Unified Cellular and Molecular Core

统一的细胞和分子核心

• 批准号: 8300025
• 负责人: JING ZHENG
• 金额: $ 16.16万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2013-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8300025
• 关键词: Antibodies; Area Analyses; Arteries; Biological Assay; Blood Vessels; Blood capillaries; Cell Separation; Cells; Collaborations; Data; Effector Cell; Endothelial Cells; Evaluation; Freezing; Funding; Green Fluorescent Proteins; Half-Life; Human; Human Resources; Image; In Vitro; Methods; Molecular; Pregnancy; Productivity; Program Research Project Grants; Protein Analysis; RNA analysis; Reader; Reagent; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; SCID Mice; Small Interfering RNA; Source; Stem cells; Stimulation of Cell Proliferation; Supervision; System; Technical Expertise; Time; Tube; Umbilical vein; angiogenesis; base; capillary; cell preparation; cell type; data acquisition; embryonic stem cell; human embryonic stem cell; immortalized cell; improved; migration; quality assurance; trophoblast

All of the projects described in this Program Project Grant either require a constant and consistent supply of uterine artery endothelial cells (UAEC) (Projects I and II) or use human umbilical vein endothelial cells (HUVEC-C; immortalized cells from ATCC) as positive control for identifying human ES derived-endothelial cells and angiogenesis bioassays (Project III)or as effector cells for trophoblast differentiation (Project IV). The role of the core is to not only relieve the project personnel of the burden of preparing frozen stocks of these cells, but also to provide a high quality of cell preparation and to provide uniformity of data acquisition between the projects. Core C will also undertake to supervise technically difficult areas of analysis In collaboration with Project 1 the core will focus on optimization of siRNA delivery systems and in collaboration with Core B (Stem Cell Core), Core C will also advise immunohistochemical studies for evaluating integration of human ES derived-endothelial cells into local blood vessels in SCID mice as proposed in Project III. Thus our specific aims are to: Aim 1: Prepare, isolate, characterize, maintain and distribute UAEC frozen stocks for Projects I and II, and also for Projects III and IV when need arises. Aim 2: Maintain and distribute HUVEC-C as required mainly for Projects III and IV but also Projects I and II when the need arises. Aim 3: Supervise in vitro angiogenesis assays including mitogenesis, migration, capillary tube formation assays in Project III using human ES cell-derived endothelial cells, HUVEC or UAEC. Aim 4: Advise and supervise real time detecting NO using fluorescent plate reader and imaging of NO and Ca2+ in cells for Projects I, II, and , and also for Project IV when the need arises. Aim 5: Develop/optimize methods for more efficient delivery of siRNA to UAEC and further combine with FACS (sorting of cells) purification based on green fluorescent protein (GFP) co-delivery to transfected cells. Aim 6: Provide ongoing support for optimization of antibody based and quantitative methods for protein and RNA analysis, including RT-PCR and siRNA as provided during the prior funding period. Aim 7: Provide an available source of fresh reagents which have short half life or are significantly more economical when purchased in bulk. In summary, by acting as a Core, we will provide much needed technical expertise, essential cell supplies and quality assurance, so contributing to the ability to directly compare data from different projects and between cell types.

本计划项目赠款中描述的所有项目要么需要持续和一致的 子宫动脉内皮细胞(UAEC)(项目I和II)或使用人脐静脉内皮细胞 (HUVEC-C；ATCC永生化细胞)作为鉴定人ES来源内皮细胞的阳性对照 细胞和血管生成生物测定(项目III)或作为滋养层细胞分化的效应细胞(项目IV)。 核心的作用不仅是减轻项目人员准备冻结库存的负担 这些电池，而且还提供高质量的电池制备和提供数据采集的一致性 在两个项目之间。核心C还将负责监督#年技术困难的分析领域。 与项目1的协作核心将侧重于siRNA递送系统的优化和协作 对于核心B(干细胞核心)，核心C还将为评估整合的免疫组织化学研究提供建议 将人类ES来源的内皮细胞转化为SCID小鼠的局部血管。因此 我们的具体目标是：目标1：准备、隔离、表征、维护和分发UAEC冻结库存 用于项目I和II，并在需要时用于项目III和IV。目标2：维护和分发 HUVEC-C主要用于项目三和四，但在需要时也包括项目一和项目二。目标3： 监督体外血管生成试验，包括有丝分裂、迁移、毛细管形成试验 项目III使用人类胚胎干细胞来源的内皮细胞，HUVEC或UAEC。目标4：为REAL提供建议和监督 使用荧光平板阅读器检测NO的时间以及项目I、II和项目II细胞内NO和钙离子的成像 ，并在需要时也适用于项目IV。目标5：开发/优化方法以实现更高效的交付 并进一步与基于绿色荧光的FACS(细胞分类)纯化相结合 蛋白质(GFP)共传递到转基因细胞。目标6：为抗体的优化提供持续支持 蛋白质和RNA分析的基础和定量方法，包括提供的RT-PCR和siRNA 在上一次供资期间。目标7：提供新鲜试剂的可用来源，这些试剂的半衰期较短 如果批量购买，性命或性价比明显更高。总而言之，通过充当核心，我们将 提供急需的技术专业知识、基本电池供应和质量保证，从而为 能够直接比较来自不同项目和不同单元格类型的数据。