Caco-2细胞与肠道菌共培养初建体外肠道共生模型

Co-culture of Caco-2 Intestinal Cell and Intestinal Floras to Build an In Vitro Intestinal Model

【目的】构建体外模拟肠道共生系统,用于评价食源性抗菌药及其耐药菌作用于肠道菌群时对人体健康造成的危害风险。【方法】21 d体外培养构建Caco-2细胞三维肠上皮,分成无菌对照组、混合的正常菌群组、沙门菌组、混合菌群-沙门菌组4组,制备混悬液,各组分别单独和混合培养4 h,荧光定量PCR法测试共培养前后各菌16SrDNA菌量变化,电阻仪测试跨膜上皮电阻(TEER)数量;免疫荧光染色观察紧密连接蛋白ZO-1变化,评价三维肠上皮屏障功能的改变。【结果】在体外共培养模型内,4种肠道菌混合培养4 h后同单独培养组间比较,细菌量增长无统计学差异(P>0.05));需氧菌的增长速度明显高于厌氧菌(P<0.01),厌氧菌:需氧菌浓度比接近100∶1,厌氧菌仍为优势菌。TEER和ZO-1免疫荧光染色结果均提示混合肠道菌群对Caco-2细胞三维肠上皮无明显破坏,侵入性沙门氏菌可显著降低TEER值,并观察到模型的ZO-1结构受到破坏。这种破坏作用在混合肠道菌群加沙门菌群组较轻。【结论】Caco-2细胞三维肠上皮同乳双歧杆菌、植物乳酸杆菌、大肠杆菌和粪肠球菌组成的混合菌群体外共培养能建立稳定的体外肠上皮模型,并具有生物学活性;正常肠道菌群对侵入性沙门菌有拮抗作用。该模型可进一步完善作为食源性抗菌药、耐药菌与肠道系统相互作用研究模型的基础。

[ Objective ] To build an in vitro intestinal model which will be used to assess the harm risk caused by foodborne antimicrobial and the resistance bacteria to human health. [ Method] The Caco-2 intestinal epithelial models after 21 days culture was built, 4 groups were divided： negative control group, mix floras group, Salmonella group, mix floras ＆ Salmonella group, the time for co-culture was 4h. The 16S rDNA fragment of each flora before and after co-culture by quantitative fluorescence PCR was quantified respectively, and the changes of Caco-2 cells intestinal integrity were evaluated by detecting Trans-epithelial electrical resistance （TEER） and the immunofluorescence staining image of tight junction protein ZO-1. [ Results ] From the 16S r DNA fragment of four intestinal floras quantified by qPCR, there were no significant changes between alone cultured and the mixed culture （P 〉 0.05）, the 2 aerobes grown significantly faster than the growth of the 2 anaerobes （P 〈 0.01）. B/E was near 100： 1, showed that the aerobes were dominant bacteria. Compared with the negative control, TEER and ZO-1 distribution in mix floras group had no significant change and revealed that there were no significant damages to the integrity of Caco-2 intestinal epithelial cells, while in Salmonella group, TEER reduced significantly, and the destruction of ZO-1 structure was observed. But the damages in mix floras ＆ Salmonella group （mixed floras pre-planted） could be some extent reduced. [Conclusion] This research built an primary in vitro co- culture intestinal epithelial model with Caco-2 cell and 4 intestinal floras-Bifidobacterium laetis, Laetobacillus plantum, Escherichia coli and Enterococcus faecalis. The model showed the certain stability and biological activity. Normal intestinal flora had inhibitory effect to invasive salmonella. Furthermore, we will refine the model to lay the foundation to study the interaction of foodborne antimicrobial drug, drug resistant bacteria and intestinal system.