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）的致病因素。然而，哪些粘膜细菌引起肠粘膜炎症尚未确定。在这项研究中，我们研究了细菌渗透和促炎细胞因子在人类结肠上皮细胞中的分泌，以应对各种粘膜细菌和益生菌。将人结肠上皮细胞（SW 480，ATCC）与分离自UC的细菌（缬沙坦杆菌、大肠杆菌、普通B、梭状芽胞杆菌）、幽门螺杆菌（ATCC 43504，TN 2GF 4）和益生菌（约氏乳杆菌、德氏乳杆菌保加利亚亚种）共培养。用细菌感染细胞培养物，并以细菌/细胞比10001重悬于DMED和含10%FBS的Ham’s F-12培养基的1：1混合物中。将共培养物进一步孵育1小时和3小时以允许细菌粘附和侵袭细胞，并收集上清液用于粘附和侵袭测定。IL-8、IL-6、IL-18和MCP-1 ...更多信息 用ELISA试剂盒测定上清液中的浓度。使用真实的时间PCR通过比较Ct方法测量m-RNA的诱导。电镜观察细胞的附着和侵袭情况。在附着试验中，所有细菌的回收率均为0.01-1.49×10^5 CFU/mL，但L.德氏扫描电镜未检测到保加利亚种。除变异镰孢菌外，所有菌株均未检测到细胞因子。暴露于来自UC的三种变异镰孢菌菌株后的IL-18浓度为（平均）19.7-52.9 pg/mL。在侵袭测定中，除益生菌外的所有细菌均以0.03-2.9 X 10^4 CFU/mL从SW 480细胞中回收。暴露于变异镰孢菌后的IL-8浓度为148-210 pg/mL，暴露于来自UC的两种变异镰孢菌菌株后的IL-18浓度为32.0-51.5 pg/mL。暴露于普通双歧杆菌后的IL-8浓度。梭菌和幽门螺杆菌（TN 2BF 4）分别为35、33和47 pg/mL。暴露于所有细菌菌株后的IL-6和MCP-1浓度低于0.150和31.3 pg/mL。IL-6、MCP-1和IL-8的mRNA在除益生菌外的所有菌株中均升高，其中在F varlum中显著升高。在透射电子显微镜中，F.在SW 480细胞中检测到变异株和幽门螺杆菌。益生菌不侵入结肠上皮细胞，因此不诱导细胞中的炎性细胞因子分泌。另一方面，一些粘膜细菌，特别是Fn侵入结肠上皮细胞，并诱导炎性细胞因子的分泌。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