Project 1 - Spleen

项目1-脾脏

• 批准号: 10211114
• 负责人: MARK A. ATKINSON
• 金额: $ 48.27万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-14 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10211114
• 关键词: 3-Dimensional; Abnormal Red Blood Cell; Anatomy; Antigens; Atypical lymphocyte; B-Lymphocytes; Back; Benchmarking; Biological Markers; Blood; Cell Compartmentation; Cells; Chromium; Clinical Pathology; Collection; Communities; Computer software; Coupled; Cryopreservation; Cytometry; Data; Deposition; Detection; Disease; Erythrocytes; Excision; Expression Profiling; Fluorescence Microscopy; Fluorescence-Activated Cell Sorting; Fluorescent in Situ Hybridization; Formalin; Foundations; Freezing; Gene Expression; Generations; Genetic Transcription; Germany; Glass; Hematopoiesis; Human; Human BioMolecular Atlas Program; Image; Immune; Immune response; Immunofluorescence Immunologic; Immunohistochemistry; Immunologic Surveillance; Immunophenotyping; Individual; Iron; Label; Light; Lymphatic System; Lymphocyte; Magnetic Resonance Imaging; Malpighian corpuscles; Maps; Marburgvirus; Measurement; Measures; Messenger RNA; Microanatomy; Microscopy; Modification; Molecular; Optics; Organ; Organ Donor; Pancreas; Paraffin; Paraffin Embedding; Particulate Matter; Pathologic; Pathology; Patients; Peripheral Blood Mononuclear Cell; Phagocytes; Phase; Polymers; Population; Procedures; Proteins; Protocols documentation; Quality Control; RNA; Reagent; Reporting; Research Personnel; Resolution; Rodent; Role; Sampling; Slide; Source; Specimen; Spleen; Splenic Red Pulp; Splenocyte; Stains; Standardization; Structure; Structure of germinal center of lymph node; Technology; Temperature; Three-Dimensional Image; Thymus Gland; Time; Tissues; Transplantation; Universities; Validation; base; blood filtration; cell type; confocal imaging; density; design; experience; fascinate; fetal; file format; human imaging; human tissue; imaging system; mRNA Expression; magnetic field; microscopic imaging; millimeter; multiphoton imaging; nanometer; particle; preservation; reconstruction; scale up; secondary lymphoid organ; single cell analysis; single-cell RNA sequencing; small molecule; spectroscopic imaging; trafficking; transcriptome sequencing

The spleen has been considered an enigmatic and mysterious organ since its original discovery in the ancient era. The spleen specifically controls phagocytic removal of abnormal red blood cells (RBCs) and particulate matter, iron storage from RBCs, initial immune response to circulating antigens, and fetal hematopoiesis. Blood filtration is anatomically associated with the red pulp whilst detection of infectious particles and initiation of an immune response occurs via formation of germinal centers in the white pulp. The microanatomy of the human spleen, however, has been primarily extrapolated from extensive studies in rodents. Recent studies by Dr. Birte Steiniger (University of Marburg, Germany), have in fact indicated that as one example, the well- delineated B-cell compartment, known as the marginal zone, between white and red pulp is distinctly absent in the human spleen. The generation of a three dimensional (3D) tissue map of normal human spleen is therefore timely from both the scientific and pathologic perspectives. One of our obvious strengths is extensive experience with procurement and handling of transplant quality human organs using high level quality control measures (AIM I). Our proposed AIMs are designed to first obtain an understanding of the overall uniformity and macro-anatomy of the human spleen using MRI (AIM IIA). We have then designed interactive tissue handling (formalin-fixed paraffin embedded [FFPE], optimal cutting temperature [OCT] compound embedded, tissue clearing, and expansion) and optical microscopy (stochastic optical reconstruction microscopy [STORM], confocal, multiphoton, light sheet fluorescence microscopy [LSFM]) pipelines, which will resolve the microanatomy of the spleen from nanometer to millimeter in resolution (AIM IIB-D), superimposed with known spleen biomarkers. Isolated/dispersed splenocytes will be compared to peripheral blood mononuclear cells (PBMCs) from the same patient's blood (AIM III), and isolated immune cell subsets will be subjected to RNA- Seq (AIM IVB). Imaging mass cytometry (IMC) will provide the ultimate co-registration of biomolecules/biomarkers to individual cells (AIM IVA). Similarly, we will use multiplexed small molecule fluorescence in situ hybridization (FISH) and ultimately high throughput multiplexed error-robust FISH (MERFISH) to co-register mRNA expression to the cognate cell types (AIM IVB). Data from RNA-Seq of individual cells will further provide new splenic biomarkers to feed back to AIMs IIB-D and IVA. The ability to reconstruct an overall 3D spleen tissue map from all proposed pipelines is based on the common file format used for all optical microscopy, IMC and FISH applications as delineated in the Data Core. With all of these individual and cooperative strengths, we are poised to complete a 3D tissue map of the normal human spleen that can be shared with all HuBMAP, HIVE and TMC investigators and ultimately, the entire scientific community.

脾自古代被发现以来，一直被认为是一个神秘而神秘的器官。 时代脾特异性地控制异常红细胞（RBC）和颗粒的吞噬清除。 物质，红细胞的铁储存，对循环抗原的初始免疫应答和胎儿造血。血液 过滤在解剖学上与红髓相关，而感染性颗粒的检测和 免疫应答通过在白色牙髓中形成生发中心而发生。人体的显微解剖学 然而，脾脏主要是从啮齿类动物的广泛研究中推断出来的。最近的研究博士。 Birte Steiniger（马尔堡大学，德国），事实上已经指出，作为一个例子，良好的- 在白色和红色髓之间的被描绘的B细胞室，称为边缘区，明显缺乏， 人类的脾脏。因此，正常人脾的三维（3D）组织图的生成是 从科学和病理学的角度来看都是及时的。我们的一个明显优势是广泛 使用高水平质量控制采购和处理移植优质人体器官的经验 目标（AIM I）。我们提出的AIM旨在首先了解整体一致性 和宏观解剖的人脾脏使用MRI（AIM IIA）。我们设计了交互式组织 处理（福尔马林固定石蜡包埋[FFPE]，最佳切割温度[OCT]化合物包埋， 组织清除和扩张）和光学显微术（随机光学重建显微术[STORM]， 共焦，多光子，光片荧光显微镜[LSFM]）管道，这将解决 显微解剖脾从纳米到毫米的分辨率（AIM IIB-D），叠加已知的 脾脏生物标志物。将分离/分散的脾细胞与外周血单核细胞进行比较 将来自同一患者血液（AIM III）的PBMC和分离的免疫细胞亚群进行RNA-聚合酶链反应（RT-PCR）。 Seq（AIM IVB）.成像质量细胞仪（IMC）将提供最终的共同登记， 生物分子/生物标志物与单个细胞的关系（AIM IVA）。同样，我们将使用多路小分子 荧光原位杂交（FISH）和最终高通量多重错误鲁棒FISH （MERFISH）以将mRNA表达共配准到同源细胞类型（AIM IVB）。数据来自RNA-Seq， 单个细胞将进一步提供新的脾生物标志物以反馈给AIM IIB-D和IVA。的能力 从所有建议的管道重建整体3D脾脏组织图是基于通用文件格式 用于数据核心中描述的所有光学显微镜、IMC和FISH应用。与所有这些 凭借个人和合作的优势，我们准备完成正常人脾脏的3D组织图 可以与所有HuBMAP，HIVE和TMC研究人员共享，最终，整个科学 社区