Probiotics regulate intestinal epithelial cell homeostasis

描述(由申请人提供)： 益生菌是通过预防或改善疾病而使宿主受益的微生物。然而，关于使用益生菌作为替代医学的科学原理，人们知之甚少。我们报道了一种这样的益生菌，Lactobacillusrhamnosus GG(LGG)，通过激活抗凋亡的Akt/蛋白激酶B和抑制细胞因子激活的促凋亡p38/丝裂原激活蛋白(MAP)来防止细胞因子诱导的小鼠和人结肠细胞的凋亡。我们进一步证明，从LGG培养上清液中回收的产物(LGG-S)显示出浓度依赖的Akt激活和抑制细胞因子诱导的细胞凋亡。本研究的长期目标是探讨益生菌对正常肠道生长发育的调控作用机制，为其在疾病治疗中的潜在应用提供机制基础。目前的研究计划集中在研究益生菌调节肠上皮细胞动态平衡的细胞和分子机制。目的：1.鉴定从LGG-S中回收的调控结肠上皮细胞存活的蛋白质。用柱层析或电洗脱法纯化LGG-S中的蛋白质，并将其用于处理小鼠和人结肠上皮细胞，以确定其对Akt激活和细胞存活的影响。2.测定氨基酸序列并克隆存在于LGG-S中的编码细胞信号和生存调节蛋白的基因(S)。为了设计简并的寡核苷酸探针，将测定调节结肠细胞存活的纯化蛋白LGG-S的氨基酸序列(目标1)。这些探针将用于筛选/杂交LGG基因组DNA文库，以获得编码全长蛋白质信息的DNA。目的3.为了确定肠上皮细胞相互作用蛋白和纯化的调节细胞存活的蛋白的分子靶点，我们将利用LGG-S中表达的GST-融合蛋白进行下拉实验或蛋白质阵列筛选结肠上皮细胞裂解产物。在结肠细胞中受LGG调控的基因将使用DNA微阵列进行鉴定。这些发现将对进一步了解益生菌对肠道健康的调控具有重要意义。

DESCRIPTION (provided by applicant): Probiotic bacteria are microorganisms that benefit the host by preventing or ameliorating disease. However, little information is known regarding the scientific rationale for using probiotics as alternative medicine. We reported that one such probiotic, Lactobacillus rhamnosus GG (LGG), prevents cytokine-induced apoptosis in both mouse and human colon cells through activating the anti-apoptotic Akt/protein kinase B and inhibiting cytokine-stimulated pro-apoptotic p38/mitogen-activated protein (MAP) activation. We further demonstrated that products recovered from LGG culture broth supernatant (LGG-s) show concentration-dependent activation of Akt and inhibition of cytokine-induced apoptosis. The long-term goal of this study is to investigate the mechanism of the probiotic bacterial regulatory effects on normal intestinal growth and development and to provide the mechanistic basis for potential therapeutic applications in diseases. The present research proposal is focused on studying the cellular and molecular mechanisms of probiotic regulation of intestinal epithelial cells homeostasis. Three Specific Aims are addressed: Aim 1. To identify proteins recovered from LGG-s that regulate colon epithelial cell survival. Proteins in LGG-s will be purified by column chromatography or electro-elution, and used to treat mouse and human colon epithelial cells to determine their effects on Akt activation and cell survival Aim 2. To determine amino acid sequence and clone the gene(s) encoding cell signaling and survival regulating proteins present in LGG-s. Amino acid sequences of purified proteins from LGG-s which regulate colon cell survival (Aim 1) will be determined for designing degenerate oligonucleotide probes. These probes will be used to screen/hybridize the LGG genomic DNA library to obtain DNA encoding the message for the full-length protein. Aim 3. To determine intestinal epithelial cell interacting proteins and molecular targets of purified proteins from LGG-s which regulate cell survival, Pull-down assays or protein arrays using proteins in LGG-s expressed as GST-fusion proteins will be performed to screen colon epithelial cellular lysates. Genes regulated by LGG in colon cells will be identified using DNA microarray. These findings will have significant relevance to further understanding the probiotic regulation of intestinal health.