Conditionally immortalized TM cell and RGC lines from Animal Model

描述（由申请人提供）：青光眼是世界上不可逆视力丧失和失明的主要原因。青光眼损伤的两个主要部位是小梁网（TM）和视网膜神经节细胞（RGC），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，导致与这种疾病相关的视力丧失。通过对培养的TM细胞和RGC的研究，已经了解了大量关于青光眼的细胞和分子生物学。然而，目前的TM细胞和RGC培养物存在显著的局限性。TM细胞生长相对缓慢，衰老迅速，限制了对早期细胞传代的相对小的细胞数量的研究。RGC是终末分化的，因此，对于进行的每个实验，必须从新生啮齿动物的眼睛制备原代RGC。虽然已经产生了永生化细胞系，但这些细胞在培养中持续增殖，并且已经失去了原代TM细胞和RGC的其他表型特征。对于新的TM细胞和RGC系存在明确的需求，其可以容易地繁殖并且仍然保留原代细胞培养物的表型特征。ImmortoMouse被开发为一种独特的资源，用于从各种组织中产生条件永生化细胞，当细胞在允许的温度下培养时，这些细胞将持续增殖，但当在非允许的温度下生长时，这些细胞将恢复其正常表型。我们的假设是，可以从ImmortoMouse中分离出条件永生化的小鼠RGC和TM细胞系，当在非允许条件下生长时，其将具有原代TM细胞和RGC的特征。以下具体目标将阐述这一假设。(SA#1）我们将开发和表征条件永生化小鼠TM细胞系，其将在允许条件下（在33°C下与γIFN一起培养）快速增殖，并且当在非允许条件下（在39°C下不含γIFN地培养）生长时将具有原代TM细胞的特征。(SA#2）我们将开发和表征条件永生化小鼠RGC系，其将在允许条件下快速增殖，并且当在非允许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很有创新性。(A)我们将分离和表征第一个条件永生化的TM和RGC细胞系。(B)我们还开发了一种新的方法，使用吞噬磁性微球纯化小鼠TM细胞。这些线将提供给各种各样的视觉研究人员：（a）更好地了解TM和RGC生物学，（B）发现和表征新的致病途径，（c）测试潜在的新治疗药物。

DESCRIPTION (provided by applicant): 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 33°C with γIFN) an will have characteristics of primary TM cells when grown under non-permissive conditions (culture at 39°C without γIFN). (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.