Exploring Oligosaccharide Synthesis in Human Mammary Gland Epithelial Cells

探索人乳腺上皮细胞中的寡糖合成

• 批准号: 8892907
• 负责人: Lars Bode
• 金额: $ 23.25万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-23 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8892907
• 关键词: Anabolism; Animal Model; Biomedical Engineering; Breast Feeding; Calculi; Cell Culture Techniques; Cell Line; Cells; Clinical; Collaborations; Complex; Computer Simulation; Culture Media; Data; Enzymes; Epithelial Cells; Exploratory/Developmental Grant; Exposure to; Fucose; Fucosyltransferase; Future; Galactose; Genes; Genomics; Gland; Goals; Harvest; Human Milk; In Vitro; Infant; Institutes; Intervention Studies; Knowledge; Letters; Mammary gland; Medicine; Messenger RNA; Milk; Modeling; Mothers; Nature; Oligosaccharides; Pathway interactions; Positioning Attribute; Prolactin; RNA Sequences; Research; Role; Sampling; Small Interfering RNA; Stem cells; System; Systems Biology; Testing; Tissue-Specific Gene Expression; Variant; base; differential expression; expression cloning; glycosylation; glycosyltransferase; in vitro Assay; in vivo; mRNA Expression; polylactosamine; public health relevance; research study; response; sialylation; stem; tissue culture; tool; transcriptome sequencing

 DESCRIPTION (provided by applicant): Human Milk ligosaccharides (HMO) are the third most abundant component of human milk. While accumulating evidence suggests that they benefit the breast-fed infant, little is known about how HMO are synthesized in the mother's mammy gland. Up until now, one of the critical barriers in elucidating HMO biosynthesis has been the lack of suitable models that synthesize oligosaccharides that resemble those found in human milk. We now provide preliminary results that place us in a unique position to overcome this critical barrier. We present a model based on mammary gland epithelial cells that were derived from progenitor cells isolated from human milk. Upon exposure to prolactin, these cells synthesize and secrete oligosaccharides that contain all structural features of HMO, including polylactosamine elongation and branching as well as fucosylation and sialylation in all linkages known to occur in HMO. Results are highly reproducible. Oligosaccharide composition in the cell culture supernatant and in the respective milk sample match and reflect interpersonal variations between different milk donors. Oligosaccharide composition in the cell culture media changes in response to cell manipulation with glycosyltransferase-specific small interfering RNA. To date, this is the first and currently only model available to explore HMO biosynthesis. Our application proposes to use this new powerful tool to determine which glycosylation-related genes are differentially expressed when mammary gland epithelial cells are exposed to prolactin (Specific Aim 1) and to identify which enzymes contribute to HMO elongation and branching, fucosylation and sialylation (Specific Aim 2). The proposed experiments and anticipated results have a high potential to significantly advance our knowledge and understanding on how HMO are synthesized in the human mammary gland. A better understanding of how HMO are synthesized in nature will help facilitate in vitro enzymatic and chemo-enzymatic HMO synthesis. In addition, the knowledge gained from the proposed studies may lay the groundwork to bioengineer a cell-based system that contains the glycosylation machinery required to synthesize not one, but a mix of a variety of different oligosaccharides that resemble the complex compositions of oligosaccharides naturally occurring in human milk. HMO produced in these in vitro and in vivo systems can be used for research in in vitro assays and animal models, for clinical intervention studies, and eventually to supplement formula to provide all infants with the benefits of HMO.

 描述（由申请人提供）： 母乳低聚糖 (HMO) 是母乳中第三丰富的成分。虽然越来越多的证据表明它们对母乳喂养的婴儿有益，但人们对 HMO 如何在母亲的乳腺中合成知之甚少。到目前为止，阐明 HMO 生物合成的关键障碍之一是缺乏合适的模型来合成类似于人乳中发现的寡糖。我们现在提供了初步结果，使我们处于克服这一关键障碍的独特地位。我们提出了一个基于乳腺上皮细胞的模型，这些细胞源自从人乳中分离的祖细胞。接触催乳素后，这些细胞合成并分泌含有 HMO 所有结构特征的寡糖，包括聚乳糖胺伸长和分支，以及已知在 HMO 中发生的所有连接中的岩藻糖基化和唾液酸化。结果具有高度可重复性。细胞培养物上清液和各自牛奶样品中的寡糖组成匹配并反映不同牛奶捐赠者之间的人际差异。细胞培养基中的寡糖组成会随着糖基转移酶特异性小干扰 RNA 的细胞操作而发生变化。迄今为止，这是第一个也是目前唯一一个可用于探索 HMO 生物合成的模型。我们的申请建议使用这一新的强大工具来确定当乳腺上皮细胞暴露于催乳素时哪些糖基化相关基因存在差异表达（具体目标 1），并确定哪些酶有助于 HMO 延伸和分支、岩藻糖基化和唾液酸化（具体目标 2）。拟议的实验和预期结果很有可能显着增进我们对 HMO 如何在人类乳腺中合成的知识和理解。更好地了解 HMO 在自然界中是如何合成的将有助于促进体外酶促和化学酶促 HMO 合成。此外，从拟议研究中获得的知识可能为生物工程基于细胞的系统奠定基础，该系统包含合成不是一种寡糖而是合成多种不同寡糖的混合物所需的糖基化机制，这些寡糖类似于人乳中天然存在的寡糖的复杂成分。这些体外和体内系统生产的 HMO 可用于体外测定和动物模型研究、临床干预研究，并最终补充配方奶粉，为所有婴儿提供 HMO 的益处。