Probiotics regulate intestinal epithelial cell homeosta

描述（由申请人提供）： 益生菌是通过预防或改善疾病而有益于宿主的微生物。然而，关于使用益生菌作为替代医学的科学依据，知之甚少。我们报道了一种这样的益生菌，鼠李糖乳杆菌GG（LGG），通过激活抗凋亡Akt/蛋白激酶B和抑制精氨酸刺激的促凋亡p38/有丝分裂原活化蛋白（MAP）激活来防止小鼠和人结肠细胞中精氨酸诱导的凋亡。我们进一步证明了从LGG培养液上清液（LGG-s）回收的产物显示Akt的浓度依赖性活化和对精氨酸诱导的细胞凋亡的抑制。本研究的长期目标是研究益生菌对正常肠道生长发育的调节作用机制，并为疾病的潜在治疗应用提供机制基础。本研究旨在探讨益生菌调节肠上皮细胞稳态的细胞和分子机制。有三个具体目标：目标1。鉴定从LGG-s中回收的调节结肠上皮细胞存活的蛋白质。LGG-s中的蛋白质将通过柱色谱法或电洗脱法纯化，并用于处理小鼠和人结肠上皮细胞以确定其对Akt活化和细胞存活的影响。确定LGG-s中存在的细胞信号传导和存活调节蛋白的氨基酸序列并克隆编码这些蛋白的基因。将确定从调节结肠细胞存活的LGG-s纯化的蛋白质的氨基酸序列（Aim 1）用于设计简并寡核苷酸探针。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。目标3.为了确定肠上皮细胞相互作用蛋白和来自LGG-s的纯化蛋白的调节细胞存活的分子靶标，将使用表达为GST融合蛋白的LGG-s中的蛋白进行下拉测定或蛋白阵列以筛选结肠上皮细胞裂解物。利用DNA微阵列技术鉴定结肠细胞中LGG调控的基因。这些发现将对进一步了解益生菌对肠道健康的调节具有重要意义。

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.