Mattar A F, Drongowski R A, Coran A G, Harmon C M
Section of Pediatric Surgery, University of Michigan, Mott Children's Hospital, Ann Arbor, MI 48109-0245, USA.
Pediatr Surg Int. 2001 May;17(4):265-8. doi: 10.1007/s003830100591.
Enteral probiotics such as Lactobacillus casei GG (LGG) have been used in the treatment of a variety of intestinal disorders in infants and children, including diarrhea, malabsorption, and Clostridium difficile colitis. We have previously demonstrated that the probiotic bacterium LGG has an inhibitory effect on bacterial translocation (BT) in a neonatal rabbit model. However, this in-vivo model is limited for investigating the cellular and molecular mechanisms responsible for probiotic inhibition of BT. The purpose of this study was to determine the efficacy of LGG in reducing the rate of Escherichia coli C25 (E. coli C25) translocation using an in-vitro enterocyte cell-culture model. Human colonic carcinoma (Caco-2) enterocytes were seeded in porous filters in the apical chamber of a two-chamber cell-culture system and grown for 14 days to confluence. The monolayers were incubated at 37 degrees C with LGG for 180 min. Non-adherent LGG was washed away prior to a 120-min incubation period with 10(5) CFU E. coli C25. E. coli that had translocated across the enterocyte monolayer were quantified by growing basal-chamber media samples on gram-negative bacteria-specific MacConkey's agar. In order to determine monolayer integrity, transepithelial electrical resistance (TEER) was measured across Caco-2 cells treated with LGG and E. coli. Statistical analysis was by ANOVA with P < 0.05 considered significant. LGG inhibited E. coli translocation at all LGG concentrations tested. The TEER ratio was not significantly altered by addition of LGG or E. coli (0.9 +/- 0.03 vs 0.8 +/- 0.05). These results demonstrate that the probiotic bacterium LGG inhibits BT of E. coli C25 in a dose-dependent manner in an in-vitro cell-culture model. This model should be valuable in investigating the cellular and molecular mechanisms involved in the inhibition of pathological enteral bacteria by probiotic agents.
诸如干酪乳杆菌GG（LGG）等肠道益生菌已被用于治疗婴幼儿的多种肠道疾病，包括腹泻、吸收不良和艰难梭菌结肠炎。我们之前已经证明，益生菌LGG在新生兔模型中对细菌移位（BT）具有抑制作用。然而，这种体内模型在研究益生菌抑制BT的细胞和分子机制方面存在局限性。本研究的目的是使用体外肠上皮细胞培养模型确定LGG在降低大肠杆菌C25（E. coli C25）移位率方面的效果。将人结肠癌细胞（Caco-2）肠上皮细胞接种在双室细胞培养系统顶腔的多孔滤器中，培养14天至汇合。将单层细胞在37℃下与LGG孵育180分钟。在与10(5) CFU大肠杆菌C25孵育120分钟之前，洗去未粘附的LGG。通过在革兰氏阴性菌特异性麦康凯琼脂上培养基底腔培养基样品来定量已穿过肠上皮细胞单层移位的大肠杆菌。为了确定单层完整性，测量了用LGG和大肠杆菌处理的Caco-2细胞的跨上皮电阻（TEER）。采用方差分析进行统计分析，P < 0.05被认为具有显著性。在所有测试的LGG浓度下，LGG均抑制大肠杆菌移位。添加LGG或大肠杆菌后，TEER比值没有显著改变（0.9 +/- 0.03对0.8 +/- 0.05）。这些结果表明，在体外细胞培养模型中，益生菌LGG以剂量依赖的方式抑制大肠杆菌C25的BT。该模型在研究益生菌制剂抑制病理性肠道细菌的细胞和分子机制方面应该具有重要价值。
