器官培养的艰难梭菌毒素B损伤结肠上皮机制的初步研究

A preliminary study using organoid culture method to investigate the mechanism underlying colonic epithelial injury due to Clostridioides difficile toxin B

目的 建立艰难梭菌感染结肠类器官模型,对艰难梭菌毒素B(TcdB)损伤小鼠结肠上皮的作用及可能机制进行初步研究。方法 处死6~8周龄C57BL/6小鼠,取结肠标本,分离、收集结肠隐窝,以基质胶包埋后加入培养基,显微镜下观察记录生长情况;在巨大芽孢杆菌中表达重组艰难梭菌毒素B(rTcdB)蛋白,经镍柱纯化后加入结肠类器官体系继续培养,观察类器官生长情况并检测干性基因表达量变化,转录组测序分析。结果 5pmol/L rTcdB作用12 h即可致结肠类器官生长缓慢及部分死亡,干性基因Lgr5、Axin2、Bmi1表达较对照组显著上调。RNA-seq测序结果显示毒素作用组较对照组差异基因有3 140个,其中上调基因有1 936个,下调基因有1 204个。Gene Ontology富集分析显示损伤组差异基因主要富集的生物过程通路分别有低氧应答和对β干扰素、γ干扰素的细胞应答。同时蛋白质互作网络(Protein-Protein Interaction Networks, PPI)筛选出9个关键基因,其中的IFIT3、IFIT1、GBP3和GBP2的编码基因也参与了α/β干扰素、γ干扰素的细胞应答通路。京都基因与基因组百科全书(KEGG)通路富集分析结果表明有47条通路差异显著富集,包括花生四烯酸代谢通路和Wnt信号通路等。结论 TcdB可能通过调节干性基因表达,增强低氧应答通路,减弱α/β干扰素、γ干扰素的细胞应答通路,调节花生四烯酸代谢通路和Wnt信号通路等介导损伤结肠上皮细胞。

Objective A Clostridioides difficile-infected colon organoid model was established to study the potential mechanism underlying the injury of mice colonic epithelium due to C. difficile toxin B(TcdB). Methods The C57BL/6 mice of 6-8 weeks old were sacrificed. The colon was removed and cut longitudinally. Then, the crypts of colon were separated with 5 mmol/L ethylenediaminetetraacetic acid(EDTA) solution. The isolated crypts were embedded in Matrigel. The growth media was added to culture, the growth of which was observed under microscope. Recombinant TcdB(rTcdB) was expressed in Bacillus megaterium harboring TcdB coding gene. rTcdB protein was purified by HisTrapHP and was added into colon organoid culture system. Then,the growth of organoids was observed. The expression of stemness genes Lgr5, Axin2, Bmi1 were detected, and RNA sequencing(RNA-seq) was analyzed. Results Exposure to 5 pmol/L rTcdB for 12 h could cause delayed growth and partial death of colon organoids, and significantly increased expression of stemness genes, such as Lgr5, Axin2, Bmi1 compared with control group.RNA-seq analysis showed that a total of 3 140 differentially expressed genes(DEGs) were identified, of which 1 936 DEGs were up-regulated and 1 204 DEGs were down-regulated. GO enrichment analysis of up-regulated DEGs and down-regulated DEGs revealed that biological process(BP) associated terms were “response to hypoxia” and “cellular response to interferon-β and interferon-γ” respectively. In addition, PPI analysis obtained 9 hub genes, some of which such as IFIT3,IFIT1, GBP3 and GBP2 were involved in cellular response to interferon-α/β/γ. KEGG enrichment analysis revealed 47 enriched pathways, and up-regulated DEGs and down-regulated DEGs enriched pathways were “arachidonic acid metabolism” and “Wnt signaling pathway” respectively, which may be involved in Tcd B-induced injury of colonic epithelium. Conclusions A organoid culture model of Tcd B-induced injury of colonic epithelium was successfully established. Tcd B may play a role in regulating expression of stemness genes, enhancing response to hypoxia, impairing cellular response to interferon-α/β/γ, regulating arachidonic acid metabolism and Wnt signaling pathway to mediate injury of colonic epithelium.