Unified Cellular and Molecular Core

统一的细胞和分子核心

• 批准号: 7189528
• 负责人: JING ZHENG
• 金额: $ 16.14万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7189528
• 关键词: American Type Culture Collection; Antibodies; Area Analyses; Arteries; Biological Assay; Birds; 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; Polymerase Chain Reaction; Productivity; Program Research Project Grants; Proteins; RNA analysis; Reader; Reagent; Research Personnel; 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; human embryonic stem cell; immortalized cell; improved; migration; programs; 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.

本计划项目补助金中描述的所有项目都需要持续不断地提供 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;来自ATCC的永生化细胞）作为阳性对照，用于鉴定人ES衍生的内皮细胞 细胞和血管生成生物测定（项目III）或作为滋养层分化的效应细胞（项目IV）。 其核心作用是不仅减轻项目人员准备冻结库存的负担， 这些细胞，而且提供高质量的细胞制备和提供数据采集的均匀性 项目之间。核心C还将负责监督技术上困难的分析领域， 与项目1合作，核心将专注于siRNA递送系统的优化， 与核心B（干细胞核心），核心C也将建议免疫组织化学研究，以评估整合 将人ES衍生的内皮细胞移植到SCID小鼠的局部血管中。因此 我们的具体目标是：目标1：准备、分离、鉴定、维持和分发UAEC冷冻库存 项目I和II，以及项目III和IV（如有需要）。目标2：维护和分发 HUVEC-C主要用于项目III和IV，但在需要时也用于项目I和II。目标三： 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ES细胞衍生的内皮细胞，HUVEC或UAEC。目标4：建议和监督真实的 对于项目I、II，使用荧光板读数器检测NO的时间和细胞中NO和Ca 2+的成像，以及 ，并在有需要时为项目IV提供服务。目标5：制定/优化更有效的交付方法 并进一步与基于绿色荧光的FACS（细胞分选）纯化联合收割机结合， 蛋白质（GFP）共递送至转染的细胞。目标6：为抗体优化提供持续支持 用于蛋白质和RNA分析的基础和定量方法，包括提供的RT-PCR和siRNA 在上一个融资期。目标7：提供一个可用的新鲜试剂来源， 使用寿命长，或者批量购买时更经济。总之，作为核心，我们将 提供急需的技术专长、基本电池供应和质量保证， 能够直接比较来自不同项目和不同细胞类型的数据。