Conditionally immortalized TM cell and RGC lines from Animal Model

来自动物模型的条件永生化 TM 细胞和 RGC 系

基本信息

批准号：
8636379
负责人：
Abbot Frederick Clark
金额：
$ 14.5万
依托单位：
UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-02-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8636379
关键词：
Address Adult Affect Anatomy Animal Model Anterior Anterior eyeball segment structure Aqueous Humor Biology Blindness Cell Count Cell Culture Techniques Cell Line Cells Cellular biology Characteristics Data Disease Eye Eye diseases Genetic Engineering Glaucoma Goals Grant High temperature of physical object Individual Interferons Large T Antigen Learning Magnetism Major Histocompatibility Complex Methods Microspheres Molecular Molecular Biology Morphology Mouse Strains Mus Muscle Fibers Neonatal Neurosciences Research Optic Nerve Pathogenesis Pathway interactions Phenotype Photoreceptors Physiologic Intraocular Pressure Population Primary Cell Cultures Primates Proliferating Property Publications Rattus Research Research Personnel Resistance Resources Retina Retinal Retinal Ganglion Cells Rodent Site Structure of sinus venosus of sclera Temperature Testing Therapeutic Agents Tissues Trabecular meshwork structure Vision Visual impairment Work anterior chamber cell type cold temperature collecting tubule structure immortalized cell innovation magnetic field mouse model novel novel therapeutics promoter public health relevance research study tool

项目摘要

Abstract Glaucoma is a leading cause of irreversible vision loss and blindness in the world. Two major sites of glaucoma damage are the trabecular meshwork (TM), which leads to elevated intraocular pressure, and retinal ganglion cells (RGCs) that progressively die in glaucoma, leading to the loss of vision associated with this disease. A great deal has been learned about the cell and molecular biology of glaucoma through research on cultured TM cells and RGCs. However, there are significant limitations with current TM cell and RGC cultures. TM cells grow relatively slowly and rapidly senesce, limiting studies to relatively small cell numbers of early cell passages. RGCs are terminally differentiated, and therefore, primary RGCs must be prepared from neonatal rodent eyes for each experiment conducted. Although immortalized cell lines have been generated, these cells continuously proliferate in culture and have lost other phenotypic characteristics of primary TM cells and RGCs. There is a definite need for new TM cell and RGC lines that can be easily propagated and still retain the phenotypic characteristics of primary cell cultures. The ImmortoMouse was developed as a unique resource to generate conditionally immortalized cells from a wide variety of tissues that will continuously proliferate when the cells are cultured at permissive temperatures, but regain their normal phenotypes when grown at non-permissive temperatures. Our hypothesis is that conditionally immortalized mouse RGC and TM cell lines from the ImmortoMouse can be isolated, which will have the characteristics of primary TM cells and RGCs when grown under non-permissive conditions. The following Specific Aims will address this hypothesis. (SA#1) We will develop and characterize conditionally immortalized mouse TM cell lines that will rapidly proliferate under permissive conditions (culture at 33oC with ¿IFN) and will have characteristics of primary TM cells when grown under non-permissive conditions (culture at 39oC without ¿IFN). (SA#2) We will develop and characterize conditionally immortalized mouse RGC lines that will rapidly proliferate under permissive conditions and will have characteristics of primary RGCs when grown under non- permissive conditions. This project is very innovative in several ways. (A) We will isolate and characterize the first conditionally immortalized TM and RGC cell lines. (B) We also have developed a novel method using phagocytized magnetic microspheres to purify mouse TM cells. These lines will be made available to a wide variety of vision researchers to: (a) better understand TM and RGC biology, (b) discover and characterize new pathogenic pathways, and (c) test potential new therapeutic agents. !

抽象的青光眼是世界上导致不可逆视力丧失和失明的主要原因。青光眼损害的是小梁网（TM），导致眼内压升高，视网膜神经节细胞（RGC）在青光眼中逐渐死亡，导致与此相关的视力丧失通过对青光眼的研究，人们对青光眼的细胞和分子生物学有了很多了解。然而，目前的 TM 细胞和 RGC 培养存在显着的局限性。 TM细胞生长相对缓慢，衰老迅速，限制了对早期细胞数量相对较少的研究 RGC 是终末分化的，因此，原代 RGC 必须从新生儿中制备。虽然已经产生了永生化细胞系，但这些细胞在每次实验中都被啮齿类动物的眼睛所观察到。在培养物中不断增殖，并且失去了原代TM细胞的其他表型特征 RGC 确实需要易于繁殖且仍能保持活力的新 TM 细胞和 RGC 系。 ImmortoMouse 被开发为保留原代细胞培养物的表型特征。从多种组织中产生有条件永生化细胞的独特资源当细胞在允许的温度下培养时，它们会持续增殖，但会恢复正常状态我们的假设是，在非允许的温度下生长时的表型是有条件地永生化的。可以从 ImmortoMouse 中分离出小鼠 RGC 和 TM 细胞系，其具有以下特征：原代 TM 细胞和 RGC 在非允许条件下生长时将实现以下具体目标。 (SA#1) 我们将开发并描述条件永生化小鼠 TM 的特征。在允许的条件下（在 33oC 下用 ¿IFN 培养）会快速增殖的细胞系，并且将具有原代 TM 细胞在非允许条件下生长时的特征（39oC 培养，无 ¿IFN）。 (SA#2) 我们将开发并鉴定条件永生化小鼠 RGC 系，这些系将迅速在允许的条件下增殖，并且在非生长条件下生长时将具有原代 RGC 的特征这个项目在几个方面都非常创新（A）我们将分离和表征。 (B) 我们还开发了一种新方法用于纯化小鼠 TM 细胞的吞噬磁性微球将可供广泛使用。各种视觉研究人员能够：(a) 更好地了解 TM 和 RGC 生物学，(b) 发现并表征新的致病途径，以及（c）测试潜在的新治疗药物。！