IMMUNO-MODULATOR EFFECT S OF COLON MUCOSAL BACTERIA AND PROBIOTICS ON MUCOSAL IMMUNITY

近年来，肠道细菌可能是溃疡性结肠炎(UC)难以捉摸的致病因素。然而，目前还没有确定是哪种粘膜细菌引起了肠黏膜的炎症。在这项研究中，我们研究了不同的粘膜细菌和益生菌对人结肠上皮细胞的细菌渗透和促炎细胞因子分泌的影响。人结肠上皮细胞(SW480，ATCC)与来自UC(F.valitrn1，E.Coli，B.uggatus，Clostridloforme)，H.Pylori(ATCC 43504，Tn2GF4)和益生菌(Lactobacillusjohnsonii，L.delBrueckii subsp.，Bulgaricus)的细菌共培养。细胞培养物被细菌感染，再悬浮在1：1的DMed和Ham的F-12培养液中，其中含有10%的FBS，细菌/细胞比为10001。共培养1h和3h，以允许细菌对细胞的黏附和侵袭，收集上清液进行黏附和侵袭实验。IL-8、IL-6、IL-18和单核细胞趋化蛋白-1…用酶联免疫吸附试验试剂盒检测上清液中更多的浓度。采用实时定量聚合酶链式反应的比较CT方法检测m-RNAs的诱导量。用电子显微镜观察细胞的附着和侵袭情况。在贴壁试验中，除德氏乳杆菌亚种外，所有细菌的回收率均在0.01-1.49×105cfu/mL之间。扫描电子显微镜未检出保加利亚乳杆菌。除杂色镰刀菌外，其余菌株均未检测到细胞因子。UC分离的3株变种镰刀菌对IL-18的平均浓度为19.7~52.9pg/mL。在侵袭实验中，除益生菌外，所有细菌均可从SW480细胞中回收，浓度为0.03~2.9×10~(-4)cfu/m L。白介素8浓度为148-210pg/mL，白介素18浓度为32.0-51.5pg/mL。布氏杆菌感染后IL-8水平的变化。肺炎衣原体和幽门螺杆菌(TN2BF4)的最低抑菌浓度分别为35、33和47pg/mL。IL-6和单核细胞趋化蛋白-1浓度分别低于0.150和31.3pg/mL。但除益生菌外，其余菌株IL-6、MCP-1和IL-8的m-RNA均升高，且F varlum的m-RNA显著升高。在透射电子显微镜下，在SW480细胞中检测到变种镰刀菌和幽门螺杆菌。益生菌不侵入结肠上皮细胞，因此不能诱导细胞分泌炎性细胞因子。另一方面，一些粘膜细菌，特别是FN侵入结肠上皮细胞，诱导炎性细胞因子的分泌。较少

Recently, intestinal bacteria might be the elusive pathogenic factors in ulcerative colitis (UC). However, which mucosal bacteria cause inflammation of the intestinal mucosa has not been determined. In this study, we investigated bacterial penetration and the secretion of proinflammatory cytokines in human colon epithelial cells in response to various mucosal bacteria and probiotics. Human colonic epithelial cells (SW480, ATCC) were cocultured with bacteria isolated from UC (F.valitrnl, E.coli, B.vulgatus, C.clostridloforme), H.pylori (ATCC 43504, TN2GF4), and probiotics (Lactobacillus johnsonii, L.delbrueckii subsp, bulgaricus). Cell cultures were infected with bacteria and resuspended in a 1:1 mixture of DMED and Ham's F-12 medium containing 10% FBS at a bacterium/cell ratio of 10001. The co-cultures further incubated for 1 h and 3h to allow bacterial adhesion and invasion to cells, and the supernatants were collected for attachment and invasion assay. The IL-8, IL-6, IL-18 and MCP-1 … More concentrations in the supernatants were determined with ELISA kits. The induction of m-RNAs was measured by comparative Ct methods using real time PCR. The attachment and invasion were also analyzed by electron microscopy. In attachment assays, all bacteria were recovered at 0.01-1.49×10^5 CFU/mL, however L. delbrueckii subsp. bulgaricus was not detected by scanning electron microscope. The cytokines were not detected in all strains except for F.varium. IL-18 concentrations after exposure to three F.varium strains from UC were (mean) 19.7-52.9 pg/mL. In invasion assays, all bacteria except for probiotics were recovered from SW480 cells at 0.03-2.9 X 10^4 CFU/mL. IL-8 concentrations after exposure to F.varizrm were 148-210 pg/mL and IL-18 concentrations after exposure to two F.varium strains from UC were 32.0-51.5 pg/mL. IL-8 concentrations after exposure to B.vulgatus. C.clostridioforine and H.pylori (TN2BF4) were 35, 33 and 47 pg/mL, respectively. IL-6 and MCP-1 concentrations after exposure to all bacteria strains were below 0.150 and 31.3 pg/mL. However, m-RNA of IL-6, MCP 1 and IL-8 were increased in all strains except for probiotics, and marked increased in F varlum. In transmission electron microscopy, F. varium and H pylori were detected in SW480 cells. Probiotics did not invade the colonic epithelial cells and therefore did not induce inflammatory cytokines secretion in the cells. On the other hand, some mucosal bacteria, especially Fn invaded colonic epithelial cells and induced inflammatory cytokines secretion. Less